Id4 Suppresses the Growth and Invasion of Colorectal Cancer HCT116 Cells through CK18-Related Inhibition of AKT and EMT Signaling.

Id4 is one of the inhibitors of DNA-binding proteins (Id) and involved in the pathogenesis of numerous cancers. The specific mechanism underlying the Id4-mediated regulation of proliferation, invasion, and metastasis of colorectal cancer (CRC) cells is still largely unclear. In the present study, results showed CRC cells had a lower baseline Id4 expression than normal intestinal epithelial NCM460 cells. In order to explore the role of Id4 in the tumorigenicity, CRC HCT116 cells with stable Id4 expression were used, and results showed Id4 overexpression arrested the cell cycle at the G0/G1 phase, inhibited the cell proliferation and the colony formation, as well as suppressed the migration and invasion. In the in vivo model, Id4 overexpression inhibited the tumor growth and metastasis in the nude mice. Furthermore, Id4 overexpression upregulated the expression of proteins associated with cell proliferation, inhibited the PI3K/AKT pathway, and suppressed epithelial-mesenchymal transition (EMT) of HCT116 cells. Moreover, Id4 significantly decreased cytokeratin 18 (CK18) expression, but CK18 overexpression in Id4 expressing HCT116-Id4 cells rescued the activation of AKT, p-AKT, MMP2, MMP7, and E-cadherin. Collectively, our study indicated Id4 may inhibit CRC growth and metastasis through inhibiting the PI3K/AKT pathway in a CK18-dependent manner and suppressing EMT. Id4 may become a target for the treatment of CRC.

Intratumoral IL-12 Gene Therapy Inhibits Tumor Growth In A HCC-Hu-PBL-NOD/SCID Murine Model.

PURPOSE: This study aimed to evaluate the efficacy and safety of intratumoral IL-12 gene therapy in an HCC-hu-PBL-NOD/SCID mouse model. MATERIALS AND METHODS: The HCC murine model was generated in NOD/SCID mice, and mice with grafted tumors were injected intraperitoneally with 2 × 107 human peripheral blood lymphocytes 14 days after modeling. After 4 days, mice were randomly divided into the 9597/IL-12 group, the 9597/plasmid group and the PBS group. The changes of tumor volume were measured and mouse peripheral blood was sampled post-treatment for ELISA and CBA analyses, and the grafted tumors were collected 28 days post-treatment for immunohistochemistry, ELISA, CBA and detection of cell cycle and apoptosis. RESULTS: The tumor volume was smaller in the 9597/IL-12 group than in the 9597/plasmid and PBS groups on days 7, 14, 21, and 28 post-treatment (P < 0.05). Higher IL-12 levels were detected in the peripheral blood and the supernatants of grafted tumor homogenates in the 9597/IL-12 group than in the 9597/plasmid and PBS groups 7, 14, 21 and 28 days post-treatment (P < 0.05). IHC revealed higher counts of CD3+T cells, CD4+T helper cells, IFN-γ Th1 cells+ and S-100 protein positive dentric cells and lower MVD in the 9597/IL-12 group than in the 9597/plasmid and PBS groups (P < 0.05). Flow cytometry showed a significantly higher proportion of HCC cells at the G0/G1 phase and a significantly lower proportion of HCC cells at the S phase in the 9597/IL-12 group than in the PBS group (P < 0.05) and a greater apoptotic rate of HCC cells in the 9597/IL-12 group than in the 9597/plasmid and PBS groups (P < 0.05). CONCLUSION: Intratumoral IL-12 gene therapy may inhibit tumorigenesis with mild adverse effects in a HCC-hu-PBL-NOD/SCID murine model through inhibiting angiogenesis, arresting cells in G0/G1 phase and inducing apoptosis.

Epstein-Barr virus-coded miR-BART13 promotes nasopharyngeal carcinoma cell growth and metastasis via targeting of the NKIRAS2/NF-κB pathway.

