Maitake mushroom extract in myelodysplastic syndromes (MDS): a phase II study

This phase II, open-label, non-randomized, safety, and efficacy trial enrolled MDS patients with IPSS low- or intermediate-1-risk disease who met criteria for MDS based on the FAB and World Health Organization classification systems [26, 27]. Additional eligibility criteria included age ≥18 years, ability to sign informed consent, bone marrow blasts ≤10 %, absolute neutrophil count (ANC) ≥0.5 K/mcL, and stable disease without history of recurrent infections, treatment with a hypomethylating or other disease-modifying agent, or prior stem cell transplant. Exclusion criteria included history of AML, known human immunodeficiency virus (HIV) infection or allergy to mushrooms. The Memorial Sloan Kettering Cancer Center (MSKCC) Institutional Review Board approved the study. Patients were enrolled after informed consent was obtained by the Leukemia Service at MSKCC.

Primary efficacy endpoints were ANC and neutrophil function as measured by changes in respiratory burst response. Changes in neutrophil count were described using the International Working Group (IWG) modified response criteria for MDS [28].

Secondary efficacy endpoints included changes in hemoglobin, platelet, and reticulocyte counts; G-CSF and GM-CSF levels, monocyte function as measured by respiratory burst response, and iron studies in part because beta-glucans are susceptible to free-radical degradation [29].

Safety was assessed with serial blood chemistry panels and symptom assessment, performed at baseline and study visits at weeks 1, 3, 7, 9, and 12. Adverse events were summarized by grade defined by Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 [30].

Following double baseline evaluation (1 week apart) for hematologic parameters, immune function studies, and symptom assessment, patients were instructed to take Maitake extract (3 mg/kg) twice daily by mouth for 12 weeks [25]. Symptom assessment, hematologic parameters, and immune studies were performed at two baseline visits and during treatment weeks 1, 3, 7, 9, and 12. Measures specific only to the first baseline visit included complete blood count and reticulocyte counts, iron status (serum iron, ferritin, and total iron-binding capacity), and chemistry panel. Iron status and chemistry panel studies were repeated at week 7 and week 12. Blood samples from healthy volunteers were assessed in parallel with patients’ immune studies over the 2-year study period. Data collected over the 12-week period of Maitake consumption were compiled and compared with baseline studies.

We compared the results of immune function studies in the MDS patients to age-matched healthy volunteers, to control for the possibility of waning age-related immune function in our MDS cohort (median age, 70). Volunteers were recruited through study flyers with assistance from the MSKCC volunteer office. Inclusion criteria were healthy individuals ≥55 years of age. Exclusion criteria were current use of corticosteroids or other immunosuppressants, known history of HIV infection, current or previous history of malignant disorder except adequately treated non-melanoma skin cancer, curatively treated in situ cancer of the cervix, or other solid tumors curatively treated with no evidence of disease for >3 years.

Neutrophil and monocyte function was measured on freshly drawn anticoagulated blood from patients and volunteers in the Weill-Cornell Cellular Immunology laboratory. Endogenous and activated intracellular ROS production was assessed by flow cytometry as previously described [17, 31]. Activators were as follows: opsonized Escherichia coli, a physiologic, whole bacterial particulate stimulus that signals through Toll-Like Receptor 4, the lipopolysaccharide (LPS) receptor; PMA, a membrane-soluble signal that bypasses receptor binding, measures capacity for ROS production, and requires an intact NADPH oxidase system; and fMLP. Aliquots of heparinized whole blood from study subjects and HC were mixed with or without activators (Phagoburst, Orpegen Pharma, Heidelberg, Germany) and briefly incubated at 37 °C. Addition of the fluorogenic substrate dihydrorhodamine (DHR-123) was used to detect formation of reactive oxidants after conversion to rhodamine-123. Red cells were removed by lysis and cells were partially fixed. DNA stain was added. Flow cytometric evaluation of respiratory burst activity was performed (FACSCalibur, Becton–Dickinson, San Jose, California), using CellQuest software for acquisition and FlowJo software (Tree Star, Ashland, Oregon) for analysis of the percentage of gated granulocyte and monocyte populations producing ROS and relative amounts by geometric mean fluorescence in each sample.

