The phenotype of ex vivo generated cytokine-induced killer cells is associated with overall survival in patients with cancer.

Cytokine-induced killer (CIK) cells are ex vivo generated heterogeneous NK-like T lymphocytes. It is not very clear whether the phenotype of CIK cells is associated with their therapeutic efficacy to cancer patients. Thus, in this study, the association of phenotype of CIK cells and the overall survival of 121 patients with hepatocellular carcinoma (HCC), 74 patients with lung cancer and 42 patients with colorectal cancer, all of whom underwent surgical resection and received autogenous CIK cell therapy, was analyzed. We found that high ratio of the CD3+CD4+ subset was associated with poorer overall survival in colorectal cancer, but not HCC or lung cancer. A high ratio of the CD3+CD8+ subset was associated with improved overall survival in all three types of cancer. A high ratio of the CD3+CD56+ NK-like subset was associated with improved overall survival in lung and colorectal cancer, but not HCC. A high ratio of the CD3-CD56+ NK subset was associated with poorer overall survival in lung and colorectal cancer, but not HCC. In conclusion, the CD3+CD8+ and CD3+CD56+ subsets, especially the CD3+CD8+ subset, may be the major phenotypes responsible for anti-tumor immunity in vivo after autogenous CIK cell therapy.

Molecular characteristics of patients with colorectal signet-ring cell carcinoma with different ABO blood groups.

Background: Colorectal signet-ring cell carcinoma (SRCC) is a rare subtype of malignant adenocarcinoma, accounting for approximately 1 % of colorectal cancer (CRC) cases. Its biomarkers and molecular characteristics remain controversial, and there are no specific therapeutic targets or strategies for its clinical treatment. Methods: A retrospective study was conducted between January 2010 and December 2021. 1058 colorectal cancer cases from the Sun Yat-sen University Cancer Center and 489 cases from the Tumor Genome Atlas Project were included in the analysis, of which 64 were SRCC. Data extraction included patient demographics, blood types and risk factors, including clinical variables and genomics (either a 19-gene panel NGS or 1021-gene panel NGS). Univariate analyses were performed to identify factors significantly associated with overall survival. Results: The blood groups of 27 (42.2 %), 18 (28.1 %), 12 (18.8 %), and seven (10.9 %) patients were classified as O, A, B, and AB, respectively. We found that O was a unique blood group characterized by a low frequency of KRAS mutations, a high frequency of heterozygosity at each HLA class I locus, and a high tumor mutational burden (TMB). Patients in blood group A with high-frequency KRAS mutations and those in blood group B with anemia and metabolic abnormalities required targeted treatment. Furthermore, genetic alterations in SRCC differed from those in adenocarcinoma and mucinous adenocarcinoma. Conclusions: Our study revealed genomic changes in SRCC patients across different blood groups, which could advance the understanding and precise treatment of colorectal SRCC.

An oncolytic adenovirus enhances antiangiogenic and antitumoral effects of a replication-deficient adenovirus encoding endostatin by rescuing its selective replication in nasopharyngeal carcinoma cells.

A replication-deficient adenovirus (Ad) encoding secreted human endostatin (Ad-Endo) has been demonstrated to have promising antiangiogenic and antitumoral effects. The E1B55k-deleted Ad H101 can selectively lyse cancer cells. In this study, we explored the antitumor effects and cross-interactions of Ad-Endo and H101 on nasopharyngeal carcinoma (NPC). The results showed that H101 dramatically promoted endostatin expression by Ad-Endo via rescuing Ad-Endo replication in NPC cells, and the expressed endostatin proteins significantly inhibited the proliferation of human umbilical vein endothelial cells. E1A and E1B19k products are required for the rescuing of H101 to Ad-Endo replication in CNE-1 and CNE-2 cells, but not in C666-1 cells. On the other hand, Ad-Endo enhanced the cytotoxicity of H101 by enhancing Ad replication in NPC cells. The combination of H101 and Ad-Endo significantly inhibited CNE-2 xenografts growth through the increased endostatin expression and Ad replication. These findings indicate that the combination of Ad-Endo gene therapy and oncolytic Ad therapeutics could be promising in comprehensive treatment of NPC.