Based on analysis of Epstein-Barr virus (EBV) BART microRNA expression profiles, we previously reported that EBV-encoded miR-BART13 is upregulated in nasopharyngeal carcinoma (NPC) plasma specimens. However, the effects and molecular mechanisms of miR-BART13 in NPC remain largely unknown. We found that miR-BART13 was significantly upregulated in NPC tissue specimens. Ectopic expression of miR-BART13 promoted NPC cell proliferation, epithelial mesenchymal transition, and metastasis in vitro, and facilitated xenograft tumor growth and lung metastasis in vivo. Molecularly, NF-κB inhibitor interacting Ras-like 2 (NKIRAS2), a negative regulator of the NF-κB signaling, was identified to be a direct target of miR-BART13 in NPC cells, and NKIRAS2 mRNA and protein expression was inversely correlated with miR-BART13 in NPC tissues, respecitvely. Furthermore, the NF-κB signaling pathway was activated by miR-BART13. By rescued experiments, reconstitution of NKIRAS2 expression abrogated all the phenotypes upregulated by miR-BART13, and attenuated activity of NF-κB signaling pathway activated by miR-BART13 in NPC cells. Our findings indicated the newly identified miR-BART13/NKIRAS2/NF-κB signaling axis may provide further insights into better understanding of NPC initiation and development, and targeting of this pathway could be further studied as a therapeutic strategy for NPC patients.

Somatostatin Octapeptide Inhibits Cell Invasion and Metastasis in Hepatocellular Carcinoma Through PEBP1.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is a major threat to human health. The condition carries a high risk of death; 45% of new cases occur in China. Surgical resection is the first choice for treatment of HCC, but 30.9% of patients experience recurrence within 6 months after the operation. To improve patient survival, we must determine how to reduce the probability of recurrence and metastasis and elucidate the underlying mechanism of disease. We therefore studied the effect of somatostatin octapeptide (octreotide) on the invasion and metastasis of HCC. METHODS: The migration and invasion cytological tests were used to detect the effect of octreotide on liver cancer cells (SK-Hep-1 and HepG2). PEBP1 RNAi was used to knockdown expression. Invasion and metastasis were measured with transwell migration and wound-healing assays. Western blotting was used to detect changes in levels of PEBP1 and invasion pathway proteins after octreotide treatment. The effect of octreotide was studied in vivo by establishing a pulmonary metastasis model using SK-Hep-1 cells in nude mice. In-vivo bioluminescence imaging and hematoxylin and eosin staining of lung tissue were used to verify the results. RESULTS: Increasing concentrations of octreotide were progressively more effective in halting the invasion and metastasis of liver cancer cells. Octreotide may upregulate PEBP1, TIMP-2, and E-cadherin while downregulating MMP-2 and Twist to inhibit cell invasion and metastasis. And downregulation of PEBP1 would also change the expression of MMP-2, TIMP-2 and Twist. The in-vivo experiments showed no cancer cell metastasis in 4 of the 6 mice in the octreotide-treatment group, while all of the mice in the control group displayed pulmonary metastasis of human HCC cells. And the survival period of the mice in the octreotide-treatment group was significantly prolonged. CONCLUSIONS: Octreotide may weaken invasion and metastasis through the upregulation of PEBP1. Octreotide may reduce the risk of recurrence and metastasis after surgery for liver cancer.

Metabolomics identifies metabolite biomarkers associated with acute rejection after heart transplantation in rats.

The aim of this study was to identify metabolite biomarkers associated with acute rejection after heart transplantation in rats using a LC-MS-based metabolomics approach. A model of heterotopic cardiac xenotransplantation was established in rats, with Wistar rats as donors and SD rats as recipients. Blood and cardiac samples were collected from blank control rats (Group A), rats 5 (Group B) and 7 days (Group C) after heart transplantation, and pretreated rats 5 (Group D) and 7 days (Group E) post-transplantation for pathological and metabolomics analyses. We assessed International Society for Heart and Lung Transplantation (ISHLT) grades 0, 3B, 4, 1 and 1 rejection in groups A to E. There were 15 differential metabolites between groups A and B, 14 differential metabolites between groups A and C, and 10 differential metabolites between groups B and C. In addition, four common differential metabolites, including D-tagatose, choline, C16 sphinganine and D-glutamine, were identified between on days 5 and 7 post-transplantation. Our findings demonstrate that the panel of D-tagatose, choline, C16 sphinganine and D-glutamine exhibits a high sensitivity and specificity for the early diagnosis of acute rejection after heart transplantation, and LC-MS-based metabolomics approach has a potential value for screening post-transplantation biomarkers.

Tumor MICA status predicts the efficacy of immunotherapy with cytokine-induced killer cells for patients with gastric cancer.