Blood samples were collected at each visit, and serum was obtained and frozen at −80 °C in multiple aliquots for each patient pending analysis by enzyme-linked immunosorbent assay. Due to a freezer malfunction, all the samples were inadvertently thawed and were not evaluated.

Maitake mushroom (G. frondosa) liquid extract was prepared from raw mushroom cultivated in controlled facilities (Yukiguni Maitake Company Ltd., Japan). After harvesting, the fruiting body was extracted with hot water and alcohol and dried to powder form (Nanba et al. [19], U.S. Patent # 5,854,404) and tested for contaminants including endotoxin (Limulus amebocyte lysate assay). Glycosyl composition analysis [32] and stimulation of G-CSF production were used as quality control markers [18]. The powder was dissolved in glycerin for liquid formulation to yield a concentration of 40 mg/mL (Yellow Emperor Inc., Eugene, Oregon) via the distributor (Tradeworks Group, Brattleboro, Vermont). Manufacturing and control methods were filed with the US Food and Drug Administration under Investigational New Drug #68853.

The sample size was based on published data of neutrophil count, 1,200/mm³ (SD 600/mm³) [33‐35]. A study with 20 evaluable patients would have a confidence interval (CI) around the change in neutrophil counts at 12 weeks of ±270/mm³. Baseline values for the neutrophil and monocyte count and function tests were calculated as the average of values between first and second baseline assessments. Percent change was calculated as the difference between the value at 12 weeks and baseline, divided by the baseline value and multiplied by 100. A one-sample t test was used to evaluate whether the change between raw score at baseline and raw score at 12 weeks was equal to zero.

The protocol suggested the use of analysis of variance (ANOVA) to test whether the means of the baseline neutrophil function tests and means of the baseline monocyte function tests were equal between HC and MDS patients receiving Maitake mushroom extract. However, due to the non-normal distribution in these groups, a rank sum test was used as the principal method of analysis. Although we considered age as a potential confounder, it was not included as a variable in the analysis due to the small sample size.

A subgroup analysis was performed on MDS ROS responses at baseline, weeks 1–3, and weeks 7–12 by repeated measures ANOVA (Prism 5.0). If significant overall variance was found, Tukey’s posttest was used to determine significance of pairwise comparisons. MDS patients’ data were grouped and evaluated as: baseline: repeated values from 2 pre-treatment studies; weeks 1–3: repeated values comprising 2 sets of studies at weeks 1 and 3; weeks 7–12: repeated values comprising 3 sets of studies at weeks 7, 9, and 12. For HC, all values were in a single group and data for the 4 patients with 2 studies were treated as repeated values.

Between April 2010 and June 2012, 23 patients met eligibility criteria and gave informed consent for the study. Two patients were removed during the double baseline screening period for having ANC values ≤0.5 at the second screening with an ANC >0.5 at initial screening, and 21 patients received Maitake extract (Fig. 1). Baseline patient characteristics are summarized in Table 1. The median age of 70 years is consistent with MDS diagnosis in the USA [36]. The time between diagnosis and study entry (approximately 2 years) allowed for adequate pre-treatment assessment of disease stability. MDS subtypes are summarized in Table 1.

A total of 18 patients, 7 women and 11 men, completed the planned 12-week treatment and were evaluable. One patient withdrew due to CTCAE 4.0 grade 1 diarrhea, and 2 other patients were removed due to disease progression. One patient with chronic myelomonocytic leukemia at baseline abruptly progressed to AML after 3 weeks of study medication and expired 2 weeks later. Another patient was removed by the treating physician due to persistent, although not worsening, leukopenia and the decision to try aggressive treatment. Leukopenia preceded Maitake treatment and was evaluated as probably unrelated.