Antitumor efficacy of a recombinant adenovirus encoding endostatin combined with an E1B55KD-deficient adenovirus in gastric cancer cells.

BACKGROUND: Gene therapy using a recombinant adenovirus (Ad) encoding secretory human endostatin (Ad-Endo) has been demonstrated to be a promising antiangiogenesis and antitumor strategy of in animal models and clinical trials. The E1B55KD-deficient Ad dl1520 was also found to replicate selectively in and destroy cancer cells. In this study, we aimed to investigate the antitumor effects of antiangiogenic agent Ad-Endo combined with the oncolytic Ad dl1520 on gastric cancer (GC) in vitro and in vivo and determine the mechanisms of these effects. METHODS: The Ad DNA copy number was determined by real-time PCR, and gene expression was assessed by ELISA, Western blotting or immunohistochemistry. The anti-proliferation effect (cytotoxicity) of Ad was assessed using the colorimetry-based MTT cell viability assay. The antitumor effects were evaluated in BALB/c nude mice carrying SGC-7901 GC xenografts. The microvessel density and Ad replication in tumor tissue were evaluated by checking the expression of CD34 and hexon proteins, respectively. RESULTS: dl1520 replicated selectively in GC cells harboring an abnormal p53 pathway, including p53 mutation and the loss of p14(ARF) expression, but did not in normal epithelial cells. In cultured GC cells, dl1520 rescued Ad-Endo replication, and dramatically promoted endostatin expression by Ad-Endo in a dose- and time-dependent manner. In turn, the addition of Ad-Endo enhanced the inhibitory effect of dl1520 on the proliferation of GC cells. The transgenic expression of Ad5 E1A and E1B19K simulated the rescue effect of dl1520 supporting Ad-Endo replication in GC cells. In the nude mouse xenograft model, the combined treatment with dl1520 and Ad-Endo significantly inhibited tumor angiogenesis and the growth of GC xenografts through the increased endostatin expression and oncolytic effects. CONCLUSIONS: Ad-Endo combined with dl1520 has more antitumor efficacy against GC than Ad-Endo or dl1520 alone. These findings indicate that the combination of Ad-mediated antiangiogenic gene therapy and oncolytic Ad therapeutics could be one of promising comprehensive treatment strategies for GC.

The efficacy of cytokine-induced killer cell infusion as an adjuvant therapy for postoperative hepatocellular carcinoma patients.

BACKGROUND: Even after surgery, hepatocellular carcinoma (HCC) has poor prognosis; adjuvant therapy is needed to improve effectively the outcome of HCC patients. We evaluated the efficacy of cytokine-induced killer (CIK) cell infusion as an adjuvant therapy for postoperative HCC patients. METHODS: A total of 410 patients were studied retrospectively (January 2002 to January 2007): 206 received surgery alone; 204 received surgery and at least four cycles of CIK cell transfusion (CIK group). Kaplan-Meier and Cox regression analyses were used to explore differences in OS between two groups. RESULTS: The CIK group overall survival rates were significantly higher than that of the surgery-alone group (log-rank test; p = 0.0007). Multivariate survival analysis showed that CIK cell treatment was an independent prognostic factor. In subgroup analysis, patients who received ≥8 cycles of CIK cell transfusion exhibited significantly better survival than the <8 cycle group (p = 0.0272). There was no significant difference in overall survival in patients with ≤5-cm tumors between the CIK and surgery-alone groups (p = 0.7567). However, in patients with >5-cm tumors, the CIK group displayed significantly better overall survival than the surgery-alone group (p = 0.0002). CONCLUSIONS: Postoperative immunotherapy with CIK cell transfusion may be an effective adjuvant treatment for improving the outcomes of HCC patients; >8 cycles of CIK cell transfusion may ensure that patients derive maximal benefits. Moreover, patients with large tumors might benefit more from CIK cell adjuvant treatment than patients with small tumors.

