Abstract
Objectives:
To evaluate the effects of Reishimmune-S, a fungal immunomodulatory peptide, on the quality of life (QoL) and natural killer (NK) cell subpopulations in patients receiving adjuvant endocrine therapy (ET) for breast cancer (BC).
Methods:
Patients who received adjuvant ET for stage I-III hormone receptor-positive BC without active infection were enrolled in this prospective pilot study. Reishimmune-S was administered sublingually daily for 6 months. QoL scores, circulating immune cell levels, including lymphocyte/NK cell subpopulations, and plasma levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α were measured at baseline and every 4 weeks. Data were analyzed using linear mixed-effect regression models.
Results:
Nineteen participants were included in the analyses. One patient with underlying asthma did not complete the study owing to the occurrence of skin rashes 15 days after the initiation of Reishimmune-S. No other adverse events were reported. Reishimmune-S supplementation significantly improved the cognitive function at 3 months and significantly decreased the fatigue and insomnia levels at 3 and 6 months, respectively. There was no significant change in the global health/QoL score between baseline and week 4 of treatment. The proportion of CD19+ lymphocytes was significantly higher at 3 and 6 months, and that of NKG2A+ and NKp30+ NK cells was significantly lower at 6 months than at baseline. In addition, fatigue positively correlated with the proportion of NKp30+ NK cells (β ± standard error: 24.48 ± 8.75, P = .007 in the mixed-effect model).
Conclusions:
Short-term supplementation with Reishimmune-S affected the circulating immune cell composition and exerted positive effects on cognitive function, fatigue, and insomnia in patients with BC undergoing adjuvant ET, providing a potential approach for the management of treatment-related adverse reactions in this patient population.
Introduction
Hormone receptor (HR)-positive breast cancer (BC) is the most common type of BC, accounting for approximately 70% of all BC cases. Increased survival of patients with resected HR-positive early BC is strongly associated with the use of adjuvant endocrine therapy (ET) with either tamoxifen or aromatase inhibitors. 1 Recent studies have shown that in addition to suppressing estrogen production and signaling, these agents can enhance the production of inflammatory cytokines, induce dysregulation of activities of circulating immune cells, including natural killer (NK) cells,2 -7 and boost immune responses.8 -10 By disrupting neuro-immune-endocrine (NIE) networks,11,12 ET may contribute to fatigue, 13 sleep disruption, 14 and inflammation around the joints and muscles, 15 leading to impaired quality of life (QoL) and affecting treatment adherence. 16 As nonadherence to adjuvant ET is correlated with impaired cancer-specific survival, 17 proper management of these adverse effects is vital for improving the health outcomes of BC survivors. However, only 44% of patients with ET-related adverse effects use medications to overcome them. 17 Furthermore, evidence-based pharmacologic treatment options are still limited for ET-related adverse effects other than pain and insomnia. 16
Recent studies have shown that NK cells mediate NIE network function 18 and chronic fatigue syndrome development. 13 NK cells are lymphocytes of the innate immune system that monitor the cell surfaces of autologous cells for aberrant expression of major histocompatibility complex (MHC) class I molecules and cell stress markers. In human circulation, NK cells are defined as a cluster of differentiation (CD)3−CD56+ cells. Depending on their environment, NK cells can acquire a variety of phenotypes, which can be further stratified by the expression of cell surface molecules. 19 The cytotoxic function of NK cells is controlled by the inhibitory and activating receptors on their surfaces. CD56dim CD16+ NK cells are the predominant peripheral cytotoxic NK cells, whereas CD56bright CD16- NK cells only have weak cytotoxic potential. The engagement of activating receptors, such as DNAX accessory molecule (DNAM-1) (CD226), 2B4 (CD244), NKG2D (CD314), and NKp30 (CD337), stimulates NK cell activity. 20 The interaction among inhibitory NK receptors, such as killer immunoglobulin-like receptors (KIRs) and NKG2A (CD159a), prevents the activation of NK cells against autologous cells. 21
Ganoderma is a medicinal mushroom with a recorded use of over 2 millennia in Asia and an extensive clinical history of safe use as a single agent.22 -24 It exhibits a broad spectrum of pharmacological activities, including immunomodulatory effects. Fungal immunomodulatory proteins (FIPs) are a group of bioactive compounds produced by fungi, such as Ganoderma, with the ability to modulate the immune system. 25 They have been garnering interest owing to their potential to serve as complementary therapies to improve QoL or performance status in a variety of patients with cancer. 23 Ganoderma microsporum immunomodulatory protein (GMI), also known as FIP-gmi, was identified in G. microsporum and belongs to the Fve-type FIP family based on its immunoglobulin-like tertiary structure characteristics. 26 Fve-type FIPs have been shown to modulate cellular immunity (such as the activities of lymphocytes, antigen-presenting cells, dendritic cells, and macrophages) through the regulation of secretion of a variety of cytokines in vivo.25,27,28
As FIPs have anti-inflammatory properties,28,29 we hypothesized that they could be beneficial for patients with BC who experience adverse effects of adjuvant ET as a result of dysregulation of the NIE networks. To verify this hypothesis, we performed a pilot prospective study to evaluate the effects of GMI on the QoL scores and NK subpopulations using a commercially available FIP, Reishimmune-S.
