| BackgroundOvarian cancer (OC) is one of the most common and leading cause of cancer death for women in gynecologic malignant tumors. Due to its late detection, tumor heterogeneity, and a high rate of metastasis, most patients have advanced stage when they are first diagnosed. Even though the development of surgery and chemotherapy are verified to have curative effects, there are still two-thirds of OC patients will develop malignant ascites (MA) during the course of disease, The MA can seriously affect the quality of life and cause series of ascites-associated symptoms (AAS), such as abdominal distention, abdominal pain, dyspnea, nausea, vomiting, anorexia, fatigue, etc. Furthermore, the MA is an independent risk factor in OC patients by reducing the overall survival. Although there were many treatment options for MA including the treatment of primary tumor, abdominal paracentesis, diuresis, sodium-restricted diets, etc., but all of these approaches have limitations in terms of efficacy and tolerability for patients. Because there is no standard treatment guideline for MA, once recurrence or chemoresistant disease developed, it will become extremely difficult and challenging. Therefore, novel and more effective treatment approaches for this challenging disease are urgently needed.In recent years, with development of the tumor molecular mechanism and biotechnology, biotherapy has become the fourth effective anti-tumor therapy followed by surgery, chemotherapy and radiotherapy. Tumor biological therapy is a new therapy through mobilizing the natural defense mechanism or giving naturally-produced substances to inhibit tumor growth. Among it, adoptive cell transfer (ACT) immunotherapy is a new treatment mode, which also have remarkable curative effect. ACT immunotherapy is based on the ex vivo selection of immune cells, such as cytokine induced killer cells (CIK), dendritic cells (DC), NK cells, tumor infiltrating lymphocytes (TILs), and their activation and amplification before re-infusion to the autologous tumor-bearing host by using biotechnology and biological agents to stimulate and enhance the body’s immune function to treat tumor. Among it, the adoptive TILs immunotherapy is the most development potential treatment.As the heterogeneity lymphocytes, the main components of TILs are lymphocyte, which exist in the tumor tissues and have antigen specificity. After activated by human recombinant interleukin-2 (rIL-2), the proliferation and anti-tumor activity of TILs will significantly enhanced 50-100 times than lymphokine activated killer (LAK) cells. Following the first report by Rosenberg that adoptive TILs therapy was an effective treatment to treat malignant melanoma, many researchers in our country and abroad also began to treat OC by using this therapy. Aoki et al. published the first human adoptive TILs trial in advanced or recurrent epithelial OC using IL-2 expanded TILs. Encouraging results were seen as a response to the TILs were seen in 5/7 patients receiving TILs without chemotherapy and in 9/10 patients reveiving TILs with chemotherapy. A pilot study from Freedman et al. in 1994 on 11 OC patients was performed with intraperitoneal injection of TILs with IL-2. Unfortunately, no significant clinical responses were seen in any of the patients. However, Zhang Junyi et al. in our hospital showed superior curative effect by using IL-2/TILs to treat 187 patients with malignant pleural effusion or ascites. The total effective rate of pleural effusion and ascites was 87% and 58.82%, respectively. The negative conversion rate of tumor cells in pleural effusion and ascites was 66.67%, and the tumor cell degeneration rate was 16.67%. Although the clinical trial using TILs treatment for OC patient was very lttle and had mixed results, the theoretical basis for the effective treatment of OC is adequate. Firstly, ovarian cancers express many tumor-associated antigens, which can serve as target for immune responses. Secondly, the presence of TILs in OC tissue correlates strongly with the survival of OC patients, especially the CD8+T lymphocytes. Lasetly, as an immunogenic tumor, ovarian cancers express multiple peptide/MHC complexes, which can be recognized by T lymphocytes.However, the main factors which can restrict the use of adoptive TILs therapy for ovarian cancer including no unified preparation method for TILs, prolonged cell culture time for TILs, the unclear mechanism of anti-tumor activity by TILs, the best quantity and quality of the infusion TILs, no unified rational criteria for patients selection, the best choice of the combined treatment application and so on. Given the potential that TILs therapy holds for OC, an improvement in lab techniques in TILs enrichment and activation and further understanding the anti-tumor activity and mechanism of TILs may improve the clinical outcomes in TILs OC trials. Thus, the further studies on the preparation method, biological characteristics, anti-tumor anctivity of TILs and the effectiveness of the adoptvie TILs therapy for OC are still the future direction of developemt.At present, the clinical and basic researches for the treatment of OC are rare, and the previous clinical trials were just small sample non randomized clinical studies. Although our preliminary clinical observation studies have found that combination TILs and rIL-2 can effectively treat the manligant pleural effusion and ascites, there is no further study to observe the mechanism of anti-tumor activity of TILs, and compare the clinical effects of TILs in combination with or without chemotherapy for OC. So, to look for a new treatment mode for MA of OC, this study planed to retrospective analysis the clinical and toxic effects of intraperitoneal TILs in combination with or without chemotherapy for NA secondary to 32 OC patients. At the same time, through the establishment of exploring a mature technical route of efficient isolating TILs and autologous OC cells at the same time from ovarian tissue or ascites, to further study the in vitro anti-tumor activity mechanisms of TILs and then provide more basises for TILs popularization and application in ovarian cancer in the furture.Objective(1) To retrospective analysis the clinical and biological effects of intraperitoneal (IP) TILs in combination with or without intravenous (Ⅳ) chemotherapy for MA secondary to OC to find the best therapy for treating ovarian MA.