Better Clinical Efficiency of TILs for Malignant Pleural Effusion and Ascites than Cisplatin Through Intrapleural and Intraperitoneal Infusion

Patients and Methods

Patients. This study was approved by the Committee Board of YantaiYuhuangding Hospital (Shandong, China) and informed consent was thoroughly read, understood and signed by all participants.

Inclusion criteria: i). Advanced malignancies were diagnosed by histological or pathological examination. ii). MSE was confirmed by imaging examination and malignant tumor cells were quantified in the serous effusion. iii). No anticancer drugs or hardener had been injected by intrapleural or intraperitoneal infusion within 1 month before the study. iv). Patients were aged between 18 and 50 years at the time of recruitment and the estimated remaining survival time was longer than 3 months. v). Test results for blood cell count, heart rate, hepatic and renal functions were within normal ranges. vi). Previous chemotherapy had been discontinued for more than 4 weeks prior to the study.

Exclusion criteria: History of allergy to biological agents; current treatment with chemotherapy; major organ dysfunctions; pregnancy or breast feeding; infections; history of refractory psychiatric diseases. Patients were withdrawn based on the following conditions: request of the patient; patient non-compliance; grade III/IV side-effects related to TIL infusion; disease progression.

Twenty-seven patients with relapsed solid tumor and MSE as the first sign of recurrence were enrolled in the study from May 2014 to June 2016. Characteristics of the patients are summarized in Table I.

Separation and culture of TILs. Approximately 500-1000 ml MSE was extracted under aseptic conditions, and centrifuged (1258 × g) for 5 min after heparin resistance coagulation. Discontinuous density gradient method was used to isolate mononuclear cells. After removal of tumor cells by the wall sticking method, TILs were separated from the suspension. The cell density was adjusted to 1×10⁶ /ml and inoculated into a culture flask with the RetroNectin (Takara, Kusatsu, Shiga, Japan) (13) package and culture solution, with phytohemagglutinin, 2-mercaptoethanol, monoclonal antibody of CD3 without serum, was added. At the same time, IL2 (6000 IU/ml) (Abcam, Cambridge, UK) was added for activation and proliferation of TILs. Subculture was carried out every 2-3 days based on the velocity of cell growth and cell density was maintained at 0.5-2.0×10⁶/ml. Antigens specific to targeted cancer type were added on day 13. TILs were harvested on the 14th day after lymphocyte subset and endotoxin examination to ensure the absence of bacteria, fungi, mycoplasma or endotoxin. Cell density of at least 1×10¹⁰/l, and cell viability of 80%, immunophenotype of CD3, CD4, CD8, CD16, CD56, CD19, CD25, CD71 and human leukocyte antigen D (HLA-DR)-related were checked before administration.

Treatment protocol. After drainage of the MSE by thoracentesis or abdominocentesis, patients received intrapleural or intraperitoneal administration of either cisplatin (60 mg) or TILs (100 ml). Patients were asked to turn over every 15 min to encourage full access of the medicine to the celom. Both groups were treated with systematic chemotherapy, i.e. 500 mg/m² pemetrexed and 75 mg/m² cisplatin on day 1 for four cycles repeated every 21 days for lung adenocarcinoma. Supportive treatments included liver and stomach protection and anti-emetic administration before infusion. Dexamethasone (5 mg) and xylocaine (5 ml) were given before or after infusion to relieve side-effects. Type-B ultrasonography was used to evaluate the treatment efficacy at the end of each treatment course every 3 weeks.

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Figure 1.

Survival curves for progression-free survival (PFS) of patients treated with tumor-infiltrating lymphocytes (TIL) and those treated with cisplatin (CP). Patients treated with TILs survived significantly longer compared to those treated with cisplatin.

Evaluation of efficacy. Complete remission (CR): accumulated effusion disappeared and remained stable for at least 4 weeks. Partial remission (PR): accumulated effusion decreased by 50%, associated with improved symptoms with no increased accumulation of fluid, and remained stable for at least 4 weeks. Stable disease (SD): less than 50% of MSE disappeared, or there was no noticeable change of symptoms. Progressive disease (PD): effusion increased with worsening of symptoms. Overall remission rate (ORR) was the sum of CR and PR. The disease control rate (DCR) was calculated by taking the sum of CR, PR and SD. Progression-free survival (PFS) refers to the period from the end of treatment to MSE increase. Overall survival (OS) refers to the period from the end of treatment to the end of follow-up or the death of the patient.

Adverse reactions were evaluated by the Common Toxicity Evaluation Criteria according to the National Cancer Institute (14). Patient QOL was assessed by KPS post-treatment as: Apparently improved: increase in KPS by ≥2; improved: increase in KPS by ≥10; stable: no obvious change in KPS; reduced: KPS decline of ≤10. Toxicity was classified grade 0-4 according to the WHO toxicity grading criteria (15). All cases were followed-up until June 2016.