Methods
Ethics Statement
Approval from the Ethics Committee of Mackay Memorial Hospital was obtained before the commencement of the study (approval number: 21CT014be). Questionnaires and blood samples were collected after obtaining written informed consent from all participants. No ethical issues were encountered during the study.
Study Participants, Study Materials, and Study Design
In this study, patients who were under regular adjuvant ET after the completion of definitive surgery and adjuvant chemotherapy for stage I-III HR-positive BC were included. Patients who did not regularly receive adjuvant ET, those with an unstable liver/renal reserve, and those with an active infection were excluded. In total, 19 patients were screened and enrolled between November 1, 2021, and June 30, 2022. After obtaining written informed consent, a baseline assessment of QoL and evaluation of NK cell subtypes and plasma levels of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) were performed 1 month and 1 day before the initiation of Reishimmune-S treatment. A chart review was conducted to retrieve clinicopathological information, including demographic characteristics and tumor node metastasis (TNM) stage. TNM staging was performed according to the guidelines of the American Joint Committee on Cancer, Eighth Edition (2017). The baseline patient characteristics are shown in Table 1.
Characteristics of Patients Included in the Study.
Abbreviations: ET, endocrine therapy; GnRH, gonadotropin-releasing hormone; HER2, human epidermal growth factor receptor 2.
Recombinant GMI (Reishimmune-S®) was obtained from MycoMagic Biotechnology Co., Ltd., New Taipei City, Taiwan. After the collection of baseline data, all participants were administered Reishimmune-S® sublingually at 0.3 mg/d for 6 months. Evaluation of the QoL questionnaire and blood samples were performed, and complete blood cell counts, lymphocyte and NK phenotype data, as well as plasma IL-6 and TNF-α levels were acquired before treatment and every 4 weeks during treatment until the end of the study. Patients were asked to maintain a diary to record drug compliance, and these diaries were reviewed every 4 weeks until the end of the study. Patients who did not complete 80% of the planned treatment at each visit were withdrawn from the study.
QoL Measurements
QoL was assessed using the European Organization for Research and Treatment of Cancer QoL Questionnaire (EORTC QLQ-C30), which consists of 30 items categorized to assess global health status and functional and symptom scores.30,31 A high score on the functional scale represents a high or healthy level of functioning, whereas a high score on the symptom scale represents a high level of symptomatology or problems. Scores for the global health status/QoL, functional scale, and symptom scale were calculated according to instructions provided with the EORTC QLQ-C30 scoring manual.30,31 All participants completed the questionnaires twice, 1 month and 1 day before the start of Reishimmune-S. The baseline QoL scores were the average of the 2 assessments. After enrollment in the study, responses to the EORTC QLQ-C30 questionnaire were collected every 4 weeks during treatment until the end of the study.
Antibody, Flow Cytometry, and Cytokine Analyses
The following antibodies were used to stain peripheral blood mononuclear cell (PBMC) suspensions: fluorescein isothiocyanate (FITC)-CD3, FITC-CD4, peridinin-chlorophyll-protein (PerCP/cyanine 5.5-CD16, phycoerythrin (PE)-CD19, PE-CD56, adenomatous polyposis coli (APC)-CD158 (KIR2D L1/S1/S3/S5), APC-CD158e1 (KIR3DL1), APC-CD159a (NKG2A), APC-CD226 (DNAM-1), APC-CD244 (2B4), APC-CD314 (NKG2D), and Alexa Fluor (AF)647-CD337 (NKp30) (BioLegend, San Diego, CA, USA), eFluor660-CD107a (eBioscience, San Diego, CA, USA), and CD8 (BD Biosciences, Franklin Lakes, NJ, USA). The subpopulation of circulating NK cells was quantified and analyzed using flow cytometry, as described previously. 32 Briefly, 5 × 105 PBMCs after red blood cell lysis were collected for incubation with antibodies for 30 minutes at 4°C, washed with phosphate-buffered saline and fixed with 4% paraformaldehyde for 20 minutes. The cells were then washed twice with phosphate-buffered saline and analyzed using FACSCalibur (BD Biosciences). Data were analyzed using Cell Quest Pro (FlowJo, LLC, Ashland, OR, USA). Human plasma IL-6 and TNF-α levels were measured with commercially available Quantikine Enzyme-Linked Immunosorbent Assay Kits (R&D Systems, Inc., Minneapolis, MN, USA) in accordance with the manufacturer’s instructions.
