Efficiency of Cytokine-Induced Killer Cells in Combination with Chemotherapy for Triple-Negative Breast Cancer / 한국유방암학회지

PURPOSE: The treatment of triple-negative breast cancer (TNBC) remains challenging, due to the absence of estrogen, progesterone, and human epidermal growth factor receptors. This study was designed to evaluate the efficiency and safety of cytokine-induced killer (CIK) cell immunotherapy, following regular chemotherapy, for patients with TNBC. METHODS: A total of 340 patients with postmastectomy TNBC, from January 1, 2010 to June 30, 2014, were included in this retrospective study. Seventy-seven patients received CIK cell immunotherapy, following regular chemotherapy (arm 1), and 263 patients received regular chemotherapy alone (arm 2). The primary aim was overall survival (OS) and disease-free survival (DFS), and the treatment responses and adverse events were also evaluated. RESULTS: The 5-year DFS and OS rates in arm 1 were 77.9% and 94.3%, compared with 69.8% and 85.6% in arm 2, respectively (p=0.159 and p=0.035, respectively). This clearly shows that there was no statistical difference in the 5-year DFS between the two groups. Multivariate analyses of arm 1 indicated that a Karnofsky performance score (KPS) ≥90 and stage I/IIA disease were significantly associated with a prolonged DFS period (hazard ratio [HR], 0.25; 95% confidence interval [CI], 0.09–0.74; p=0.012; and HR 0.21; 95% CI, 0.06–0.82; p=0.024, respectively), but a KPS ≥90 and stage I/IIA disease were not independent prognostic factors for OS. Toxicity was mild in patients who received the CIK therapy. CONCLUSION: The data suggested that CIK cell immunotherapy improved the efficiency of regular chemotherapy in patients with TNBC, and the side effects of CIK cell immunotherapy were mild.

The expression of Tregs and CD8+T cells in malignant melanoma and its relationship with prognosis / 天津医药

Objective To investigate the different distribution of regulatory T cells (Tregs) and CD8+T cells in the local immune microenvironment of mucosal malignant melanoma and cutaneous malignant melanoma, and analyze the relationship between the two indicators and the prognosis of patients. Methods Immunohistochemistry staining was used to assess the ex?pression of Foxp3+Tregs and CD8+T cells in tumor microenvironment of 58 patients with malignant melanoma. The correlation between two factors, clinicopathological characteristics, and prognosis were analyzed. Results There is no correlation be?tween the expression of Foxp3 and CD8. The number of Foxp3+Tregs was significantly higher in mucosa malignant melanoma than that in cutaneous malignant melanoma (t=2.648, P=0.011). The proportion of Foxp3highTregs was significantly higher in pa?tients with tumor diameter≥3 cm, lymph node metastasis and distant metastasis than that in patients with tumor diameter<3 cm, no lymph node metastasis and no distant metastasis (P<0.05). In addition, in patients with ulceration that proportion was significantly higher in CD8high group than that in patients without ulceration (33.3%vs 5.9%, P<0.05). The median progres?sion-free surial (PFS) was 12 months in Foxp3high group, which was significantly longer than that of Foxp3low group (31 months, P<0.05). The median PFS was significantly higher in CD8high group (25 months) than that of CD8low group (12 months, P<0.05). Subgroup analysis showed that the median PFS was 7 months in Foxp3high CD8low group, which was significantly lower than that of Foxp3highCD8high group (25 months) and Foxp3lowCD8low group (18 months, P=0.003). Univariate analysis showed that median PFS was different in patients with different tumor location, different number of Foxp3+Treg, different number of CD8+T cells, and distant metastases. Conclusion The number of Tregs is closely associated with metastasis in patients with malig?nant melanoma. Compared with cutaneous malignant melanoma, our results indicate that the poor prognosis of mucosal malig?nant melanoma may be associated with the infiltration of more Tregs.

