Interleukin‐10 gene transfer activates interferon‐γ and the interferon‐γ‐inducible genes Gbp‐1/Mag‐1 and Mig‐1 in mammary tumors

Expression of IL‐10 as a transgene inhibits murine mammary tumor growth and metastasis. Using differential display methodology, we sought genes whose expression was modulated by IL‐10. We compared mRNA isolated from parental murine mammary 66.1 tumors, as well as tumors derived from neo^r‐transfected cells and 6 different IL‐10‐expressing cell lines. We identified 2 cDNA products that were up‐regulated in all 6 IL‐10‐expressing tumors in comparison to parental and 66‐neo tumors. One cDNA corresponds to the murine guanylate‐binding protein gene Gbp‐1/Mag‐1. The other cDNA corresponds to the chemokine Mig‐1 (monokine induced by IFN‐γ). Both genes were originally identified in IFN‐γ‐activated macrophages or macrophage cell lines. We now report that cultured mammary epithelial tumor cell lines also express both genes in response to treatment with IFN‐γ and LPS. Furthermore, IFN‐γ mRNA is elevated in IL‐10‐expressing tumors in comparison with parental or neo‐transfected tumors. Thus, high‐level expression of IL‐10 as a transgene results in activation rather than suppression of IFN‐γ as well as 2 IFN‐γ‐inducible genes. Up‐regulation of host IFN‐γ is critical to anti‐tumor activity since IL‐10 no longer inhibits tumor growth in hosts with a deletion in the IFN‐γ gene. Additionally, Gbp‐1/Mag‐1 and Mig‐1 gene induction no longer occur in IFN‐γ mutant mice. Int. J. Cancer 80:624–629, 1999. © 1999 Wiley‐Liss, Inc.     

Clinical features and outcomes in patients with extraskeletal Ewing sarcoma

BACKGROUND:

Ewing sarcoma can arise in either bone or soft tissue. The purpose of this study was to investigate whether patient characteristics, treatment strategies, and outcomes differ between skeletal Ewing sarcoma and extraskeletal Ewing sarcoma (EES).

METHODS:

Patients <40 years of age with Ewing sarcoma or peripheral primitive neuroectodermal tumor reported to the United States Surveillance, Epidemiology, and End Results Program database from 1973 to 2007 were evaluated based on skeletal (n = 1519) versus extraskeletal (n = 683) site of origin. Patient characteristics were compared using Fisher exact tests. Overall survival was estimated via the Kaplan‐Meier method and compared using log‐rank tests and Cox proportional hazard models.

RESULTS:

Patients with EES had a higher mean age (19.5 vs 16.3 years; P < .001) and were less likely to be male (53.4% vs 63.3%; P < .001) or white (84.8% vs 92.5%; P < .001) compared with patients with skeletal tumors. Extraskeletal tumors were more likely to arise in axial locations (72.9% vs 54.2%; P = .001) but were less likely to arise specifically in the pelvis (19.8% vs 26.6%; P < .001). Metastatic status or tumor size did not differ by group. Five‐year overall survival was superior for localized EES compared with localized skeletal tumors (69.7% vs 62.6%; P = .02). The hazard ratio for death in patients with localized skeletal tumors compared with localized EES was 2.36 (95% confidence interval, 1.61‐3.44) beyond 24 months from initial diagnosis.

CONCLUSIONS:

Patient characteristics and outcomes differ among patients with EES compared with patients with skeletal Ewing sarcoma. These findings may have important implications for patient care. Cancer 2011. © 2011 American Cancer Society.     

Up‐regulation of interleukin‐17 expression by human papillomavirus type 16 E6 in nonsmall cell lung cancer

BACKGROUND:

Human papillomavirus (HPV) 16/18 infection is associated with nonsmoking lung cancer. In this study, the authors investigated a putative correlation between interleukin (IL)‐17 expression and HPV infection in clinical nonsmall cell lung cancer (NSCLC) tissues and examined the effects of HPV infection on a human NSCLC cell line.

METHODS:

IL‐17 expression was investigated in 79 NSCLC tumor tissues by immunohistochemistry. Growth rate, IL‐17 mRNA, and secreting protein levels were also examined in HPV 16/18 E6‐transfected H1299 human NSCLC cells.