In this study, we determine the relationship between the expression of major histocompatibility complex class I chain-related gene A (MICA) in gastric cancer tumors after D2 gastrectomy and the clinical outcome of a CIK-containing adjuvant therapy. Ninety-five consecutive patients with gastric cancer after D2 gastrectomy who received adjuvant chemotherapy combined with CIK cell therapy were enrolled. The MICA expression of their tumors was determined by immunohistochemistry (IHC). High expression of MICA protein was documented by IHC in 38 of 95 tumor samples (40.0 %). The MICA status was significantly associated with the age and stage, p = 0.008 and 0.023, respectively. Analysis of NKG2D on in vitro expanded CIK cells showed that the percentages of NKG2D+ in CD3+/CD56+, CD3-/CD56+, and CD3+/CD8+ cells populations were 97.2 ± 1.4, 97.9 ± 1.8, and 95.6 ± 2.1 %, respectively. For patient with high MICA-expressing tumors, the median DFS and OS were longer than for the patients with tumors with low expression of MICA; 46.0 versus 41.0 months (p = 0.027), and 48.0 versus 42.0 months (p = 0.031), respectively. In a multivariate analysis, stage and MICA expression were independent prognostic factors for DFS and OS. Our findings show that adjuvant chemotherapy plus CIK therapy treatment is a promising modality for treating gastric cancer patients after D2 gastrectomy. Especially, those who have tumors with high expression of MICA were more likely to benefit from such a treatment strategy. Subsequent studies in clinical trial cohorts will be required to confirm the clinical utility of these markers.

MHC I-related chain a expression in gastric carcinoma and the efficacy of immunotherapy with cytokine-induced killer cells.

Cytokine-induced killer (CIK) cells have shown promising activity against gastric cancer in vitro and in vivo. Previous studies showed that cell signaling through MHC I-related Chain A (MICA)-Natural killer group 2, member D (NKG2D) results in CIK cell activation leading to cytolytic activities against tumor cells. In this study, we investigate the MICA status in patients with gastric carcinoma, and determine the potential relationship between MICA and clinical outcome of a CIK containing therapy. Two hundred and forty-three patients with gastric cancer who had received curative D2 gastrectomy were enrolled. The MICA expression of their tumors was determined by immunohistochemistry (IHC). Disease-free survival (DFS) and overall survival (OS) were evaluated. One hundred and forty-eight patients received adjuvant chemotherapy alone, and 95 patients received adjuvant chemotherapy combined with autologous CIK cell therapy. Patients who received adjuvant chemotherapy plus CIK had significantly longer DFS, 42.0 months vs. 32.0 months (P = 0.012), and OS, 45.0 months vs. 42.0 months (P = 0.039), by log-rank test. MICA high-expression, IHC scores of 5-7, was found in tumors from 89 of 243 patients (36.6%). The MICA expression was significantly correlated with the stage (P = 0.007) and there was a borderline association with histological grade (P = 0.054). In the adjuvant chemotherapy plus CIK group (n = 95), patients with high MICA expression had longer DFS, 46.0 months vs. 41.0 months (P = 0.027), and OS, 48.0 months vs. 42.0 months (P = 0.031). In the adjuvant chemotherapy alone group (n = 148), the median DFS and OS had no significant correlation with the MICA status. In a multivariate analysis stage, CIK therapy, and the interaction of MICA status and CIK therapy were independent prognostic factors for DFS and OS. Our study indicated that adjuvant chemotherapy plus CIK immunotherapy is a promising modality for treating gastric cancer patients after D2 gastrectomy. MICA status was associated with the outcome measures in CIK therapy, validation in prospective clinical trials is required to assess the value of this biomarker in the clinical decision-making process.

Combined Analysis of EGFR and PTEN Status in Patients With KRAS Wild-Type Metastatic Colorectal Cancer.