The Expression of TSLP and Its Relationship with the Number of Infiltrating Regulatory T Cells in Lung Cancer / 中国肿瘤临床

Objective: To investigate the expression of TSLP in human lung cancer tissue and the correla-tion between TSLP expression and number of regulatory T cells (Tregs). Methods: The expression of TSLP mRNA and protein was detected in different pathological lesions of the lung by Q-RT-PCR and immunohisto-chemistry. Immunohistochemistry was used to detect Foxp3+ Tregs. The correlation of TSLP with the number of Tregs was analyzed. Results: TSLP gene was expressed in tumor tissues (n=37), latero-tumor tissues (n=29) and non-tumor lung tissues (n=24), without statistical difference (P=0.148). TSLP protein was expressed in the cytoplasm and was observeed in 69.57% of tumor tissues, 13.33% of benign lesions and 30.00% of non-tumor lung tissues, with a significant difference (P<0.05). The expression of TSLP protein was correlated with tumor size (P=0.000) and lymph node metastasis (P=0.018). The number of Tregs in TSLP positive group was more than that in TSLP negative group (P<0.05). Conclusion: The expression of TSLP in lung tu-mor tissues is increased and is correlated with the number of Tregs, indicating that TSLP could induce Treg to play an important role in tumor immunotolerance.

Effect of RetroNectin on the Proliferation of CIK Cells and the Possible Mechanisms / 中国肿瘤临床

Objective: To investigate the effect of RetroNectin on CIKs cells and the related mechanisms. Methods: Peripheral blood mononuclear cells (PBMC) were collected from patients and divided into two groups: group Ⅰ and group Ⅱ. Samples in group Ⅰ were seeded into culture flask precoated with RetreNec-tin and CD3mAb to induce CIKs. While samples in group Ⅱ were seeded into common culture flask. The pro-liferation of CIKs was detected by cytometric analysis. The cytotoxic activity of CIKs was determined by LDH assays. The phenotype changes and cell cycle of CIKs were identified by flow cytometry. The apoptosis of cells was detected by Annexin V/PI. Western blot was employed to detect the level of protein Vav1. The CD49d and CD49e were blocked by anti-CD49d and anti-CD49e and the proliferation of cells was tested by cytometric analysis after the blockage. The phenotype changes of cells were identified by flow cytometry after the blockage. Results: RetroNectin enhanced the proliferation of CIKs (P<0.05). Flow cytometric analysis showed that RetroNectin significantly increased the number of CD25+ T cells (P<0.05). RN-CIK was more ac-tive than CIK in killing HCT-8 cell lines in vitro (P<0.05). RetroNectin could block the CIKs at G_1 phase (P<0.05) and resist apoptosis. There was no significant difference in the proliferation between the two groups af-ter the blockage with CD49d and CD49e (P>0.05). The expression of protein Vavl was associated with CD25+T cells. Conclusion: RetroNectin enhances the proliferation of CIKs by influencing the cell cycle, resist-ing apoptosis possibly through the site of CD49d and CD49e, and inducing T cell activation as the second sig-naling through Vav1.

Clinicalefficacy of activated-HLA haploidentical peripheral blood stem cells in treatment of advanced solid tumors / 中国肿瘤生物治疗杂志

Objective: To evaluate the anti-tumor and side effects of activated-HLA haploidentical peripheral blood tem cells (haplo PBSCs) in the treatment of advanced refractory solid tumor patients. Methods: Forty-two patients with advanced refractory tumor, who were diagnosed in our hospital from Oct. 2004 to Oct. 2007, were enrolled in this study (all patients signed informed consent), including 12 with ovarian cancer, 9 with renal cancer, 8 with lung cancer, 8 with breast cancer, 2 with colon cancer, 2 with gastric cancer, and 1 with melanoma. The donors were healthy direct relatives of the patients; the donors' haplo-PBSCs were mobilized, collected, and activated by rhIL-2 in vitro. The clinical efficacy and side effects of haplo-PBSCs therapy were assessed by CT/PET-CT scanning, RESIST standard, KPS score, and clinical response rates, etc. Results: All 42 patients received one episode of haplo-PBSCs treatment. The progression-free survivals (PFS) were 6 months and the clinical beneficial rate (CR+PR+SD) was 73.8%. The beneficial rate of life quality was 76.2% and the KPS increased by 20 (0-30) points on average after haplo-PBSCs treatment. The patients with KIR unmatched in GVH direction had better outcomes than those with KIR matched or KIR unmatched in HVG direction (P<0.05), and the clinical beneficial rate, PFS and total beneficial rate were 94.1% vs 60.0%, (13.4±1.3) vs (8.0±0.9) months, and 89.5% vs 65.2%, respectively (all P<0.05). The donor/recipient relation as the mother/child had a better outcome than that as the father/child (P<0.05). Patients with renal cancer or ovarian cancer had better outcomes than those with other cancers, with clinical beneficial rates being 90.0% and 81.8%, respectively. Conclusion: Activated haplo-PBSCs therapy can induce non-specific anti-tumor effect, and improve the clinical symptom and life quality of advanced tumor patients.

