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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.
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Humans , Arm , Cytokine-Induced Killer Cells , Disease-Free Survival , Drug Therapy , Estrogens , Immunotherapy , Multivariate Analysis , Progesterone , Prognosis , ErbB Receptors , Retrospective Studies , Triple Negative Breast NeoplasmsABSTRACT
Objective:This study aimed to analyze and compare the prognosis and the prognostic factors of combined small cell lung cancer (CSCLC) and pure small cell lung cancer (PSCLC) retrospectively. Methods:The clinicopathological characteristics of the 343 small cell lung cancer patients who were diagnosed in Tianjin Medical University Cancer Institute and Hospital between January 2006 and December 2012 were collected and reviewed. Survival analysis was performed and prognostic factors were assessed. Results:The median OS (overall survival) and PFS (progression free survival) of CSCLC were 31 and 21 months, respectively, and the median OS and PFS of PSCLC were 15 and 9 months, respectively. The Kaplan-Meier survival curves revealed that the prognosis of CSCLC was significantly better compared with that of PSCLC. COX analysis showed that disease stage, pathology, and therapy were indepen-dent prognostic factors of small cell lung cancer. Univariate analysis indicated that the small cell lung cancer group benefited from the surgery, particularly the CSCLC. NLR , therapy, and disease stage influenced the prognosis of PSCLC, and disease stage and therapy in-fluenced the prognosis of CSCLC. Multivariate analysis revealed that disease stage and therapy were independent risk factors of CSCLC in regard to OS. Conclusion:The prognosis of CSCLC was better compared with that of PSCLC. Limited-stage small cell lung cancer should undergo surgery, particularly the CSCLC.
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10.3969/j.issn.1000-8179.2013.12.001
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Objective:To explore the status of STAT3 phosphorylation in myeloid-derived suppressor cells (MDSCs) of breast cancer and its function in the immunosuppressive effect of MDSCs on proliferation and cytokine secretion of T cells. Methods:CCD33+cells were isolated from healthy umbilical cord, blood-derived, peripheral blood mononuclear cells and were co-cultured with breast cancer cell line MDA-MB-231 in vitro using Transwell plates to induce MDSCs. The untreated CD33+cells were used as con-trols. Idoxuridine (IDO) suppressor expression and STAT3 phosphorylation were examined using Western blot assay. The proliferation and cytokine secretion of T cells, which were co-cultured with MDSCs, were determined by methyl thiazol tetrazolium assay and en-zyme-linked immunosorbent assay. 1-MT and JSI-124 were used to investigate the function of IDO and pSTAT3 in MDSC-mediated T cell immunosuppression. Results:The protein levels of IDO and pSTAT3 in MDSCs were significantly upregulated. MDSCs obviously suppressed T-cell proliferation, which was reversed by 1-MT or JSI-124 (P<0.05). MDSCs could promote TGF-βand IL-10 secretions, but could also remarkably inhibit IFN-γsecretion (P<0.05). After incubation with 1-MT or JSI-124, the increase in TGF-βand IL-10, as well as the decrease in IFN-γ, was significantly reversed. Conclusion:The upregulated pSTAT3 induced the IDO increase in MDSCs. JSI-124 can block MDSC-mediated immunosuppressive effect on T cells in breast cancer.
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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.
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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.
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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.
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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.
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This study was aimed to study the potential effects of alloreactive NK cells (allo-NKs) in therapy of relapsed lung cancer after haploidentical hematopoietic stem cell transplantation using donor lymphocyte infusion (DLI). The F1 donors derived-NK cells were purified with MACS magnetic separation system, in which the proportion of the alloreactive Ly49A(+) cells was detected by flowcytometry and alloreactivity was measured by LDH method. The relapse model of lung cancer after haploidentical-HSCT was established. The distribution kinetic of infused donor lymphocytes in vivo was analyzed. The inhibition of relapse tumor, infiltration of lymphocytes in situ and fluctuation of 22 kinds of cytokines in serum after DLI were compared among different groups. The results showed that the infused donor cells of allo-NK group were accumulated mostly in lung, spleen and kidney for more than 48 hours with considerable higher levels according to the distribution kinetic curve. The sizes of relapse tumors between chemotherapy + PBS group and chemotherapy + DLI group showed no difference. However, the relapsed tumors in allo-NK + DLI group were significantly smaller than that in chemotherapy + DLI group or allo-NK + PBS group, in which increased infiltration of lymphocytes were defined in situ. The levels of cytokines such as MCP-1, IL-17, IL-12 and MCP-5 in serum of allo-NK + DLI group ascended compared with control group, though the level of IL-10 declined simultaneously. It is concluded that allo-NKs prolong the survival time of infused donor lymphocytes in vivo, promote the secretion of inflammatory cytokines and Th1-type of cytokines, and further improve the antitumor effects of DLI against relapse after transplantation.