To determine the relationship between the expression of phosphatase and tensin homologue (PTEN) and epidermal growth factor receptor (EGFR) in metastatic colorectal cancer (mCRC) and the clinical outcome of cetuximab-containing chemotherapy. A total of 158 consecutive mCRC patients with wild-type KRAS status who received chemotherapy with or without cetuximab, and for whom tumor tissue was available, were enrolled. The EGFR and PTEN expression was determined by immunohistochemistry (IHC). A total of 158 mCRC patients with wild-type KRAS status were enrolled in the study; 51 patients received chemotherapy combined with cetuximab, 107 patients received chemotherapy alone. Patients who received chemotherapy combined with cetuximab had longer overall survival (OS) compared with patients who received chemotherapy alone. High EGFR expression was detected in 60 patients (38.0%), while normal PTEN expression was detected in 60 patients (59.5%). The PTEN status was significantly related with the histological grade. For patients who received chemotherapy combined with cetuximab the median OS of patients with high-expression of EGFR was longer than the OS of patients with low EGRF expression; 25.0 versus 19.0 months, P = 0.002. For patient with normal PTEN the median OS were longer than the median OS for patients with loss of PTEN; 24.0 versus 19.0 months, P = 0.026. The overall response rate (ORR) had a borderline association with EGFR and PTEN expression (P = 0.055 and 0.048, respectively). In a multivariate analysis, ECOG PS, EGFR status, chemotherapy ± cetuximab, and the interaction of EGFR or PTEN and chemotherapy ± cetuximab were independent prognostic factors for OS. Our findings show that chemotherapy combined with cetuximab demonstrated encouraging antitumor activity for mCRC patients with wild-type KRAS status. Especially, those who have high EGFR expression or normal PTEN expression were more likely to benefit from such a treatment strategy. Subsequent studies in clinical trial cohorts will be required to confirm the clinical utility of these markers.

Efficacy of adjuvant chemotherapy combined with immunotherapy with cytokine-induced killer cells for gastric cancer after d2 gastrectomy.

BACKGROUND AND AIMS: It remains a challenge to prevent the local recurrence or distant metastasis of gastric carcinoma after D2 gastrectomy. Cytokine-induced killer (CIK) cells have shown promising activity against solid tumors in vitro and in vivo. We investigated the effect of adjuvant chemotherapy combined with autologous CIK therapy after D2 gastrectomy compared with adjuvant chemotherapy alone after D2 gastrectomy in patients with stage II-III gastric cancer. METHODS: From January 2009 to December 2011, 226 patients with stage II-III gastric cancer who had had curative D2 gastrectomy were enrolled. Eighty-nine patients (CIK group) received adjuvant chemotherapy combining with autologous CIK therapy and 137 patients (control group) received adjuvant chemotherapy alone. Disease-free survival (DFS) and overall survival (OS) were evaluated. RESULTS: Patients in the CIK group had longer DFS and OS than patients in the control group (DFS 41.0 months vs. 32.0 months, OS 45.0 months vs. 44.0 months, by log-rank test P = 0.006 and P = 0.028, respectively). In subgroup analysis, no significant differences in DFS and OS were observed between the two groups for the patients with stages II disease. Patients with stage III disease in the CIK group had longer median DFS and OS than patients in the control group (P = 0.031 and P = 0.038, respectively). In multivariate analysis, the stage and the interaction of stage and CIK therapy were independent prognostic factors for DFS and OS. CONCLUSIONS: The data suggest that adjuvant chemotherapy combined with autologous CIK therapy can improve prognosis for gastric carcinoma patients after D2 gastrectomy, especially for the patients with stage III disease.

Decreased expression of liver-type fatty acid-binding protein is associated with poor prognosis in hepatocellular carcinoma.

BACKGROUND/AIMS: The purpose of this study was to assess liver-type fatty acid-binding protein (L-FABP) expression and its association with clinicopathological features in hepatocellular carcinoma (HCC). METHODOLOGY: L-FABP mRNA expression in 57 samples of HCC and corresponding adjacent liver tissue and 8 normal liver tissue samples were examined by real-time reverse transcriptase (RT)­PCR analyses. Tissue microarray technique and immunohistochemistry (IHC) were used to detect the expression of L-FABP in 163 HCCs. The association between L-FABP expression and the clinicopathological factors and prognosis was analyzed. RESULTS: The average expression of L-FABP mRNA was 0.233 in the HCC tissues, 1.407 in the peri-carcinoma tissues, and 1.0 in the normal liver tissues. IHC analysis showed that there were 47% (76/163) HCCs exhibited weak or even no immunoreactivity of L-FABP. The L-FABP expression in HCC showed significant associations with preoperative levels of AFP (p=0.039), tumor size (p=0.026), histological grade (p=0.000), differential degree (p=0.000), vascular invasion (p=0.016), capsular invasion (p=0.029) and recurrence (p=0.004). Patients with L-FABP high-expression showed better prognosis than patients with L-FABP low-expression (p=0.008). CONCLUSIONS: L-FABP was downregulated in HCC and could be served as a promising prognostic marker for HCC patients.