Comparative study on anti-tumor immune response of autologous cytokine-induced killer (CIK) cells, dendritic cells-CIK (DC-CIK), and semi-allogeneic DC-CIK.

BACKGROUND AND OBJECTIVE: Cytokine-induced killer (CIK) cells and autologous dendritic cells-CIK (DC-CIK) cells co-cultured with autologous dendritic cells (DCs) and CIK cells are commonly used for immunotherapy recently. We compared the anti-tumor immune response of CIK cells, autologous DC-CIK cells, and semi-allogeneic DC-CIK cells to explore a more effective anti-tumor adoptive immunotherapy approach. METHODS: Peripheral monocytes were isolated from patients with renal carcinoma, lung cancer, or maxillary squamous cell carcinoma and their healthy adult children. Isolated cells were cultured and induced as DCs and CIK cells in vitro. CIK cells from patients were co-cultured with autologous DCs and DCs from their children respectively, generating DC-CIK cells and semi-allogeneic DC-CIK cells. The anti-tumor activities of autologous CIK cells, autologous DC-CIK cells, and semi-allogeneic DC-CIK cells were measured by LDH assay. Intracellular staining was used to test the secretion of cytokines. Flow cytometry was applied for detecting the phonotype changes of these three types of cells. Cell proliferation and cell apoptosis were detected by 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and Annexin V/PI respectively. RESULTS: Compared with autologous CIK cells and DC-CIK cells, semi-allogeneic DC-CIK cells significantly enhanced the anti-tumor activity and IFN-gamma secretion, reduced IL-4 secretion, increased the ratio of CD3(+)CD56(+) cells and CD3(+)CD8(+) cells, decreased the number of CD4(+)CD25(+) cells, promoted cell proliferation, and lessened cell apoptosis. CONCLUSIONS: Semi-allogeneic DC-CIK cells had a stronger anti-tumor effect than did autologous CIK cells and DC-CIK cells. Our results provided experimental evidence for clinical application of DC-CIK cells.

Adenovirus-mediated delivery of human IFNgamma gene inhibits prostate cancer growth.

Interferon gamma (IFNgamma) is regarded as a potent antitumor agent, but therapy with IFNgamma is hampered by its short half-life and significant side effects. We developed a replication defective adenovirus carrying the human IFNgamma gene and evaluated the effects of adenovirus-mediated IFNgamma (Ad-IFNgamma) gene transfer on human prostate cancer cell lines in vitro and on xenografts in vivo. Our results showed infection of prostate cancer cells with Ad-IFNgamma led to production of an active cytokine and resulted in an antiproliferative effect on the prostate cancer cells. Intratumoral injection of Ad-IFNgamma significantly inhibited the growth of DU-145 cell xenografts in vivo, while no significant toxicity effect was observed. RT-PCR analysis indicated transgene expression mainly enriched in tumors in vivo, and slightly distributed in livers. These findings suggest adenovirus-mediated IFNgamma gene transfer is a promising approach in the treatment of advanced prostate cancer.

Adenoviral expression of a truncated S1 subunit of SARS-CoV spike protein results in specific humoral immune responses against SARS-CoV in rats.

The causative agent of severe acute respiratory syndrome (SARS) has been identified as SARS-associated coronavirus (SARS-CoV), but the prophylactic treatment of SARS-CoV is still under investigation. We constructed a recombinant adenovirus containing a truncated N-terminal fragment of the SARS-CoV Spike (S) gene (from--45 to 1469, designated Ad-S(N)), which encoded a truncated S protein (490 amino-acid residues, a part of 672 amino-acid S1 subunit), and investigated whether this construct could induce effective immunity against SARS-CoV in Wistar rats. Rats were immunized either subcutaneously or intranasally with Ad-S(N) once a week for three consecutive weeks. Our results showed that all of the immunized animals generated humoral immunity against the SARS-CoV spike protein, and the sera of immunized rats showed strong capable of protecting from SARS-CoV infection in vitro. Histopathological examination did not find evident side effects in the immunized animals. These results indicate that an adenoviral-based vaccine carrying an N-terminal fragment of the Spike gene is able to elicit strong SARS-CoV-specific humoral immune responses in rats, and may be useful for the development of a protective vaccine against SARS-CoV infection.