RESULTS:

Immunohistochemical data showed that 48.1% of lung tumors had IL‐17 staining, which was significantly associated with patients' sex (P = .03), HPV infection (P = .002), and tumor stage (P = .03). Significant correlations of IL‐17 with IL‐6 (P < .001) and IL‐17 with Mcl‐1 (P < .001) expression were also observed. Cell growth rate was increased, and IL‐17/Mcl‐1 expression levels were elevated in HPV 16 E6‐transfected H1299 cells. The transfected E6 oncoproteins can significantly up‐regulate expression levels of IL‐17 and antiapoptotic protein Mcl‐1.

CONCLUSIONS:

The study suggests that HPV infection‐induced IL‐17 levels can stimulate Mcl‐1 expression through the PI3K pathway and promote lung tumor cell progression through a p53‐ and IL‐6‐independent pathway. Cancer 2010. © 2010 American Cancer Society.     

Late effects of chemotherapy and radiotherapy in osteosarcoma and Ewing sarcoma patients: The Italian Sarcoma Group Experience (1983-2006)

BACKGROUND:

Patients with osteosarcoma and Ewing sarcoma have achieved longer survival over the past decades, but late side effects of chemotherapy and radiotherapy have become important concerns.

METHODS:

The authors reviewed all patients with localized osteosarcoma or Ewing sarcoma who had been enrolled in the Italian Sarcoma Group neoadjuvant protocols from 1983 through 2006. Data were updated in December 2010 to determine 3 endpoints: the incidence of a secondary primary cancer (designated as “second malignant neoplasm” [SMN]), infertility, and cardiotoxicity.

RESULTS:

Data were available on 883 patients with osteosarcoma and 543 patients with Ewing sarcoma. In the osteosarcoma group, there were 39 SMNs (4.4%) in 36 patients; in the Ewing sarcoma group, 15 patients (2.8%) experienced a single SMN each. The cumulative 10‐year and 20‐year incidence of an SMN (±standard error) was 4.9% ± 0.9% and 6.1% ± 1.2%, respectively, in the osteosarcoma group and 3.4% ± 0.9% and 4.7% ± 1.6%, respectively, in the Ewing sarcoma group. The most common SMN in the osteosarcoma group was breast cancer (n = 11), and the most common SMN in the Ewing sarcoma group was radiotherapy‐induced osteosarcoma (n = 6). After 20 years, the risk of developing an SMN increased, whereas the risk of a recurrence of the primary tumor decreased. Permanent sterility was more common in males than in females. Doxorubicin cardiotoxicity occurred in 18 patients with osteosarcoma (2%) and in 7 patients with Ewing sarcoma (1.3%).

CONCLUSIONS:

The awareness of late side effects in long‐term survivors of primary bone cancers should encourage longer follow‐up. Cancer 2012. © 2012 American Cancer Society.     

Ewing sarcoma demonstrates racial disparities in incidence‐related and sex‐related differences in outcome

BACKGROUND:

Previous reports of Ewing sarcoma cohorts suggested that there is a difference in incidence according to racial origin. However, to the authors' knowledge, this finding has never been tested in a population‐based database, and the impact of race on clinical outcome and the significance of known risk factors stratified to racial groups have not been reported.

METHODS:

Patients who had Ewing sarcoma diagnosed between 1973 and 2005 were identified in the Surveillance, Epidemiology, and End Results database. Patient demographic and clinical characteristics; incidence; year of diagnosis; tumor location, tumor size, and disease stage at diagnosis; treatment(s); cause of death; and survival were extracted. Kaplan‐Meier, log‐rank, and Cox regressions were used to analyze the significance of prognostic factors.

RESULTS:

Race‐specific incidence indicated that Caucasians have the highest incidence (0.155), followed by Asians/Pacific Islanders (0.082), and African Americans (0.017). The difference in incidence between Caucasians and African Americans was 9‐fold and significant (P < .001). The incidence of Ewing sarcoma increased over the past 3 decades among Caucasians (P < .05). Survival was not impacted by race. Local disease stage, primary tumor location in the appendicular skeleton, and tumor size ≤8 cm conferred a significant survival benefit. Women demonstrated improved survival among the Caucasian patients (P < .03).