Effects of MICA expression on the prognosis of advanced non-small cell lung cancer and the efficacy of CIK therapy.

OBJECTIVE: To investigate the clinical significance of the expression of MHC class I chain-related gene A (MICA) in patients with advanced non-small cell lung cancer and explore the relationship between MICA expression and the efficacy of cytokine-induced killer cell (CIK) therapy for treating advanced non-small cell lung cancer. METHODS: We obtained data on 222 patients with advanced non-small cell lung cancer, including data on MICA expression, age, gender, ECOG score, pathological type, stage, treatment history (including 38 patients who were given autologous CIK cell infusion), and overall survival (OS). MICA expression in lung cancer tissue was evaluated by immunohistochemical staining. Analyses of MICA expression, and CIK therapy association with survival outcomes were performed using Cox proportional models, Kaplan-Meier methods, and the log-rank test. RESULT: s MICA was expressed in both membrane and cytoplasm. MICA expression correlated with the stage of lung cancer, ECOG score, gender and age. Multivariate COX regression analysis showed that the expression of MICA was an independent prognostic factor of advanced non-small cell lung cancer (p = 0.002). In subgroup analysis, we divided the 222 patients into CIK and control groups. In the CIK group, the medium OS (mOS) of patients with a high expression of MICA was longer than in those with low expression of MICA (27 months vs. 13 months). In the control group, the mOS in patients with a high expression of MICA was shorter than in patients with low MICA expression (9 months vs. 18 months). COX regression analysis showed that the MICA expression affects the effect of CIK therapy (p<0.0001). CONCLUSION: 1) The high expression of MICA is one of the indicators of a poor prognosis for advanced non-small cell lung cancer patients. 2) The high expression of MICA might be one of the predictive factors for successful CIK therapy.

[Expression and significance of the NK cell receptors in primary hepatocellular carcinoma and paracancerous tissues].

AIM: To investigate the expression of the activating and inhibitory receptors on the surface of NK cells of primary hepatocellular carcinoma and its adjacent tissues, and the relationship between these two receptors and occurrence and development of primary liver cancer was analyzed. METHODS: The number and activity of the NK cells, the expression of the activating and the inhibitory receptors on the surface of those cells were detected flow cytometry and immunohistochemistry, which were obtained from 52 cases of primary hepatocellular carcinoma and its adjacent tissues. The relative analysis was done between those results and clinical relative factors. RESULTS: In the tissues of primary hepacellular carcinoma, the number of NK cells is lower than that in the adjacent tissues obviously (P<0.01); the expression of activating receptors, NKG2D and NKP44, is also lower than that in the adjacent tissues obviously (P<0.05); the expression of inhibitory receptors, CD158b and CD159a, is significantly higher than that in the adjacent tissues (P<0.05). A negative correlation was found between the expression of NKG2D, NKP30 and NKP44 and the clinical stage of the liver cancer. The expression of NKG2D, NKP30 and NKP44 was higher in patients with early and middle stages (P<0.05). The content of the inhibitory receptors of NK cells, CD158b and CD159a, is higher in tissues from patients with advanced cancer stage (P<0.05). That's also correlated with the level of AFP and the HBsAg. There is no significant statistical difference between the expression of NK receptors and the distant metastasis, tumor differentiation as well as the tumor size (P>0.05). CONCLUSION: The decrease of NK cell numbers and the activating NK cell receptors and the increase of the inhibitory receptors would be relevant to the incidence of primary hepacellular carcinoma.

KRAS mutation and NF-κB activation indicates tolerance of chemotherapy and poor prognosis in colorectal cancer.

