The 56-year old Chinese patient underwent radical resection and was diagnosed with renal clear cell carcinoma (stage II) in 2015. Diagnosis of the disease was based on magnetic resonance (MR) imaging and pathological findings of kidney cancer (6x4 cm, no lymphovascular invasion, sarcomatoid features, or necrosis), and there was no significant increase in tumor markers. After surgery, IL-2 and IFN-alpha-2b were administered for 2 months each. Lung metastasis was confirmed in April 2016 (Figure 1), and a tyrosine kinase inhibitor (Sunitinib) was administered for 3 months. The drug was discontinued after progression of lung metastasis was found. After cryosurgery for lung metastasis, an immune checkpoint inhibitor (nivolumab) was used for 2 months. Gross hematuria occurred in January 2017. Ultrasonography revealed space-occupying lesions in the bladder, and transurethral cystectomy was performed under general anesthesia. Postoperative pathology showed metastatic clear cell carcinoma. Later, imaging revealed metastases to the right pleura, right kidney, liver, left ribs, and right tonsil. The patient showed systemic fatigue, back and lower limb pain, difficulty walking, recurrent daily fever (around 38.5 C), poor appetite, and emaciation. Peripheral blood erythrocyte count dropped below 3x1012/L and hemoglobin below 100 g/L; the Karnofsky score was 30. After hospital admission, the patient received parenteral nutrition, blood transfusion, analgesics, and other symptomatic treatments. MBV treatment was started on April 12, 2017, after consultation with the patient and his family. This case report was approved by the ethics committee of Fuda Cancer Hospital, Jinan University and was in accordance with the Declaration of Helsinki; the patient's written informed consent including the images for publication was received for this study.
The MBV includes 6 kinds of heat-inactivated bacteria, namely 1) Pertussis bacillus (concentration 9 billion/mL), 2) Diphtheria bacillus endotoxin (concentration 20 Lf/mL), 3) Tetanus bacillus endotoxin (concentration 5 Lf/mL), 4) Typhoid bacillus (concentration of 300 million/mL), 5) Bacillus paratyphoid A/B (concentration 150 million/mL each), and 6) 10% Staphylococcus aureus solution (concentration 1 billion/mL). Strains were purchased from National Institutes for Food and Drug Control. Zhejiang Weixin Biological Pharmaceutical Co., Ltd., was entrusted with technical service and culture of bacterial media. Additional additive included 0.1% Poly I:C (tlrl-pic; InvivoGen, San Diego, CA, USA) and 30% fat emulsion for injection, blended in a certain proportion.
The most common adverse reactions recorded included local (injection site swelling and blisters) and systemic (chills, fatigue, and fever) reactions. Liver function was evaluated based on the levels of alanine transaminase (ALT), aspartate transaminase (AST), total bilirubin (T.BIL), and gamma-glutamyl transpeptidase (GGT) multiple times during MBV treatment. Bone marrow hematopoietic function was evaluated based on the levels of peripheral blood erythrocytes and hemoglobin.
Peripheral blood (2 mL) was collected before and multiple times during MBV treatment for the detection of immune function. Flow cytometry (Facscanto  II; BD, Franklin Lakes, NJ, USA) was used for analysis. The analyzed indexes included the number and function of lymphocytes in the peripheral blood. The BD Multitest 6-color TBNK reagent (no 644611; BD) was used to detect the number of CD3+CD4+ cells (95% range: 441-2,156 cells/mL), CD3+CD8+ cells (95% range: 125-1,312 cells/mL), total CD3+ cells (95% range: 603-2,990 cells/mL), and CD3-CD16+CD56+ cells (95% range: 95-640 cells/mL). The BD Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II (no 551809; BD) was used to detect the expression of IL-2 (95% range: 8-12.5 pg/mL), IL-4 (95% range: 3.5-6 pg/mL), IL-6 (95% range: 2.7-8.5 pg/mL), IL-10 (95% range: 1.8-4 pg/mL), TNF (95% range: 1.7-2.5 pg/mL), and IFN-gamma (95% range: 1.5-4 pg/mL). The tests were performed according to the protocols given in the instruction manuals. Results above or within the reference range were considered to indicate normal immune function. One or more values below the reference range was considered to indicate immune dysfunction.
Since no tumor markers can be used to evaluate tumor activity, CTCs were measured as a biomarker for the identification of patients at high risk of relapse and to monitor immune responses to therapy. According to our previously published reports, CD45-CK+CD326+ cells were defined as CTCs. In normal conditions, the reference number of CTCs is 0-1 cells/7.5 mL blood. CTCs >5 indicate poor prognosis and high risk of recurrence or metastasis. CTCs <5 indicate low risk of recurrence or metastasis. The detection procedure included collection of 7.5 mL blood from the patient and isolation of mononuclear cells using human peripheral blood lymphocyte separation solution (Haoyang Biological Manufacture Co., Ltd., Tianjin, China). Isolated cells were enriched by binding to magnetic CD326 (Ep-CAM) MicroBeads (Miltenyi Biotec Ltd., Bergisch Gladbach, Germany) using magnetic-activated cell sorting. Enriched cells were labeled with monoclonal antibodies targeting CD45, CD326, and CK (Miltenyi Biotec Ltd.) and counted by flow cytometry.
Changes in MR tumor imaging were monitored to evaluate the curative effect of MBV. According to the RECIST 1.1 guideline, therapeutic effect is categorized as a CR, disappearance of tumor detection of all target lesions; partial response (PR), total reduction in the diameter of the target lesions >=30%; stable disease (SD), tumor regression fails to reach PR or progressive disease (PD); or PD, total progression of the tumor diameter >=20%.