Statistical Analysis
To analyze repeated measurements during Reishimmune-S treatment, the relationships between (1) time points and QoL itemized scores, (2) time points and circulating immune biomarkers (immune cell subpopulations, such as leukocytes, lymphocytes, and NK cells, and circulating cytokine levels), (3) circulating immune biomarkers and QoL itemized scores were evaluated using linear mixed-effect regression models. 33 There were 4 repeated measurements at baseline, 1 month, 3 months, and 6 months. In the mixed effect model, we estimated the 3 regression coefficients for 1 month versus baseline, 3 months versus baseline, and 6 months versus baseline (Tables 2 and 3). An unstructured covariance matrix and random intercept model were applied based on the lowest Akaike information criterion value for the model fit. Data were analyzed using SAS (version 9.4; SAS Institute Inc., Cary, NC, USA). All statistical tests were 2-sided. P values of < .05 were considered to indicate statistical significance.
EORTC QLQ-C30 QoL Itemized Scores After Use of Reishimmune-S.
All data were analyzed using a mixed-effect model for repeated measures.
Abbreviations: QoL, quality of life; SD, standard deviation; SE, standard error.
The average of 2 measurements before treatment.
All data is presented as mean score ± SD.
P < .05 by comparison with baseline data using mixed effect model.
1 β ± SE = 4.77 ± 2.13.
2 β ± SE = −6.95 ± 3.28.
3 β ± SE = −12.64 ± 5.42, by mixed effect model.
Presentations of Leukocytes, Lymphocytes, NK Cells, and Cytokines Before and After Reishimmune-S Use.
All data were analyzed using a mixed-effect model for repeated measures.
Abbreviations: NK: natural killer; SD: standard deviation, TNF: tumor necrosis factor-α; IL-6: interleukin-6.
Measurement before treatment.
P < .05 in comparison with baseline data using a mixed-effect model.
Data are presented as fractions of total blood cells count.
Data were presented as fraction in total lymphocytes count.
Data are presented as fraction of CD3−CD56+NK cells count.
Results
Patient Demographics
Nineteen female patients with BC receiving adjuvant ET were included in the study between November 2021 and June 2022. The clinicopathological characteristics of the patients are listed in Table 1. Fifteen of the 19 patients completed 6 months of Reishimmune-S therapy without showing obvious adverse reactions. No impairment of liver or renal function was observed in any of the patients (n = 19). One patient with underlying bronchial asthma dropped out of the study because of skin rashes 15 days after the initiation of Reishimmune-S treatment. Three patients dropped out of the study due to travel and relocation after Reishimmune-S use for 2.8, 4.2, and 5.6 months.
Reishimmune-S Improves Cognitive Function Scores and Reduces Symptom Scores for Fatigue and Insomnia
As shown in Table 2, the EORTC QLQ-C30 function scale assessment showed a significant increase in the cognitive function scores at 3 months (mean score ± standard deviation [SD] at baseline vs Reishimmune-S for 3 months: 82.5 ± 13.9 vs 87.3 ± 15.1; β ± standard error [SE] = 4.77 ± 2.13; P = .03 with the mixed-effect model). In the symptom scale assessments, significant reductions were observed in fatigue scores at 3 months and insomnia scores at 6 months of Reishimmune-S use (for fatigue, mean score ± SD at baseline vs 3 months: 20.8 ± 15.7 vs 13.7 ± 14.4, β ± SE = −6.95 ± 3.28; P = .04 in the mixed-effect model; for insomnia, mean score ± SD at baseline vs 6 months: 32.5 ± 27.5 vs 22.2 ± 20.6, β ± SE = −12.64 ± 5.42, P = .02 in the mixed-effect model). Despite the early improvement in the fatigue score and cognitive function, there was no significant difference in scores between baseline and month 6 of treatment (for fatigue score: baseline vs 6 months: 20.8 ± 15.7 vs 20.0 ± 16.4; P > .05 in the mixed-effect model; for cognitive function, baseline vs 6 months: 82.5 ± 13.9 vs 84.4 ± 14.7; P > .05 in the mixed-effect model). Given the small number of patients, there was no significant change in the global health status/QoL (mean score ± SD at baseline vs 6 months: 76.1 ± 16.8 vs 81.1 ± 10.2; P > .05 in the mixed-effect model).