BACKGROUND: Evidence shows a strong relationship between KRAS mutations and the NF-κB signaling pathway. In colorectal cancer, however, the study of this subject has been very limited and results are inconsistent. AIMS: To examine the relationship between KRAS mutations and NF-κB activation and their effect on chemotherapy response and survival of colorectal cancer patients. MATERIALS AND METHODS: NF-κB activation was analyzed by immunohistochemistry in 167 primary colorectal cancer specimens in which the KRAS mutation status was confirmed. Clinical and pathologic data were extracted from the medical records and reviewed. RESULTS: Of 167 tumors screened, 63 (37.7 %) had NF-κB activation, 59 (35.3 %) had KRAS mutations, and 30 (18.0 %) had both NF-κB activation and KRAS mutations. The frequency of NF-κB activation in tumors with KRAS mutations was significantly higher than in tumors with wild type KRAS; 50.8 versus 30.6 %, P = 0.012. Patients with both KRAS mutations and NF-κB activation had a lower objective response to first-line chemotherapy than patients with other tumors, 23.8 versus 49.4 % (P = 0.035). Compared to patients with both KRAS mutations and NF-κB activation, overall survival of patients in other groups was significantly higher; median overall survival was 28.4 months (95 % CI 21.0-35.8) versus 46.3 months (95 % CI 39.4-53.2), hazard ratio 0.259 (95 % CI 0.125-0.538), P = 0.005. CONCLUSIONS: NF-κB activation was associated with KRAS mutation, and both KRAS mutation and NF-κB activation were indicative of high tolerance of chemotherapy and poor prognosis for colorectal cancer patients. Tumors with KRAS mutations and NF-κB activation may be a unique subtype of colorectal cancer.

NF-κB activity is downregulated by KRAS knockdown in SW620 cells via the RAS-ERK-IκBα pathway.

The relationship between KRAS and NF-κB in colorectal cancer is not clear. Western blotting was used to determine whether KRAS knockdown in SW620 cells altered the levels of NF-κB-p65 and other molecules. Furthermore, we investigated the association between the KRAS status and NF-κB expression in 167 colorectal cancers tumor tissues and their correlation with overall survival (OS) of patients with KRAS mutations and activated NF-κB. RAS, p-ERK, p-IκBα and p65 expression was decreased in SW620 cells with KRAS knockdown. The MEK inhibitor U0126 downregulated p-ERK, p-IκBα and p65 levels in SW620 cells. p65 activation in tumors with KRAS mutations was higher (50.8%) than in tumors with the wild-type KRAS gene (30.6%) (P=0.012). Compared to patients with other types of tumors, OS was lower (median 28.4 months) in patients with KRAS mutations and NF-κB activation, vs. a median of 46.3 months in patients with other types of tumors (P=0.005). NF-κB activation was reduced in SW620 cells with KRAS knockdown, possibly via the RAS-ERK-IκBα pathway. The presence of both KRAS mutations and the active form of NF-κB in CRC tumors indicates poor patient prognosis.

[Significance of the expression of lymphocytes and cytokines infiltrating in HCC].

AIM: To reveal the classification of lymphocytes and expression of cytokines infiltrating in HCC, and investigate the significance of changes of immune microenvironment in HCC. METHODS: 76 tumor and non-tumor tissues of HCC were collected to detect the amount of immune cells in the tissues by FCM and immunohistochemistry (IHC). Th1/Th2 cytokines in tissues were detected by Cytometric bead array (CBA), and TGF-1, VEGF detected by ELISA. RESULTS: There were more CD3(+); T cells, CD4(+); Th cells, Treg cells, and CD45RO(+); memory T cells in tumors than in non-tumor tissues. On the contrary there were less CD8(+); CTLs in tumors. There was negative correlation between Treg and Th or CTL cells. CD69, which is the early activating factor, expressed less on CD3(+); T cells and NK cells in tumors than non-tumor tissues. Whereas HLA-DR, which is the late activating factor, expressed more on CD3(+); T cells in tumors than non-tumor tissues. More IL-10, TGF-1 and VEGF were secreted in tumors than non-tumor tissues. CONCLUSION: Along with the decrease of effective immune cells and increase of suppressor immune cells and cytokines, the lymphocytes infiltrating in the tumor were immune incompitence, which contributed to the tumorigenesis.

Irradiated haploidentical donor leukocyte infusions as an adoptive immunotherapy strategy to induce host-versus-tumor effects.

