Design. This study was designed as a randomized controlled trial to compare the G. lucidum treated group with untreated patients. In the treatment group, patients were treated with 2000 mg of G. lucidum spore powder three times a day for 6 weeks (obtained from the Jiangxi Tianhai Group, Batch number 2014AA022206-LC02). Placebo was applied in untreated group for 6 weeks. At the end of the treatment period, CD3+, CD4 + T cell subsets and inflammatory factors such as Foxp3 + and TGF-β were detected. Routine laboratory data (neutrophil and lymphocyte absolute count, serum ALB and GLB value) were collected during follow-up. PFS was calculated from the first cycle of treatment to the time of documented tumor progression or death.
Participants. The study protocol and all procedures were approved by The Institutional Review Board of the Third Affiliated Hospital of Harbin Medical University. All participants were recruited to the study after signed informed consent and were receiving post-operative adjuvant chemotherapy for breast and lung cancer at the Third Affiliated Hospital of Harbin Medical University from June 2015 to September, 2016. All patients eligible for the study who met the inclusion criteria were assessed by periodic imaging, hematology examination including immune function (on the 21st and 42nd day) and biochemical indices. Outcomes including PFS were determined by clinical evidence.
The eligibility criteria for recruitment to the study were as follows (a) provision of signed informed consent; (b) > 18 years of age; (c) diagnosed with stage I-IIIA breast cancer and non-small cell lung cancer (NSCLC); (d) ECOG score between 0 and 1; (e) Estimated survival period > 3 months; (f) no other underlying diseases leading to immune-compromise; (g) previous surgical treatment and undergoing standard adjuvant chemotherapy. The exclusion criteria were as follows (a) allergic to the G. lucidum; (b) Uncontrolled and symptomatic brain metastases; (c) abnormal levels of serum alanine transaminase (ALT), aspartic acid transaminase (AST), blood urea nitrogen (BUN), or creatinine; (d) hematological disease with hemoglobin levels < 9 g/dL and platelets < 80000/mL.
Immunological Indicators:
Flow cytometry analysis. Forearm venous blood samples were separately collected in 5 mL vacutainers containing EDTA and unfractionated heparin. Flow cytometry was performed according to the manufacturer’s instructions. A BD LSRII flow cytometer (BD Biosciences, Heidelberg, Germany) was used to flow cytometry analysis. The TSNE algorithm from FlowJo software (FlowJo, Ashland, OR, USA) was used to dimensional reduce cell clustering. The following fluorescent antibodies were used for analysis; anti-CD3 PerCP(SK7), anti-CD8 FITC-(SK1), anti-CD25 APC (2A3), anti-CD4 V500(RPAT4), anti-HLA-DR PerCP-Cy5.5 (L243) (obtained from Becton-Dickinson, San Diego, CA, USA), Anti-CD28-APC(CD28.2) (obtained from eBioscience, San Diego, CA) and CD16-FITC, CD56-PE, CD29-FITC, CD45RA-FITC, CD45RO-PE, CD19-FITC (obtained from Beckman-Coulter, USA). T-cell subtypes were differentiated following phenotypic analysis (Fig. 1). This included identification of the following cell subtypes; Total T lymphocytes (CD3+), helper T cells (CD4 + CD8−), cytotoxic T cells (CD4 − CD8 + CD28+), inhibitory T cells (CD4-CD8 + CD28-) and Tregs (CD4 + CD25 high). Expression of the late activation marker, HLA-DR, was determined. Naive (CD45RA + CD45RO−) and memory T cells (CD45RA-CD45RO+) were also distinguished in CD4 + and CD8 + subsets.
RNA isolation and RTqPCR analysis. Total RNA was extracted by TRIzol reagent according to the manufacturer’s protocol. The PrimeScript RT Reagent Kit (Takara) was used to synthesize cDNA. QPCR was conducted using a SYBR Green Master mix (Roche) three times. A standard curve method was used to evaluate the relative expression of genes and β-Actin designated as an internal expression reference gene. The gene-specific PCR primers are summarized in Supplementary Table 1. Of these primers, TGF- 1 and COX-2 demonstrated negative associations with tumor immunity. HIF-1 was associated with the hypoxic response. IL-12R demonstrated consistent association with Th1 cells. IL-2 demonstrated positive associations with Th1 cells, whilst IL-6, IL-10 and TGF-1 were negatively associated with Th2 cells [15].
Clinical data collection. Laboratory data was periodically collected including neutrophil/lymphocyte absolute counts and serum ALB/ GLB. NLR values were defined by dividing the absolute neutrophil count by the absolute lymphocyte count. AGR was reported as the ALB value divided by the GLB value.
Safety and Toxicity. Supervised safety and toxicity was reported for renal (sodium, potassium and urea creatinine) and liver function (total protein, albumin, total bilirubin, alkaline phosphate and alanine transaminase) during follow-up. Adverse effects were assessed using the NCICTC, version 4.0 scale. The Response Evaluation Criteria in Solid Tumors (RECIST, version 1.1) was used to evaluate cancer progression.
Data analysis
All data analyses were performed by clinical oncologists based on descriptive statistical analysis of demographic variables that were used to assess the clinical characteristics of the study samples. The data of T cell subsets was analyzed using FlowJo software (version 7.6.1, FlowJoLLC, Ashland, OR, USA). Differences in T-cell subsets and cytokines in the same patients were statistically compared using a paired t test. The Spearman’s test was conducted to determine correlation between cytokine genes and T cell-associated markers. The impact of Ganoderma spore powder on AGR and NLR with PFS was analyzed using the Kaplan–Meier method with a two-tailed Log-rank test to determine statistical significance. The Cox proportional hazard model for the uni- and multivariate analysis was established to assess the prognostic variables based on PFS. Comparison of P < 0.05 (bilateral) was statistically significant. Data statistics were analyzed by SPSS, version 22 and GraphPad Prism, version 7.