Adjuvant cellular immunotherapy in patients with resected primary non-small cell lung cancer.

Postoperative non-small cell lung cancer (NSCLC) patients require adjuvant therapy to improve their prognosis. In this study, we investigated the efficacy of a sequential combination of autologous cellular immunotherapy (CIT) and chemotherapy for postoperative NSCLC. This retrospective study included 120 postoperative NSCLC patients: 60 cases received only chemotherapy; 33 cases received chemotherapy and sequential CIT with cytokine-induced killer (CIK) cells; and 27 cases received chemotherapy and sequential CIT with alternate CIK and natural killer (NK) cells. Survival analysis showed significantly higher overall survival rates in the CIT group compared with the control group. Overall survival was higher in patients who received CIT with alternate CIK and NK cells than those who received treatment with only CIK cells. Multivariate analysis showed that adjuvant CIT was an independent prognostic factor for overall survival of patients with NSCLC. In subgroup analyses, adjuvant CIT significantly improved the overall survival of patients with less than 60 y old and positive lymph node. In conclusions, these data indicate that adjuvant CIT, especially with alternate application of CIK and NK cells, is an effective therapeutic approach to prolong survival of patients with NSCLC, particularly for patients ≤60 y old with positive lymph nodes.

Phase I trial of adoptively transferred tumor-infiltrating lymphocyte immunotherapy following concurrent chemoradiotherapy in patients with locoregionally advanced nasopharyngeal carcinoma.

Adoptive cell therapy (ACT) for cancers using autologous tumor-infiltrating lymphocytes (TILs) can induce immune responses and antitumor activity in metastatic melanoma patients. Here, we aimed to assess the safety and antitumor activity of ACT using expanded TILs following concurrent chemoradiotherapy (CCRT) in patients with locoregionally advanced nasopharyngeal carcinoma (NPC). Twenty-three newly diagnosed, locoregionally advanced NPC patients were enrolled, of whom 20 received a single-dose of TIL infusion following CCRT. All treated patients were assessed for toxicity, survival and clinical and immunologic responses. Correlations between immunological responses and treatment effectiveness were further studied. Only mild adverse events (AEs), including Grade 3 neutropenia (1/23, 5%) consistent with immune-related causes, were observed. Nineteen of 20 patients exhibited an objective antitumor response, and 18 patients displayed disease-free survival longer than 12 mo after ACT. A measurable plasma Epstein-Barr virus (EBV) load was detected in 14 patients at diagnosis, but a measurable EBV load was not found in patients after one week of ACT, and the plasma EBV load remained undetectable in 17 patients at 6 mo after ACT. Expansion and persistence of T cells specific for EBV antigens in peripheral blood following TIL therapy were observed in 13 patients. The apparent positive correlation between tumor regression and the expansion of T cells specific for EBV was further investigated in four patients. This study shows that NPC patients can tolerate ACT with TILs following CCRT and that this treatment results in sustained antitumor activity and anti-EBV immune responses. A larger phase II trial is in progress.

A nomogram for predicting the benefit of adjuvant cytokine-induced killer cell immunotherapy in patients with hepatocellular carcinoma.

The benefits of adjuvant cytokine-induced killer (CIK) cell immunotherapy for hepatocellular carcinoma (HCC) remain mixed among patients. Here, we constructed a prognostic nomogram to enable individualized predictions of survival benefit of adjuvant CIK cell treatment for HCC patients. Survival analysis showed that the median overall survival (OS) and progression-free survival (PFS) for patients in the hepatectomy/CIK combination group were 41 and 16 months, respectively, compared to 28 and 12 months for patients in the hepatectomy alone group (control). Based on multivariate analysis of the entire cohort, independent factors for OS were tumor size, tumor capsule, pathological grades, total bilirubin, albumin, prothrombin time, alpha-fetoprotein, and tumor number, which were incorporated into the nomogram. The survival prediction model performed well, as assessed by the c-index and calibration curve. Internal validation revealed a c-index of 0.698, which was significantly greater than the c-index value of the TNM (tumor-node-metastasis) staging systems of 0.634. The calibration curves fitted well. In conclusions, our developed nomogram resulted in more accurate individualized predictions of the survival benefit from adjuvant CIK cell treatment after hepatectomy. The model may provide valuable information to aid in the decision making regarding the application of adjuvant CIK cell immunotherapy.