AIMS AND BACKGROUND: Previous research has shown that irradiated splenocytes preserve the antitumor effect and induce relatively weaker graft-versus-host disease in parent C57BL/6~CB6F1 transplantation. The present study was designed to investigate the antitumor effect of 5-Gy-irradiated haploidentical donor leukocyte infusions (DLI) without prior bone marrow transplantation and the possibly involved mechanism in (H-2d/k)~(H-2b/d) infusion model systems. METHODS: Hepa 1-6 tumor-bearing mice were used to evaluate the antitumor effect and the possible mechanism of irradiated haploidentical DLI treatment. Changes in tumor volume, lymphocyte proliferation and cytotoxicity, IFN-gamma and IL-2 secretion and donor cell survival in vivo were analyzed. RESULTS: After irradiated haploidentical DLI treatment of the poorly immunogeneic Hepa 1-6 tumor mouse model, the donor cells were rejected and disappeared within 4 days. Surprisingly, an antitumor response was still observed. The infusion treatment effectively inhibited tumor growth and prolonged the survival of recipients, and this effect could be enhanced by combined treatment with cyclophosphamide and impaired by deleting donor-derived T cells. Moreover, the infusion treatment increased the levels of type 1 cytokines including IFN-gamma and IL-2, and enhanced the proliferation of lymphocyte subsets, particularly CD8 + T and NK cells. Specifically, multiple infusions proved to enhance the antitumor effect without inducing graft-versus-host disease. CONCLUSIONS: As an adoptive therapy, irradiated haploidentical DLI without bone marrow transplantation might be a promising and safe treatment for cancer.

[Application of serum proteomic mass spectrum analysis in breast cancer].

OBJECTIVE: To analyze the characteristics of serum proteins mass spectra in healthy controls, benign breast tumors, and CA15-3 negative or CA15-3 positive breast cancer patients by surface enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS). METHODS: Tissue samples of 113 cases of breast cancer (93 case of CA15-3 negative, 20 case of CA15-3 positive), 103 cases of benign breast tumor and 92 cases of healthy controls were examined and analyzed by SELDI and protein chip (CM10) techniques. Biomarker Pattern Software (BPS) was used to detect the protein peaks significantly different between them and establish a diagnostic pattern which was further evaluated by a blind test. RESULTS: Twelve significantly different protein peaks were found in serum samples between breast cancer patients and healthy controls. Eleven significantly different peaks were found between benign breast tumor patients and healthy controls. By combined analysis of those three different protein mass spectra, the peak 15 952 was found to be significantly different between breast cancer group and healthy controls, and the peak 7985 was significantly different among breast cancer group, benign breast tumor group and health controls. The blind test with the differential proteins for the serum samples of 93 cases of CA15-3 negative breast cancer and 36 cases of benign breast tumors showed that the sensitivity was 80.6% and specificity was 91.7%. The blind test in 20 cases of CA15-3 positive breast cancer and 36 cases of benign breast tumors showed that the sensitivity was 75.0% and specificity was 91.7%. Four significantly different protein peaks were found between the benign breast tumor patients and CA15-3 negative breast cancer patients. No significantly different protein were found between CA15-3 negative and CA15-3 positive patients. CONCLUSION: Significantly different protein peaks can be screened out in breast cancer, benign breast tumor patients and healthy controls by SELDI-TOF-MS analysis.

Efficacy of transcatheter arterial chemoembolization combined with cytokine-induced killer cell therapy on hepatocellular carcinoma: a comparative study.

BACKGROUND AND OBJECTIVE: Cytokine-induced killer (CIK) cells have high anti-tumor activity for hepatocellular carcinoma (HCC). Whether CIK cell therapy can eradicate residual cancer cells and prevent or postpone tumor relapse after transcatheter arterial chemoembolization (TACE) should be testified. This study was to evaluate the efficacy of CIK cell therapy combined with TACE on HCC. METHODS: A total of 146 consecutive patients with unresectable HCC were divided into combination group (72 patients treated with CIK cell therapy combined with TACE) and TACE group (74 patients treated only with TACE). The progression-free survival (PFS) and overall survival (OS) were analyzed. RESULTS: The 6-month, 1-year, and 2-year PFS rates were 72.2%, 40.4%, 25.3% in combination group, and 34.8%, 7.7%, 2.6% in TACE group. The median time to progression was 11 months [95% confidence interval (CI), 8-14 months] in combination group and 5 months (95% CI, 4-7 months) in TACE group. The estimated 6-month, 1-year, and 2-year OS rates were 90.3%, 71.9%, 62.4% in combination group, and 74.6%, 42.8%, 18.8% in TACE group. The median OS was 31 months (95% CI, 27-35 months) in combination group and 10 months (95% CI, 7-13 months) in TACE group. The times of TACE, ECOG performance status, and CIK cell therapy were independent prognostic factors for PFS and OS. CONCLUSION: Adjuvant immunotherapy with CIK cells could greatly improve the efficacy of TACE on HCC, and plays an important role in prolonging the PFS and OS of HCC patients after TACE.