(2) To set a mature technical route of efficient isolating TILs and OC cells at the same time from ovarian tissue or ascites.(3) To research the proportion change of CD3+, CD8+/CD3+and CD4+/CD3+TILs before and after activation and amplification by rIL-2 in vitro.(4) To explore the changes of specific and nonspecific cytotoxic effects of TILs on tumor cells before and after activation and amplification by rIL-2.(5) To explore the tumor markers and ascites formation factors of autologous OC cells induced by TILs after activation and amplification by rIL-2.(6) To explore the apoptosis rate, apoptosis protein of autologous OC cells induced by TILs after activation and amplification by rIL-2.Materials and Methods1. Clinical section(1) Between January 2001 and December 2011, the clinicopathologic parameters of 32 patients with MA arising from OC were analyzed in this study at Nanfang Hospital of Southern Medical University.(2) Among the 32 ovarian cancer patients,8 patients received intraperitoneal (IP) administration of TILs therapy alone (TILs group) and 11 patients received a combination of chemotherapy and IP TILs therapy (combination group), while the remaining 13 patients received chemotherapy alone (chemotherapy group).(3) All the complete medical histories, KPS scores, AAS (anorexia, insomnia, dyspnea, nausea, vomiting, abdominal pain, abdominal distention and fatigue), routine blood examinations, CA125 and albumin levels in patients were conducted before and after treatment.(4) To detect the quality of life of patients after treated with different treatments, that is to analyze the changes of AAS and KPS before and after treatment.(5) To compare the control rate of ascites and solid tumors after treated with different methods in ovarian cancer.(6) To observe the survival outcomes of OC patients with malignant ascites after treated with different treatments.2. Experimental section(1) Fourteen fresh tumor tissue or ascites of first diagnosed OC patients were obtained from Nanfang Hospital of Southern Medical University between January 2014 and December 2014.(2) The TILs and OC cells were isolated by using joint enzyme digestion method, adherent method and Ficoll discontinuous density gradient centrifugation etc.(3) Flow cytometry was performed on TILs to detect the proportion change of CD3+ CD8+/CD3+ and CD4+/CD3+ TILs before and after activation and amplification by rIL-2 in vitro.(4) The specific and nonspecific cytotoxic effect of TILs on tumor cells before and after activation and amplification by rIL-2 were detected by MTT method.(5) The tumor marker CA125 and ascites formation factors VEGF and MMP-9, which were secreted by OC cells were detected by enzyme-linked immunosorbent assay method (ELISA) before and after treated by the activation and amplification TILs.(6) Apoptosis kits were used to detect the changes of apoptosis rate of OC cells after treated with the activation and amplification TILs.(7) The expression of apoptosis related protein Caspase3 in OC cells after treated by activation and amplification TILs was detected by immunohistochemical method.Results1. Clinical results(1) The clinic characters of OC patients with malignant ascitesThe median age of 32 OC patients with malignant ascites was 56 years. There were 21 patients had a serous adenocarcinoma in all patients.1 pateint and 31 patients had a FIGO II and III-IV staging, respectively. The positive of peritoneal cytology was found in 27 patients, negative in 5 patients. There were no statisitical significances in age, clinical stage, debulking surgery, histological subtype, histological grade, the cytology and depth of ascites between the three groups (P>0.05).(2) The clinic characters of IP TILsIn the TILs group, the mean ages of TILs were 10.3±2.8 days, and the total treatment doses of IP TILs ranged from 9×109 to 39×109 cells (24.3±11.4×109). Compare with the TILs group, the mean ages of TILs were 10.5±3.6 days, and the total treatment doses of IP TILs ranged from 3×109 to 54×109 cells (25.2± 17.0 ×109) in the combined group, all of these had no statistically difference.(3) The quality of life of OC patients after treated with different treatmentsThe patient’s quality of lives in 32 patients had dramatically improved after treatment. The mean KPS and AAS scores of 32 patients were significantly improved from 62.2±10.4 and 1.4±0.6 to 74.4±13.7 and 0.6±0.7, respectively. All the differences had statistical significance (P<0.001). There were no differences of KPS, AAS and albumin before and after treatment in TILs group. In the chemotherapy group, only the KPS, abdominal distension, anorexia and fatigue were improved after treatment. Except the dyspnea, nausea and vomiting, all the remaining AAS and albumin were improved after treatment in combination group (P <0.05).