A preliminary study on the effects of feto-maternal microchimerism in activated human leukocyte antigen haploidentical mobilized peripheral blood stem cells on treatment of solid tumors / 中华内科杂志

Objective To study the effect of feto-matemal microchimerism in the treatment of activated human leukocyte antigen (HLA) haploidentical mobilized peripheral blood cells against solid tumors. Methods Genomic DNA samples of 25 pairs of HLA haploidentical donors and recipients were extracted. The donor-derived HLA-DRB loci were detected with nested PCR-sequence specific primer(SSP) typing. The mixed lymphocyte proliferation action between the patients and respective donors, the engraftment of donor's cells and the serum levels of Th1/Th2 type of cytokines were measured with MTT,FISH and EIJSA method respectively. The survival time of patients with or without feto-matemal microchimerism were compared as well. Results Using nested PCR-SSP typing, the positive rates of feto-maternal microchimerism in the 25 pairs of HLA haploidentical donors and recipients were 40% in the maternal/children pairs and 0 in the paternal/children pairs. The chimerism positive patients showed less proliferation activity when cocultured with respective donors as compared with unrelated ones (P=0.03).Only one chimerism positive patient experienced the engraft of donor's cell 3 months after treatment as the donor derived XX chromosome was identified with FISH. When the data of chimerism positive patients were deleted, the serum levels of IFNγ 1 month after treatment dropped dramatically from 171.4 (26. 3～258.4) ng/L to 29. 4(1.2～39.9)ng/L. The survival time in chimerism positive patients of the maternal/children pairs was significantly longer than that in chimerism negative patients, which was (31.2±4. 3) months and (11.1±3.3) months, respectively (P=0.036). Conclusion Feto-maternal microchimerism might induce anergy in the HLA haploidentical donors, favor the engraftment of donor's progenitors and maintenance of positive microenvironment and prolong the survival time.

Separation and Identification of the Exosomes Derived from A Human Tumor Cell Line / 天津医药

Objective: To separate and identify the exosomes derived from MCF-7 tumor cells,and observe their function on IL-2 induced lymphocyte proliferation.Methods:The exosomes were separated by serial centrifugation,concentration and density gradient centrifugation, and then identified by transmission electron microscope.The effects of exosomes on IL-2 induced proliferation were studied by MTT method.Results:Total 20 mL exosomes from 2×10~7 MCF-7 cells with concentration of protein were(1.84±0.01) g/L. The diameters of these purified bi-layer membrane exosomes were 55-70 nm, which were consistent with the internal vesicles in multi-vesicular endosomes.The result of MTT test showed that proliferation of donor peripheral blood lymphocytes was inhibited by tumor exosomes at a dose-dependent manner in response to IL-2.Conclusion:It is feasible to separate high-purity exosomes from tumor cell culture medium supernatant by serial centrifugation and concentrate method. These tumor derived exosomes have inhibitory effect on IL-2 induced proliferation.

Fermentation, Purification and Identification of Recombinant Human Interleukin-18 / 天津医药

Objective: To investigate the expression of recombinant human intedeukin-18 (hIL-18) in 3.7 L fermenter with the constructed engineer train Pichia pastoris X-33/hIL-18, and the procedures of expression and purification thereof.Methods: The train X-33/hIL-18 was inoculated in BMGY medium and then inoculated into the fermenter until the A600 of the culture reached about 6. The supernatant of fermentation was isolated and purified with centrifugal fiher devices, hydrophobic chromatography column and anion exchange chromatography column. Results: The recombinant hIL-18 was expressed in 3.7 L fermenter with batch feed methanol and the concentration reached 202 mg/L. After the purification, the purity could be more than 97%. The recombinant hIL-18 was shown to induce interferon-gamma (IFN-γ) production by human peripheral blood mononuclear cells (PBMCs), and enhanced NK cell cytotoxicity synergistically with IL-2. Conclusion: A great deal of the recombinant hIL-18 with higher purity could be harvested by Pichiapastoris expression system. This study showed a new potential for further study of its function and activities.