[Screening for K-ras mutations in colorectal and lung cancers by using a novel real-time PCR with ADx-K-ras kit and Sanger DNA sequencing].

OBJECTIVE: to map out the frequency and types of K-ras gene mutations present in colorectal and lung cancer patients; to evaluate the clinical applicability of a novel real-time double-loop probe PCR using the ADx-K-ras kit, and to compare its performance with the result by using traditional Sanger DNA sequencing in detection of somatic mutations of the tumor genes. METHODS: a total of 827 formalin-fixed paraffin-embedded (FFPE) blocks including 583 from the colorectal and 244 from the lung cancer patients were assayed. Genomic DNA of the sample tissues was extracted, purified and subjected to PCR amplification of K-ras gene codon 12 and 13 and DNA sequencing was carried on using both the traditional Sanger sequencing method and the ADx's K-ras mutation detection kit, respectively. The mutation rates for K-ras gene at codon 12 and 13, and the mutation frequencies detected by using both methods were analyzed. RESULTS: 533 out of 583 (91.4%) colorectal cancer samples and 144 out of 244 lung cancer samples (59.0%) were detected using the traditional Sanger DNA sequencing technique, and 583 out of 583 (100.0%) colorectal plus 244 out of 244(100.0%) lung cancers were detected, respectively by using the ADx-K-ras kit. Of the 583 colorectal cancer samples, 192 (32.9%) showed mutations by using the ADx-K-ras kit in comparing with a result of 160 samples (27.4%) with K-ras gene mutation by using the traditional Sanger DNA sequencing technique. Of the 244 lung cancer samples, 26 (10.7%) showed K-ras gene mutations by using ADx-K-ras kit, while in 144 samples detected by using the traditional Sanger DNA sequencing technique, only 12 samples (8.3%) showed K-ras gene mutations. In colorectal cancer analyzed, GGT→GAT at codon 12 was the most common event with 35.1% (66/188) mutations, followed by GGC→GAC at codon 13 with 26.6% (50/188) and GGT→GTT at codon 12 with 18.6% (35/188), while GGT→GCT at codon12 was the most rare with only 1.6% (3/188) of the total mutation cases. In patients with lung cancer analyzed, GGT→GTT at codon 12 was the most common mutation, accounting for 40.9% (9/22), and GGT→GCT at codon 12 the most rare with only about 4.5% (1/22) of the total mutation cases. CONCLUSIONS: K-ras gene mutations were present in colorectal cases, and significantly more frequent than that in lung cancer. There were significant statistical differences between the two methods. ADx-K-ras real-time PCR showed much higher successful detection rates and mutation ratios compared to Sanger sequencing. As a result, the real-time PCR with ADx-K-ras kit proves to have a good clinical applicability and a strong advantage over the traditional Sanger DNA sequencing. It is a effective and reliable tool for clinical screening of somatic gene mutations in tumors.

The combined administration of partially HLA-matched irradiated allogeneic lymphocytes and thalidomide in advanced renal-cell carcinoma: a case report.

Renal-cell carcinoma (RCC) is susceptible to immune therapy including the use of the nonmyeloablative allogeneic transplantation (NST). However, NST can produce severe toxicity, might not be appropriate for many patients with metastatic RCC. Other novel allogeneic immunotherapies are designed to induce an autologous immune response directed against the malignancy. In single-arm phase II trials, thalidomide has demonstrated a modest activity in the treatment of advanced RCC. Here we present a case report in which a patient with advanced RCC in the absence of transplant conditioning, that was receiving thalidomide, was infused with partially HLA-matched irradiated allogeneic lymphocytes. In this patient a complete response with weak acute graft-versus-host disease (GVHD) was observed. No evidence of the disease was present over the subsequent 36 months survival of the patient, suggesting that the infusions may have played a major role in the antineoplastic effect. A potential mechanism of this protocol may involve a host-versus-graft reactions-mediated antitumor effect against the malignancy. In addition, the present results suggest that a combination protocol with alternate treatment (e.g., chemotherapy) schedules merit further investigation in the management of various malignancies.