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Animals , Mice , Cytokines , Blood , Hematopoietic Stem Cell Transplantation , Methods , Killer Cells, Natural , Cell Biology , Lung Neoplasms , Therapeutics , Lymphocyte Transfusion , Methods , Mice, Inbred BALB C , Mice, Inbred C57BL , Neoplasm Recurrence, Local , Therapeutics , Transplantation Conditioning , MethodsABSTRACT
This study was aimed to investigate the feasibility of low dose of fludarabine, cyclophosphamide combined with donor derived alloreactive NK cells as a new nonmyeloablative conditioning regimen in the haploidentical hematopoietic stem cell transplantation (haploidentical HSCT). F1 derived-NK cells were enriched with MACS magnetic separation system, in which the proportions of the Ly49C+ and Ly49A+ cells were detected by flow cytometry and the alloreactivity was measured by LDH method. The haploidentical HSCT models were constructed, and the myeloablativity in vivo, donor engraftment and the intensity of GVHD were compared between different myeloablative and nonmyeloablative conditioning regimens, including 9 Gy TBI, 6.5 Gy TBI, flu + cy, and flu + cy + allo-NK. The results showed that the flu + cy + allo-NK conditioning was nonmyeloablative, but the rate of donor chimerism after haploidentical HSCT was significantly higher as compared with other nonmyeloablative methods, which were (28.70 +/- 5.90)% in bone marrow and (46.40 +/- 5.00)% in spleen at day 21 post-transplantation. When compared with the flu + cy conditioning, the intensity of GVHD was slight in the flu + cy + allo-NK group, in which only a half of C57BL/6 recipients experienced weight loss, and no distinct pathological damages observed in the liver, intestine, kidney and skin samples. It is concluded donor derived-alloreactive NK cells can facilitate engraftment of the haploidentical hematopoietic stem cells and mitigate GVHD. The flu + cy + allo-NK conditioning provides a new method for those elder patients with high-risk solid tumor undergoing haploidentical-HSCT.
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Animals , Female , Mice , Cyclophosphamide , Graft vs Host Disease , Haplotypes , Hematopoietic Stem Cell Transplantation , Methods , Killer Cells, Natural , Transplantation , Mice, Inbred BALB C , Mice, Inbred C57BL , Models, Animal , Transplantation Chimera , Transplantation Conditioning , Methods , Vidarabine , Whole-Body IrradiationABSTRACT
<p><b>OBJECTIVE</b>To construct a recombinant adenovirus vector expressing TbetaR-II extracellular domain-RANTES fusion gene and evaluate its anti-tumor effects.</p><p><b>METHODS</b>Mouse origin TbetaR-II extracellular domain and RANTES gene were amplified by RT-PCR. The TbetaR-II extracellular domain-RANTES fusion gene was amplified by overlapping PCR method. TbetaR-II extracellular domain-RANTES fusion gene was cloned into pDC316 vector. The recombinant adenovirus vector expressing the fusion gene was constructed by adMax adenovirus vector creation system. Recombinant adenovirus vector expressing the fusion gene was transfected into LA795 cells. The expression of recombinant adenovirus was checked by Westen blot. The levels of TGF-beta1, RANTES in supernatant were checked by ELISA. The transfected cells were counted and growth curve was obtained. Apoptosis of transfected cells was detected by Annexin V FITC method. The chemotactic activity of supernatant of transfected cells to splenic lymphocytes was assayed. Transfected cells (1 x 10(5)) were inoculated into T739 mice and to observe the tumor growth and survival time. Ad-TbetaR-II extracellular domain, Ad-RANTES and Ad-TbetaR-II extracellular domain-RANTES fusion gene(1 x 10(10) pfu) were injected into the tumor in T739 mice. The tumor size and tumor weight were recorded and tumor growth inhibition rate was counted and statistically analyzed.</p><p><b>RESULTS</b>TbetaR-II extracellular domain and RANTES gene were amplified by RT-PCR and TbetaR-II extracellular domain-RANTES fusion gene amplified by overlapping PCR, were identified by DNA sequence analysis. Restriction enzyme digestion analysis showed that the recombinant vector was constructed correctly. The recombinant adenovirus vector expressing the fusion gene was constructed successfully using the AdMax Adenovirus Vector Creation System. Its titer was 8 x 10(10) pfu/ml. Ad-TbetaR-II extracellular domain-RANTES fusion gene was transfected into LA795 cells and had specific protein fragment proved by Western Blot. The concentration of TGF-beta1 was decreased and RANTES was increased in supernatant of transfected cells. The growth curve showed that recombinant adenovirus vector expressing the fusion gene could delay tumor development and induce apoptosis, with an apoptosis rate in vitro of 16.9%. The supernant of infected cells showed chemotactic activity to splenic lymphocytes. Tumor growth and survival time were prolonged significantly in group tranfected with recombinant adenovirus vector expressing the fusion gene, and tumor growth was effectively inhibited after injecting recombinant adenovirus vector expressing the fusion gene, with a tumor growth inhibition rate of 37.6%.</p><p><b>CONCLUSION</b>A recombinant adenovirus vector expressing TbetaR-II extracellular domain-RANTES fusion gene has been constructed successfully. The recombinant adenovirus vector can bind TGF-beta1 effectively, counteract immune suppression mediated by TGF-beta, enhance immune function, induce significant antitumor immune respone, inhibit tumor growth, and prolong the survival time of tumor-bearing mice.</p>