Clinical activity of adjuvant cytokine-induced killer cell immunotherapy in patients with post-mastectomy triple-negative breast cancer.

PURPOSE: Triple-negative breast cancer (TNBC) is a high risk form of this disease, even after surgery, due to the absence of targets for hormone treatment and anti-Her-2 therapy. Chemotherapy is the main therapeutic strategy for such patients with breast cancer, although the outcome is often unsatisfactory. Thus, the development of combination adjuvant therapies is essential for improved prognosis in patients with TNBC. In this study, we investigated the efficacy of a sequential combination of cytokine-induced killer cell (CIK) infusion and chemotherapy for patients with post-mastectomy TNBC. EXPERIMENTAL DESIGN: From 2008 to 2012, 90 patients with post-mastectomy TNBC were included in this retrospective study: 45 cases received chemotherapy alone or with sequential radiotherapy; a further 45 cases received chemotherapy with/without radiotherapy and sequential CIK infusion. RESULTS: Survival analysis showed significantly higher disease-free survival (DFS) and overall survival (OS) rates in the CIK treatment group compared with the control group (P = 0.0382, P = 0.0046, respectively; log-rank test). Multivariate survival analysis showed that CIK adjuvant treatment was an independent prognostic factor for OS of patients with TNBC. In subgroup analyses, CIK adjuvant treatment significantly increased the DFS rate of patients with pathologic grade 3, and significantly increased the OS rate of patients in N1, N2, N3, IIB, III TNM (tumor-node-metastasis) stages, and with pathologic grade 3. CONCLUSIONS: These data indicate that adjuvant CIK treatment combined with chemotherapy is an effective therapeutic strategy to prevent disease recurrence and prolong survival of patients with TNBC, particularly those with lymph node metastasis, advanced TNM stage, and poor pathologic grade. Clin Cancer Res; 20(11); 3003-11. ©2014 AACR.

Decreased expression of the ARID1A gene is associated with poor prognosis in primary gastric cancer.

BACKGROUND: The ARID1A gene encodes adenine-thymine (AT)-rich interactive domain-containing protein 1A, which participates in chromatin remodeling. ARID1A has been showed to function as a tumor suppressor in various cancer types. In the current study, we investigated the expression and prognosis value of ARID1A in primary gastric cancer. Meanwhile, the biological role of ARID1A was further investigated using cell model in vitro. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the role of ARID1A gene in primary gastric cancer pathogenesis, real-time quantitative PCR and western blotting were used to examine the ARID1A expression in paired cancerous and noncancerous tissues. Results revealed decreased ARID1A mRNA (P = 0.0029) and protein (P = 0.0015) expression in most tumor-bearing tissues compared with the matched adjacent non-tumor tissues, and in gastric cancer cell lines. To further investigate the clinicopathological and prognostic roles of ARID1A expression, we performed immunohistochemical analyses of the 224 paraffin-embedded gastric cancer tissue blocks. Data revealed that the loss of ARID1A expression was significantly correlated with T stage (P = 0.001) and grade (P = 0.006). Consistent with these results, we found that loss of ARID1A expression was significantly correlated with poor survival in gastric cancer patients (P = 0.003). Cox regression analyses showed that ARID1A expression was an independent predictor of overall survival (P = 0.029). Furthermore, the functions of ARID1A in the proliferation and colony formation of gastric cell lines were analyzed by transfecting cells with full-length ARID1A expression vector or siRNA targeting ARID1A. Restoring ARID1A expression in gastric cancer cells significantly inhibited cell proliferation and colony formation. Silencing ARID1A expression in gastric epithelial cell line significantly enhanced cell growth rate. CONCLUSIONS/SIGNIFICANCE: Our data suggest that ARID1A may play an important role in gastric cancer and may serve as a valuable prognostic marker and potential target for gene therapy in the treatment of gastric cancer.