Reishimmune-S Significantly Increases the Proportion of CD19+ Lymphocytes and Affects NK Cell Phenotypes
We evaluated the changes in the proportion of circulating immune cells induced by Reishimmune-S using flow cytometry. As shown in Table 3, a significant increase was observed in the proportion of CD19+ lymphocytes after 3 and 6 months of Reishimmune-S treatment (mean proportion of CD19+ lymphocytes in total lymphocytes ± SD: 0.22 ± 0.10, 0.31 ± 0.17, and 0.31 ± 0.18 at baseline, 3 months, and 6 months, respectively; P < .05 in the mixed-effect model in comparison with the baseline). The proportion of CD19+ lymphocytes increased by approximately 1.4-fold, whereas the proportion of lymphocytes in circulating white blood cells did not change significantly.
Consistent with previous reports, CD3−CD56+ NK cells, predominantly CD56dim cells (~88%-90%), accounted for 11% to 14% of circulating lymphocytes. Although the proportion of CD3−CD56+ NK cells among the total lymphocytes remained unchanged, the proportions of NKG2A+ and NKp30+ NK cells significantly decreased after 6 months of Reishimmune-S use (for NKG2A + NK cells, the mean proportion in CD3−CD56+ NK cells ± SD at baseline vs 6 months: 0.40 ± 0.19 vs 0.30 ± 0.15, P < .05 in the mixed-effect model; for NKp30+ NK cells, the mean proportion in CD3−CD56+ NK cells ± SD at baseline vs 6 months: 0.43 ± 0.23 vs 0.26 ± 0.12, P < .05 in the mixed-effect model). NKG2A+ and NKp30+ NK cells showed changes of approximately 0.75- and 0.6-fold, respectively. Next, we examined the correlation between changes in relative immune cell proportions and itemized QoL scores. As shown in Table 4, a positive correlation was observed between the fatigue symptomatology score and the NKp30+ NK cell proportion (β ± SE = 24.48 ± 8.75, P = .007 in the mixed-effect model).
Correlation Between EORTC QLQ-C30 QoL itemized Scores and Representative Immune Cells & Cytokines.
Abbreviations: EORTC, European Organization for Research and Treatment of Cancer; QoL, quality of life; NK, natural killer; SE, standard error; TNF, necrosis factor-α; IL-6, interleukin-6.
P < .05.
Discussion
This pilot study demonstrated that the short-term use of Reishimmune-S, a commercially available FIP, could temporarily but significantly improve cognitive function and alleviate fatigue at 3 months and sleep disturbances at 6 months in survivors of BC receiving adjuvant ET. Along with these changes, a significant increase in the proportions of CD19+ lymphocytes and a decrease in the proportions of both NKG2A+ and NKp30+NK cells were observed after 3 to 6 months of Reishimmune-S treatment. A positive correlation was observed between the proportions of NKp30+ NK cells and fatigue.
Fatigue is the most common distressing symptom experienced by survivors of cancer. Over 50% of aromatase inhibitor users report moderate-to-severe fatigue, which is closely related to several clinical features, including insomnia. 34 Although the mechanisms underlying cancer-related fatigue remain to be determined, accumulating data indicate that inflammation may play a key role 35 and that fatigue may be further enhanced by the suppressed estrogen levels during ET. 36
Complementary and integrative medicine is a widely accepted approach to alleviate the adverse effects of cancer treatment. In a study using spore powder extracts of Ganoderma lucidum in patients showing BC-related fatigue and receiving ET, patients reported improvements in the fatigue subscale, physical well-being, anxiety, depression, sleep, cognitive function, and global QoL; furthermore, the levels of circulating immune markers of chronic fatigue, such as TNF-α and IL-6, decreased significantly after 4 weeks of treatment. 37 Consistent with these findings, we found that Reishimmune-S, a purified fungal immune peptide from G microsporum, improves cognitive function, alleviates the severity of fatigue, and reduces insomnia, with alterations in the proportions of NK subpopulation over longer observation periods. However, regarding the fatigue subscale score, our patients demonstrated an improvement only at 3 months, with no sustained effect at 6 months. This observation might be explained by the small number of patients or other confounding factors at longer follow-ups. Unlike the significant improvement in global QoL reported by Zhao et al, 37 the average global health status QoL score in our study increased from 76.1 ± 16.8 to 81.1 ± 10.2 at 6 months; however, the change was not significant in the mixed-effect model. The lack of significance in our study may be partially explained by a difference in patient characteristics. All participants in our study were stably under adjuvant ET treatment for years, with a mean duration of 4.8 years (Table 1). However, 60% of patients in the study by Zhao et al 37 received ET for <3 years. The baseline global health status QoL score in our study was 76.1 ± 16.8 (Table 2), whereas that in Zhao et al’s study 37 was 55.8 ± 22.9.
