A 74-year-old Japanese man was referred to our hospital with multiple left neck masses, a history of weight loss, and a slight fever. His body mass index was 18 kg/m2. He had an approximately 2-cm palpable but painless mass in his left neck. An abdominal mass and enlarged para-aortic lymph node were detected by abdominal ultrasonography. Laboratory testing showed serum C-reactive protein 2.5 mg/dl, serum hemoglobin concentration 5.8 g/dl, serum protein 5.9 g/dl, and serum albumin 2.7 g/dl, suggesting malnutrition and anemia caused by inflammation or bleeding. Carcinoembryonic antigen (CEA) was 31.6 ng/ml. The patient was admitted for further examination. Abdominal computed tomography (CT) scans showed a thickened gastric wall and multiple lymphadenopathies around the para-aortic lesion, portal section, mediastinum, and left supraclavicular soft tissue. 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) revealed abnormal FDG uptake in the same lesions detected by CT (Fig. 1a). Gastroenterological endoscopy revealed a tumor lesion with widespread ulceration in the gastric cardia, with lower esophageal invasion (Fig. 2a). A biopsy of the tumor lesion indicated a pathological diagnosis of poorly differentiated adenocarcinoma. Open left cervical lymph node biopsy showed histological features identical with the gastric tumor, indicating left clavicle lymph node metastasis of GC. Based on these clinical and pathological findings, the clinical stage was T4aN3bM1, stage IV, according to the Union for International Cancer Control (UICC 7th edition).
In consideration of his progressive disease, the patient received a blood transfusion for chemotherapy and S-1 plus cisplatin (S-1/CDDP) was then initiated at Hamanomachi Hospital. S-1 (100 mg/day) was administered orally for three consecutive weeks, and cisplatin (90 mg/body) was administered intravenously on day 8, followed by a 2-week rest period. The left neck tumor and abdominal mass had regressed rapidly after first chemotherapy as well as CEA decrease, and we administered combined CAK cell therapy and S-1/CDDP at our clinic.
CAK cells consisted of activated T cells expressing high levels of the activating receptor, natural-killer group 2, member D (NKG2D), and activated natural killer (NK) cells (Fig. 3). The procedure for CAK cell generation has been described previously [8]. Briefly, peripheral blood mononuclear cells (PBMCs) were collected using a blood cell separator (Haemonetics CCS, Haemonetics Corporation, Braintree, MA, USA) and stimulated with human recombinant interleukin (IL)-2 (rIL2, 200 U/ml; Primmune Inc. Kobe, Japan) and 5 μg/ml antibody to CD3 (MACS GMP CD3 pure; Miltenyi Biotec Inc. Auburn, CA, USA). After 7 days in culture, PBMCs were transferred to a culture-bag system (KBM550, Kohjin Bio, Osaka, Japan) and expanded for 7 days to obtain sufficient numbers of CAK cells. All cultures were inspected for contamination with endotoxin, β-glucan, and peptide-glycan using a Toxinometer ET-6000 (Wako Pure Chemical Industries, Ltd., Osaka Japan), according to Food and Drug Administration guidelines. Mycoplasma contamination was checked using a Mycoalert kit (Lonza Rockland Inc., ME, USA). The cell processing and immunotherapy procedures were approved by the ethics committee of our institution, and the patient provided written informed consent for the procedure. A total of 1.32 × 1010 CAK cells were transferred 14 times over 2 years. After four courses of S-1/CDDP administration, the patient developed chemotherapy-related anorexia and stomatitis (Common Terminology Criteria for Adverse Events grade 3), and chemotherapy was switched to weekly paclitaxel at the request of the patient. Paclitaxel (120 mg/body) was administered weekly, three times every 4 weeks. CAK cell therapy was well tolerated, and the only treatment-related adverse event was low-grade fever. After 2 years of combined chemotherapy (S-1/CDDP, paclitaxel) and CAK cell immunotherapy, the lesions other than the stomach lesion had markedly regressed according to CT and FDG-PET (Fig. 1b), and CEA had decreased from 30.2 ng/ml to 2.2 ng/ml (Fig. 4). We confirmed the maximum reduction in the gastric lesion by gastroenterological endoscopy (Fig. 2b), prompting us to consider conversion surgery. Confirming peritoneal lavage cytology negative and no peritoneal dissemination or liver metastasis, we proceeded to laparoscopy-assisted total gastrectomy for GC with D1+ lymphadenectomy and Roux-en-Y reconstruction. The patient’s postoperative course was uneventful, and he was discharged home on postoperative day 18, tolerating an oral diet. The histological findings of the resected specimen (Fig. 5) showed tumor invasion into the subserosal layer and only one lymph node metastasis in 46 dissected lymph nodes. The resected specimen showed the emergence of irregular pyknotic nuclei and many infiltrating mononuclear CD3+ cells by immunohistochemistry (Fig. 6a, b). These findings suggest the effects of chemotherapy and immunotherapy, respectively. Accumulation of form cells was detected in some dissected lymph nodes, suggesting regression of the metastatic lesions. Based on clinical and these pathological findings, the postoperative stage was ypT2ypN1cM0, ypStage IIA, according to the Union for International Cancer Control (UICC 7th edition).
The patient was treated with adjuvant chemotherapy consisting of paclitaxel plus S-1 for 1 year and immunotherapy with tumor lysate-pulsed DC-activated killer (T-DAK) cell immunotherapy for 5 years. Paclitaxel (120 mg/body) was administered weekly, twice every 4 weeks for four courses, after which it was replaced by S-1 because of difficulty in securing vascular access. S-1 (80 mg/day) was administered orally for 2 weeks, followed by a 2-week rest period. The methods used to prepare the T-DAK cells have been described previously [10]. Firstly, immature DCs were prepared from plastic-adherent PBMCs using recombinant human granulocyte/monocyte colony-stimulating factor (100 ng/ml; Primmune KK, Kobe, Japan) and recombinant human IL-4 (500 U/ml; Primmune KK) for 7 days. Tumor cells obtained from the tumor masses were lysed by five freeze–thaw cycles and then adjusted to 1 mg protein concentration. The tumor lysate was added to immature DCs at a final concentration of 50 μg/ml and then incubated for 12 h, followed by the addition of tumor necrosis factor and interferon-γ. Secondly, non-adherent cells among the patient’s PBMCs were cultured with autologous tumor-pulsed DCs for 1 week in low dose IL-2, then expanded in OKT3-coated flasks followed by culture in oxygen-permeable bags (KBM550BA-M; Kojin-Bio, Saitama, Japan). A total of 3.0 × 1010 T-DAK cells were infused 29 times over 5 years. The patient’s clinical tolerance during and after the adjuvant therapy was excellent, and no adverse reactions were observed. The above cell processing and immunotherapy procedures for T-DAK cell immunotherapy were approved by the ethics committee of our institution.
The patient was followed up carefully and remained well with no signs of recurrence at 5 years and 6 months after surgery.