CONCLUSIONS:

To the authors' knowledge, this is the first report focusing on racial disparity in incidence of Ewing sarcoma. Caucasians were affected more frequently, although outcomes were similar between races. It is noteworthy that being a woman constituted a survival benefit only among the Caucasian patients. Further studies will need to clarify the reasons for racial disparities in incidence and for sex differences in survival. Cancer 2009. © 2009 American Cancer Society.     

Systemic administration of reovirus (Reolysin) inhibits growth of human sarcoma xenografts

BACKGROUND:

Despite advancement in therapies, overall survival rates for relapsed pediatric sarcomas are dismal. Newer therapies are needed to effectively salvage these patients. Oncolytic viruses (such as reovirus) and other genetically altered viruses (such as herpes simplex viruses and adenoviruses) have shown efficacy in a variety of solid tumors including sarcomas. Reolysin is an unmodified oncolytic reovirus that selectively replicates in Ras‐activated cancer cells while not causing any significant human illness in its wild form.

METHODS:

By using a panel of pediatric sarcoma cell lines in vitro and flank xenografts in vivo, Reolysin was evaluated as a single agent and in combination with cisplatin and radiation therapy. Electron microscopy and immunohistochemistry was used to demonstrated a cytopathic effect in treated tumors.

RESULTS:

Reolysin inhibited the proliferation and viability of sarcoma cell lines at a dose of 1 to 10 virus particles per cell. In vivo, 5 × 10⁹ plaque‐forming units (PFU) administered via the tail vein every other day for 3 doses every 21 days inhibited the growth of tumor xenografts with improvement in event‐free survival. In the SKES1 Ewing sarcoma line, there was therapeutic enhancement when reovirus was administered in combination with radiation or cisplatin. In the RH30 line and the OS33 line, therapeutic enhancement was demonstrated with radiation and cisplatin, respectively.

CONCLUSIONS:

These results suggest that Reolysin alone or in combination with other cytotoxic agents may be effective therapy in pediatric sarcomas. Cancer 2011. © 2010 American Cancer Society.     

Malignant sarcoma of the pelvic bones: treatment outcomes and prognostic factors vary by histopathology

BACKGROUND:

Treatment of malignant sarcomas of the pelvis poses a challenge for local disease control and oncologic outcome. Many reports have described the dismal outcomes. Most studies are retrospective series coming out of single centers, thus biased toward patient selection and are of limited statistical power.

METHODS:

The authors used the Surveillance, Epidemiology, and End Results database to analyze 1185 pelvic sarcoma cases from 1987 to 2006. Kaplan‐Meier and Cox regression were used to analyze the significance of prognostic factors. The analysis was repeated for different histopathological subtypes to determine specific prognostic factors in each case.

RESULTS:

Incidence of pelvic sarcoma in 2006 was 89 per 100,000 persons; it has significantly increased since 1973 (P < .05). The overall 5‐year survival for all the patients with pelvic sarcoma was 47%, with osteosarcoma having the worst 5‐year survival at 19% and patients with chordoma having the best 5‐year survival at 60%. Independent prognostic factors included age, stage, grade, size of primary lesion, histopathology, and treatment‐related factors. Comparing the patients only with high‐grade lesions, patients with Ewing sarcoma have the best prognosis.

CONCLUSIONS:

This is an analysis of patients with pelvic sarcomas derived from a population‐based registry. Survival and prognostics vary with histopathological diagnoses. Although surgical resection was associated with superior outcomes for osteosarcoma and chondrosarcoma, there was no significant difference in outcomes of patients with Ewing sarcoma treated with surgery and/or radiotherapy. Cancer 2011. © 2010 American Cancer Society.     

Putative tumor suppressor miR‐145 inhibits colon cancer cell growth by targeting oncogene friend leukemia virus integration 1 gene

BACKGROUND:

Tumor suppressor microRNA miR‐145 is commonly down‐regulated in colon carcinoma tissues, but its specific role in tumors remains unknown.