Maitake was generally well tolerated. CTCAE 4.0 grade 1 eosinophilia was noted in 4 patients, and two of these patients also experienced grade 1 diarrhea. One patient experienced grade 1 nausea. One patient with grade 1 diarrhea withdrew consent due to the persistent, albeit low-grade nature of the diarrhea.

Compared with the normal range neutrophil values from MSKCC Clinical Laboratories, 14 (78 %) patients were normal and 4 patients had a low ANC (<1.5 K/mcL) at baseline. Most patients had normal levels of monocytes, neutrophils, and lymphocytes, but 10 (56 %) had a low white blood cell (WBC) count (<4 K/mcL). Three patients had low neutrophil counts with normal monocyte and lymphocyte counts; one patient had a low neutrophil count, normal lymphocyte count, and high monocyte count (>1.3 K/mcL); one patient had normal neutrophil and monocyte counts and a low lymphocyte count (<0.5 K/mcL); five patients had a low WBC (<4 K/mcL) with normal neutrophil, monocyte, and lymphocyte counts (Table 2).

Baseline analysis was carried out using pre-treatment samples from 17 patients. Due to insufficient gated events, one MDS patient’s blood sample was excluded. There was no significant difference in neutrophil and monocyte endogenous intracellular ROS production when baseline values of MDS patients were compared with HC (Table 3). ROS response to fMLP and PMA were also comparable for MDS and HC. In contrast MDS patients’ monocytes demonstrated a significantly lower ROS response to E. coli stimulation compared with HC (mean difference −14.3; 95 % CI −21.1, −7.4; p = 0.002; Table 3).

Most study patients had normal neutrophil counts at baseline. When total neutrophil and monocyte counts were examined over time, no patient met IWG criteria for Hematologic Improvement, although three patients showed transient increases in ANC values that were not sustained. Mean monocyte count (Table 3) was not significantly increased at 12 weeks. A treatment-related decrease in mean neutrophil count (−0.3; 95 % CI −0.5, 0.0; p = 0.044) was not clinically meaningful (Table 3). A small significant decrease in mean hemoglobin, correlating with changes in hematocrit and red blood cell (RBC) count, was observed from 11.5 (±1.4) to 11.0 (±1.4) g/dL as shown in Table 2. Mean corpuscular hemoglobin did not decrease (data not shown). No patient became anemic (Hgb < 9 g/dL). The significant increase in mean eosinophil count (1.37 K/mcL; p = 0.011) was considered CTCAE Grade 1, while basophil count was unchanged (Table 2).

After 12 weeks of Maitake treatment, MDS patients showed an overall increase in mean endogenous neutrophil ROS production ex vivo, indicating enhanced basal function (3.2; 95 % CI 1.3, 5.1; p = 0.005; Table 3). Basal monocyte function also increased at 12 weeks compared with pre-treatment levels (p = 0.021). Stimulated ROS production in monocytes increased in response to both E. coli (p = 0.0004) and fMLP (p = 0.03). Response to PMA did not change in either cell type, and neutrophil responses to fMLP and E. coli were not affected. These responses were comparable with those of HC at baseline. Figure 2 shows a histogram of pre- and post-treatment monocyte responses to fMLP, PMA, and E. coli in a single patient compared with endogenous activity at one baseline visit and again at 12 weeks. Individual MDS patients showed variable changes over the 5 treatment week visits. Figure 3 shows MDS monocyte responses to fMLP and to E. coli at pre-treatment baseline visits, at weeks 1–3, and at weeks 7–12 compared with HC (see Methods). MDS patient monocytes stimulated with fMLP showed increased ROS activity at weeks 7–12 compared with baseline (p < 0.01) and with HC (p < 0.05) by repeated measures ANOVA and Tukey’s posttest. Response to E. coli was reduced compared with HC at baseline, but not at 12 weeks (Table 3). Repeated measures by ANOVA and Tukey’s posttest also show an increase at weeks 7–12 in MDS patients compared with baseline (p < 0.05).