(4) Therapeutic evaluation of different treatmentsThe objective response rate of MA was 90.9% in the combination group, which was higher than the TILs group (37.5%, P=0.009) and chemotherapy group (53.8%, P=0.047). However, tumor objective response was 63.6% in the combination group, which was higher than the TILs group (25%,.P=0.002) and had no significant difference with chemotherapy group (30.8%).There were 8 and 4 cases of patients had Ⅰ-Ⅲ degree chemotherapy related adverse, including bone marrow suppression, nausea and vomiting, in chemotherapy group and combination group, respectively. There was no IP TILs related adverse.(5) The survival outcomes of OC patients with malignant ascites after treated by different treatmentsMedian TTP in combination group (13 months) was significantly longer than TILs group (1 months, P<0.001) and the chemotherapy group (6 months, P=0.027). The median OS of patients in combination group (25 months) was also significantly longer than the TILs group (7 months,P<0.001), but not the chemotherapy group (18 months).2. Experimental results(1) Successfully developed a mature technical route of efficient isolating TILs and OC cells at the same time from ovarian tissue or ascitesThis experiment adopted a low cost of joint enzyme (collagenase, hyaluronidase, and DNAse) digestion method, filtration method, half an hour adherence method, Ficoll discontinuous density gradient centrifugation and rIL-2 activation and amplification method to efficient isolating OC cells and TILs at the same time from ovarian tissue or ascites.(2) The growth of TILs and primary OC cellsThe TILs and OC cells were successfully separated from 14 OC patients. With the time extension, the cellular morphology of TILs changed from initial round to round, branching and rods. The TILs will form sample colony growth when it at exuberant growth period. The primary OC cells grew very slowly in the form of clouds and piles. The shapes of OC cells had round, quasi-circular and irregular polygon.(3) Subgroup analysis of TILs after activation and amplification by rIL-2The proportion of CD3+, CD8+/CD3+ and CD4+/CD3+ TILs were significant increased after rapid amplification by rIL-2 on the 14th day when compared to the initial TILs. CD3+was increased from 46.31±15.53% to 85.54±5.54%(P<0.001). CD8+/CD3+ was increased from 16.31±7.49% to 42.32±14.01%(P<0.001). CD4+/CD3+ was increased from 18.59±13.06% to 31.88±10.99%(P=0.007). The proportion of CD8+/CD3+TILs was significantly higher than CD4+/CD3+TILs after rapid amplified by rIL-2 (P=0.037).(4) The anti-tumor activity of TILs was significantly increased after activation and amplification by rIL-2The cytotoxic effects of the TILs on autologous tumor cells, K562 and Raji cells were very low before activation and amplification. After activated by rIL-2 on the 14th day, the specific and nonspecific cytotoxic effects of TILs were significantly increased and higher on autologous OC cells (54.60±11.20%) than K562 (23.42±6.92%) and Raji cells (19.68±5.73%). All the difference had statistical significance (P<0.001).(5) The TILs could effectively suppress the molecular markers of OC cells after activation and amplification by rIL-2The tumor marker and ascites formation factors secreted by OC cells were significantly inhibited by TILs after activation and amplification 14 days by rIL-2. After treatment with TILs, the CA125 secreted by OC cells in culture supernatant was decreased from 377.21±258.44 U/ml to 165.05±132.82 U/ml (P<0.001). VEGF was decreased from 247.22±226.69 pg/ml to 100.22±98.26 pg/ml (P=0.003). MMP-9 was decreased from 211.00±56.59 pg/ml to 64.23±57.63 pg/ml (P<0.001). All the differences had statistical significance.(6) The TILs could induce OC cells apoptosis after activation and amplification by rIL-2The apoptosis rate of OC cells induced by TILs was significantly increased after activated by rIL-2 on the 14th day. Compared with the control group, the apoptosis rate of OC cells was significantly increased from 4.21±4.14% in control group to 24.49±17.29% in treatment group (P<0.001). At the same time, the express score of apoptosis associated protein Caspase3 was significantly increased from 1.57±1.40 to 5.21±3.70 (P=0.001), and the apoptosis positive rate was increased from 14.3%(2/14) to 57.1%(8/14) (P=0.018).Conclusions1. In the MA of advanced OC patients who are unable or unwilling to accept chemotherapy, TILs can improve the quality of life in some extent, even the parognosis of some patients.2. Compared with the sigle use of chemotherapy, the combined TILs and chemotherapy treatment is not only safe and low toxity, but also effectively controls the MA of OC patients and significantly improves patients’quality of life.3. Our laboratory successfully developed a mature technical route of efficient isolating TILs and OC cells at the same time from ovarian tissue or ascites.4. The TILs could reach the clinically effective treatment dose (x109 cells) after activation and amplification by rIL-2 in vitro within 2 weeks.5. All the CD3+, CD8+/CD3+and CD4+/CD3+ TILs could be significantly increased after activation and amplification by rIL-2.6. The TILs after activation and amplification by rIL-2 have well specific cytotoxic effect on tumor cells.7. The TILs could induce autolougous OC cells apoptosis by Caspase3 apoptotic pathway.8. The TILs could effectively control the synthesis and secretion of the ascites formation factors VEGF and MMP-9 on OC cells after activation and amplification by rIL-2. This may be one of the reasons that the ascites in OC patients can be effectively controlled by IP TILs treatment.9. So TILs treatment could become one of the effective methods to improve the prognosis of ovarian cancer by making use of the TILs in surgical tumor tissue or ascites. |
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