METHODS:

In this study, the authors identified the Friend leukemia virus integration 1 gene (FLI1) as a novel target of miR‐145. FLI1 is involved in t(11;22)(q24:q12) reciprocal chromosomal translocation in Ewing sarcoma, and its expression appears to be associated with biologically more aggressive tumors.

RESULTS:

The authors demonstrated that miR‐145 targets a putative microRNA regulatory element in the 3′‐untranslated region (UTR) of FLI1, and its abundance is reversely associated with FLI1 expression in colon cancer tissues and cell lines. By using a luciferase/FLI1 3′‐UTR reporter system, they found that miR‐145 down‐regulated the reporter activity, and this down‐regulation was reversed by anti–miR‐145. Mutation of the miR‐145 microRNA regulatory element sequence in the FLI1 3′‐UTR abolished the activity of miR‐145. miR‐145 decreased FLI1 protein but not FLI1 mRNA, suggesting a mechanism of translational regulation. Furthermore, the authors demonstrated that miR‐145 inhibited cell proliferation and sensitized LS174T cells to 5‐fluorouracil–induced apoptosis.

CONCLUSIONS:

Taken together, these results suggest that miR‐145 functions as a tumor suppressor by down‐regulating oncogenic FLI1 in colon cancer. Cancer 2011. © 2010 American Cancer Society.     

Imatinib plus low‐dose doxorubicin in patients with advanced gastrointestinal stromal tumors refractory to high‐dose imatinib

BACKGROUND:

In KIT‐expressing Ewing sarcoma cell lines, the addition of doxorubicin to imatinib increases apoptosis, compared with imatinib or doxorubicin alone. On the basis of these in vitro data, the authors conducted a phase 1‐2 trial of doxorubicin with imatinib in patients with gastrointestinal sarcoma tumors refractory to high‐dose imatinib therapy.

METHODS:

Patients with metastatic gastrointestinal sarcoma tumor resistant to imatinib at 400 mg by mouth (p.o.) twice a day were eligible for this multicenter study, and received imatinib (400 mg p.o. every day [q.d.]) concomitantly with doxorubicin 15‐20 mg/m²/weekly for 4 cycles (monthly cycles), followed by imatinib (400 mg p.o. q.d.) maintenance in nonprogressive patients. Spiral computed tomography and positron emission tomography with F18‐fluorodeoxyglucose were done basally and after 2 months of therapy to evaluate response. An in vitro study assessed the effect of combining imatinib and doxorubicin.

RESULTS:

Twenty‐six patients with progressive gastrointestinal sarcoma tumor were entered in the study. Treatment was well tolerated. Three (14%) of 22 evaluable patients had partial responses per Response Evaluation Criteria in Solid Tumors, and 8 (36%) had clinical benefit (partial response or stable disease for ≥6 months). Median progression‐free survival (PFS) was 100 days (95% confidence interval [CI], 62‐138), and median survival was 390 days (95% CI, 264‐516). Interestingly, PFS was 211 days (95% CI, 52‐370) in patients with wild type (WT) KIT and 82 days (95% CI, 53‐111) in non‐WT patients (10 mutant, 6 not assessed). A synergistic effect on cell line proliferation and apoptosis was found with imatinib and doxorubicin combination.

CONCLUSIONS:

Low‐dose chemobiotherapy combination showed promising activity in heavily pretreated gastrointestinal sarcoma tumor patients, especially in those with WT‐KIT genotype. Cancer 2010. © 2010 American Cancer Society.     

Pediatric sarcoma in Central America

BACKGROUND:

Children with cancer in middle‐income countries have inferior outcomes compared with similar children in high‐income countries. The magnitude and drivers of this survival gap are not well understood. In the current report, the authors sought to describe patterns of clinical presentation, magnitude of treatment abandonment, and survival in children with sarcoma in Central America.

METHODS:

A retrospective review was conducted of hospital‐based registries from national pediatric oncology referral centers. Patients with newly diagnosed osteosarcoma, Ewing sarcoma, rhabdomyosarcoma (RMS), and soft tissue sarcoma (STS) between January 1, 2000 and December 31, 2009 were included. Survival analyses were performed first using standard definitions of overall survival (OS) and event‐free survival (EFS) and then with abandonment included as an event (abandonment‐sensitive OS and abandonment‐sensitive EFS).