Intratumoral expression of IL-17 and its prognostic role in gastric adenocarcinoma patients.

In this study, we characterized the intratumoral expression of IL-17 and CD8(+) TILs in gastric adenocarcinoma patients after resection and determined the correlation between the survival probability of gastric adenocarcinoma patients and the expression of IL-17 in tumor. Expression of IL-17 and CD8 was assessed by immunohistochemistry, and the prognostic effects of intratumoral IL-17 expression and CD8(+) TILs were evaluated by Cox regression and Kaplan-Meier analysis. Immunohistochemical detection revealed the presence of IL-17 and CD8(+) cells in gastric adenocarcinoma tissue samples (90.6%, 174 out of 192 patients and 96.9%, 186 out of 192 patients, respectively). We have also found that intratumoral IL-17 expression was significantly correlated with age (p=0.004) and that the number of CD8(+)TILs was significantly correlated with UICC staging (p=0.012) and the depth of tumor invasion (p=0.022). The five-year overall survival probability among patients intratumorally expressing higher levels of IL-17 was significantly better than those expressing lower levels of IL-17 (p=0.036). Multivariate Cox proportional hazard analyses revealed that intratumoral IL-17 expression (HR: 0.521; 95% CI: 0.329-0.823; p=0.005) was an independent factor affecting the five-year overall survival probability. We conclude that low levels of intratumoral IL-17 expression may indicate poor prognosis in gastric adenocarcinoma patients.

[Inhibition of tongue cancer development in nude mice transfected with adenovirus carrying human endostatin gene].

BACKGROUND & OBJECTIVE: The squamous cell carcinoma of tongue is one of the most common malignant tumors of oral cavity. Surgical therapy is now the mainstay of combined treatment for tongue squamous cell Carcinoma with chemotherapy and radiotherapy. The overall 5-year survival rate was about 50%. The antiangiogenesis therapy has become a new approach of the treatment of tongue carcinoma. This paper was designed to study the characteristics of endostatin expression in tongue cancer cell line (Tca8113), human embryonic epithelial cell line (ECV) and the inhibition of carcinogenesis in nude mice, xenografted with Tca8113, after transfected with recombined adenovirus (Ad/hEnd) which was cloned with human endostatin gene in EI mutated region. METHODS: (1) To determine the expression and distribution of endostatin in Tca and ECV cells transfected with Ad/hEnd using immunohistochemistry. To determine the endostatin in supernatants of Tca cells transfected with Ad/hEnd using ELISA method. To examine the characteristics of endostatin gene expression in Tca8113 and ECV cells by Western blot analysis. (2) To determine the inhibition rate of proliferation and apoptosis rate of ECV cells by WST-1 test and flow cytometry (FCM), respectively.(3) To observe the inhibition of tumor growth in xenografted nude mice with Tca8113 cells by Ad/hEnd administration. RESULTS: (1) Immunohistochemistry detection indicated that the endostatin was expressed in cytoplasm of Tca8113 cells and ECV cells transfected with Ad/hEnd. Endostatin expression in the supernatant was dose-dependent with the highest to 597 ng/ml. The expression of endostatin in Tca cells was detectable from 1 day to 7 day. Ad/hEnd inhibited ECV cell growth in dose-dependent manner. (2) FCM showed that Ad/hEnd arrested ECV cells in S and G(2) phase and induced apoptosis.(3) The tumor growth curve showed that Ad/hEnd significantly repressed xenograft tumor growth with Tca cell in nude mice; the inhibition rate on Ad/hEnd administrated groups was 45.8% in the 3rd week. CONCLUSION: Ad/hEnd expressed efficiently in Tca8113 and ECV cells. Ad/hEnd can change the cell cycle distribution of ECV cells and induce apoptosis and inhibit proliferation of ECV cells. Ad/hEnd could inhibit the growth of tongue carcinoma in xenograft nude mice.