The correlation between the proportion of NKp30+ NK cells and fatigue in our study suggests an immune-based mechanism for chronic fatigue syndrome. NKp30 is also known as a natural cytotoxicity triggering receptor (NCR)3. However, previous studies on the associations between chronic fatigue syndrome and NCR-expressing NK cells, such as NKp30 and NKp44, have yielded mixed results. 13 Although NK cells possess a repertoire of activating and inhibitory receptors, no single receptor dominates the activation status of these cells. 38 Thus, the integration of signaling from these receptors determines the activation and cytotoxicity of NK cells. Patients who received Reishimmune-S showed reductions in the proportions of NK cells with activating receptors (NKp30) and NK cells with inhibitory receptors (NKG2A). NK cells constitutively express killer immunoglobulin-like receptors (KIRs), such as NKG2A, to recognize self-MHC-1 molecules and guide NK cells during development. However, despite the activity of inhibitory receptors, NK cells require activating signals to exert effector function. 19 Understanding how Reishimmune-S affects the interplay between the 2 groups of receptors on NK cells and their functions was beyond the scope of this study. In this regard, an experimental design that includes a cytotoxicity assay for NK cells may be helpful.
This study had several limitations. First, the study lacked randomization and a placebo control. Although we used the results of 2 independent QoL assessments prior to the start of Reishimmune-S administration as the baseline control, the possibility of placebo effects could not be excluded. Second, QoL and treatment-related fatigue are influenced by patient age, educational level, and polypharmacy. However, the number of participants in this study was too small to account for these psychosocial variables. Third, all participants in the study were stably under adjuvant ET for a mean duration of 4.8 years. Whether such an approach could result in a more significant improvement in the global QoL in poorly adherent patients receiving ET requires further investigation. Fourth, the present study focused on the QoL of survivors of BC receiving ET; therefore, the generalizability of the findings to other cancer types remains to be determined.
To the best of our knowledge, this is the first study to investigate the impacts of Reishimmune-S on survivors of BC receiving ET. As adverse reactions to treatment regimens, such as ET, may impair adherence,39 -41 BC survival outcomes, 17 the development of non-pharmacological interventions for treatment-related fatigue in this population represents an important clinical challenge. Reishimmune-S, a recombinant GMI product, may hold promise for improving fatigue management and cognitive function as well as alleviating sleep disturbance in this population.
Footnotes
Acknowledgements
We thank Dr. Caleb Gonshen Chen at the Hematology Division, Mackay Memorial Hospital, Taipei, Taiwan, and Dr. Hsu-Yuan Fu at MycoMagic Biotechnology Co., Ltd. (New Taipei City, Taiwan) for their helpful discussions and advice. We also thank MycoMagic Biotechnology for supplying Reishimmune-S. We are grateful to Ms. Chih-Hui Hsu for providing statistical consulting services from the Biostatistics Consulting Center, Clinical Medicine Research Center, and National Cheng Kung University Hospital.
Correction (September 2024):
Article updated to add additional affiliation “Department of Medicine, Mackay Medical College, New Taipei, Taiwan” for the author “Ying-Wen Su” and to correct the Dose of Reishimmune-S from 2.3 mg/d to 0.3 mg/d in the “Study Participants, Study Materials, and Study Design” section.
Author Contributions
Conception and design of study: Ying-Wen Su. Acquisition of data (laboratory or clinical): Ying-Wen Su, Wen-Yu Huang, Po-Sheng Yang. Data analysis and/or interpretation: Ying-Wen Su, Sheng-Hsiang Lin. Drafting of manuscript and/or critical revision: Ying-Wen Su, Sheng-Hsiang Lin, Po-Sheng Yang. Approval of final version of manuscript: Ying-Wen Su, Wen-Yu Huang, Sheng-Hsiang Lin, Po-Sheng Yang.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was initiated by the principal investigator Ying-Wen Su and supported by MycoMagic Biotechnology Co., Ltd., New Taipei City, Taiwan (grant number: IAC-11001).