RESULTS:

In total, 785 new cases of pediatric sarcoma were reported (264 diagnoses of osteosarcoma, 175 diagnoses of Ewing sarcoma, 240 diagnoses of RMS, and 106 diagnoses of STS). The rate of metastatic disease at presentation was high (osteosarcoma, 38%; Ewing sarcoma, 39%; RMS, 29%; and STS, 21%). The treatment abandonment rate also was high, particularly among patients with extremity bone sarcomas (osteosarcoma, 30%; Ewing sarcoma, 15%; RMS, 25%; and STS, 15%). Of 559 patients who experienced a first event, 59% had either recurrent or progressive disease. The 4‐year OS rate (±standard error) was 40% ± 3%, and the EFS rate was 30% ± 2%; however, these rates decreased further to 31% ± 2% and 24% ± 2%, respectively, when abandonment was taken into account.

CONCLUSIONS:

The current results indicated that high rates of metastases and treatment abandonment and difficulty with upfront treatment effectiveness are important contributors to the poor survival of children with pediatric sarcomas in Central America. Initiatives for early diagnosis, psychosocial support, quality improvement, and multidisciplinary care are warranted to improve outcomes. Cancer 2013. © 2012 American Cancer Society.     

Identification of borderline thyroid tumors by gene expression array analysis

BACKGROUND:

A subset of follicular lesions of the thyroid is encapsulated similar to follicular adenomas but with partial nuclear features suggestive of papillary thyroid carcinoma (PTC), raising the possibility of biologically borderline tumors.

METHODS:

Gene expression profiling and advanced significance analyses were performed on 50 histologically unequivocal benign and malignant tumors, and a list of 61 differentially expressed genes was generated. By using this 61‐gene list, unsupervised hierarchical and K‐means cluster analyses were performed on 40 additional tumors, including 15 histologically borderline tumors, 11 benign tumors, and 14 PTCs.

RESULTS:

Analysis revealed 3 distinct tumor groups—benign, malignant, and intermediate. Tumors in the intermediate group (n = 15) were mostly histologic borderline tumors and had an expression profile overlapping with the benign and malignant groups. Twenty‐seven genes were expressed differentially between the benign and intermediate groups, including the cyclic AMP response element‐binding protein/p300‐interactivator with glutamic acid/aspartic acid‐rich carboxy‐terminal domain 1 or CITED1 gene and the fibroblast growth factor receptor 2 or FGFR2 gene. Fourteen genes were expressed differentially between the intermediate group and malignant tumors, notably overexpression of the met proto‐oncogene and of the high‐mobility group adenine/thymine‐hook 2 or HMGA2 gene in malignancies. Mutations of the v‐raf murine sarcoma viral oncogene homolog B1 or BRAF gene were identified in 4 of 14 malignant tumors but not in benign or intermediate tumors. Patients who had either histologically or molecularly borderline tumors did not have metastasis or recurrences.

CONCLUSIONS:

Gene expression profiling supported the finding that encapsulated thyroid follicular lesions with partial nuclear features of PTC are biologically borderline tumors that are distinct molecularly from benign and malignant tumors. Cancer 2009. © 2009 American Cancer Society.     

Hyperthermia‐treated mesenchymal stem cells exert antitumor effects on human carcinoma cell line

BACKGROUND:

Mesenchymal stem cells (MSCs) possess the potential for differentiation into multilineages. MSCs have been reported to play a role as precursors for tumor stroma in providing a favorable environment for tumor progression. Hyperthermia destroys cancer cells by raising the temperature of tumor‐loaded tissue to 40°C to 43°C and causes indirect sensitizing effects when combined with chemo‐ and/or radiotherapy. However, how hyperthermia affects the tumor‐supportive stroma is unknown. Here, the authors investigated the effects of hyperthermia‐treated MSCs, from different sources, on the human ovarian cancer cell line SK‐OV‐3.

METHODS:

MSCs from adipose tissue and amniotic fluid were untreated or heat‐treated (HS‐MSCs). The culture supernatant of each treatment group was collected and transferred to the SK‐OV‐3 cells.

