Before enrolling for randomization after surgery, patients were stratified according to their stage, curability, and whether or not they received induction chemotherapy. Those who had received surgery at first were stratified by pathological stage: group I, stage IB; group II, stage II; group III, stage IIIA; and group IV, stages IIIB and IV. Those who had received induction chemotherapy were stratified to group V, stage IIIA and group VI, stages IIIB and IV.
Preparation of activated killer T cells and dendritic cells from regional lymph nodes
Tumor-draining lymph nodes include those from the intra-pulmonary to the mediastinal lymph nodes. 1–2 g of regional lymph nodes located as near to the primary tumors as possible with no metastasis was used. If there was no metastasis, intra-pulmonary or hilar lymph nodes were used, and if metastasis had already taken place as far as the mediastinal lymph nodes, mediastinal lymph nodes without metastasis were chosen.
Halves of the two or three tumor-draining regional lymph nodes (TDLN) with no tumor metastasis were rinsed with 50 ml of RPMI-1640 medium containing antibiotics, while their other halves were submitted for pathological examination for the presence of metastasis. Those in which no metastasis was found were transferred to a sterile Petri dish and minced aseptically into 1-mm3 tissue fragments. Each one showing evidence of metastasis was discarded. The tissue preparation was then suspended in 50 ml KBM-400 or Alyse serum-free lymphocyte medium containing 400 IU/ml human recombinant interleukin 2, transferred to a 75-cm2 culture flask, and incubated at 37 °C in air containing 5 % CO2. When the TDLN started to release AKT-DC—usually 2–3 weeks after the initiation of the culture—the tissue and cells were transferred to a culture bag specifically designed for lymph node tissue cultures. Half the volume of fresh medium was added every 2–3 days as long as the cells continued to proliferate exponentially. The AKT- DC generated were separated from the TDLN tissue by filtering through a nylon mesh and were then transferred to another bag.
The AKT-DC suspension was split 2–3 times every 3–4 days into new bags each containing 800 ml of fresh medium. Then, cells containing AKT-DC were harvested, washed twice using 200 ml of saline suspended
in the cryoprotective agent CP-1 with 4 % human albumin, and stored, 5–10 × 109 cells/ bag at −80 °Cuntil used. Usually, the TDLN cultures continued to release AKT-DC for 2–3 months. When the TDLN
stopped releasing cells, 1–2 × 109 peripheral blood lymphocytes (PBL) obtained by lymphocyte apheresis with a COBE Spectra System were added. TDLN together with a PBL culture were carried out until
obtained a sufficient number of AKT-DC (1–3 × 1011 cells) for 12–14 courses of immunotherapy.
Those who were allocated to group A (immunotherapy arm) received 4 monthly courses of postsurgical chemo- therapy. Adoptive immunotherapy using AKT-DC was added 1 week after each course of chemotherapy and was then continued once a month for the first 6 months after resection and then every 2 months until 2 years after surgery. This amounted to a total of 12–15 courses in 2 years. Group B (control arm) received four courses of postsurgical chemotherapy. Stage IIIA patients received two courses of induction chemotherapy before surgery.
Out of a total of 762 courses, 52 (6.8 %) were accompanied with chills and shivering, and 47 courses (6.2 %) were followed by fever (>38). Of 50 patients treated with immunotherapy, 28 had no adverse reactions and 22 had at least one adverse reaction of chills, shivering and/or fever. Chills and shivering started about 30 min after the start of each cell transfer, continued for 10–20 min, and were followed by fever up to 38– 40 °C. These fevers lasted for 2–3 h, and the body temperature then gradually declined to a normal level within the same day. No adverse reaction other than chills or fever was observed.
There were 19 cases of recurrence in group A (lungs, 6; lymph nodes, 5; bones, 3; and others, 5) and 33 in group B (lungs, 9; lymph nodes, 8; bones, 8; brain, 5; and others, 3). Seven patients in group A became tumor free after recurrence following treatments combined with immunotherapy: two cases of EGFR-TKI, three cases of resection of lung metastasis, one case of ALK fusion gene inhibitor, and 1 of radiation; and continued to be tumor free until the time of the final analysis. In one case in group B, CR was attained after chemotherapy. Therefore, 38 patients in group A and 19 patients in group B were tumor free at the time of analysis. Recurrence was seen in 14 and 21 cases of adenocarcinoma and in 3 and 7 squamous cell carcinoma cases in groups A and B, respectively.
The Kaplan–Meier estimates of overall survival. The 2- and 5-year overall survival rates were 93.4 % and 81.4 % in group A, and 66.0 % and 48.3 % in group B, respectively. The median survival time of group B was 47.5 (from 26.3 to not reached) months and was never reached in group A. The 2- and 5-year recurrence-free survival rates were 68.5 % and 56.8 % in group A, 41.4 % and 26.2 % in group B, respectively.
Immunotherapy has the potential to improve the postsurgical prognosis of lung cancer patients, but a large-scale multi-institutional RCT is awaited for further confirmation of this study.
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Scientific article publishing date :9/28/2014
Immucura identifier BSC21_193EN