RESULTS:

The morphological analysis and cell proliferation assay showed a reduced viability of the tumor cells in the conditioned medium with the HS‐MSCs. Further investigations revealed that the conditioned medium of the HS‐MSCs induced a higher nuclear condensation and a greater number of sub‐G1 cells among the tumor cells. Analysis of the mRNA expression demonstrated that the conditioned medium of the HS‐MSCs induced up‐regulation or down‐regulation of several tumor‐associated molecules. Finally, the cytokine array of each conditioned medium showed that angiogenin, insulin‐like growth factor binding protein 4, neurotrophin 3, and chemokine (C‐C motif) ligand 18 are involved as main factors.

CONCLUSIONS:

This study showed that the conditioned medium of the HS‐MSCs exerted a suppressive effect on tumor progression and malignancy, suggesting that hyperthermia enables tumor stromal cells to provide a sensitizing environment for tumor cells to undergo cell death. Cancer 2009. © 2009 American Cancer Society.     

Optimal amount of monocyte chemoattractant protein‐1 enhances antitumor effects of suicide gene therapy against hepatocellular carcinoma by M1 macrophage activation

Suicide gene therapy combined with chemokines provides significant antitumor efficacy. Coexpression of suicide gene and monocyte chemoattractant protein‐1 (MCP‐1) increases antitumor effects in murine models of hepatocellular carcinoma (HCC) and colon cancer. However, it is unclear whether the doses administered achieved the maximum antitumor effects. We evaluated antitumor effects of various amounts of recombinant adenovirus vector (rAd) expressing MCP‐1 in the presence of a suicide gene in a murine model of HCC. HCC cells were transplanted subcutaneously into BALB/c nude mice, and transduced with a fixed amount of Ad‐tk harboring the suicide gene, HSV‐tk, and various doses of Ad‐MCP1 harboring MCP‐1 (ratios of 1:1, 0.1:1, and 0.01:1 relative to Ad‐tk). Growth of primary tumors was suppressed when treated with Ad‐tk plus Ad‐MCP1 (1:1 and 1:0.1) as compared with Ad‐tk alone. The antitumor effects against tumor rechallenge tended to be high in the Ad‐tk plus Ad‐MCP1 group (1:0.1). The effects were dependent on production of Th1 type‐cytokines. Delivery of an optimal amount of rAd expressing MCP‐1 enhanced the antitumor effects of suicide gene therapy against HCC by M1 macrophage activation, suggesting that this is a plausible form of cancer gene therapy to prevent HCC progression and recurrence. (Cancer Sci 2008; 99: 2075–2082)     

The CXCR4-CXCL12 axis in Ewing sarcoma: promotion of tumor growth rather than metastatic disease

Background

Chemokine receptor CXCR4, together with its ligand CXCL12, plays critical roles in cancer progression, including growth, metastasis and angiogenesis. Ewing sarcoma is a sarcoma with poor prognosis despite current therapies, particularly for patients with advanced-stage disease. Lungs and bone (marrow), organs of predilection for (primary/metastatic) Ewing sarcoma, represent predominant CXCL12 sources.

Methods

To gain insight into the role of the CXCR4-CXCL12 axis in Ewing sarcoma, CXCR4, CXCL12 and hypoxia-inducible factor-1α protein expression was studied in therapy-naïve and metastatic tumors by immunohistochemistry. CXCR4 function was assessed in vitro, by flow cytometry and proliferation/ cell viability assays, in the presence of recombinant CXCL12 and/or CXCR4-antagonist AMD3100 or under hypoxic conditions.

Results

Whereas CXCR4 was predominantly expressed by tumor cells, CXCL12 was observed in both tumor and stromal areas. Survival analysis revealed an (expression level-dependent) negative impact of CXCR4 expression (p?<?0.04). A role for the CXCR4-CXCL12 axis in Ewing sarcoma growth was suggested by our observations that i) CXCR4 expression correlated positively with tumor volume at diagnosis (p?=?0.013), ii) CXCL12 was present within the microenvironment of virtually all cases, iii) CXCL12 induced proliferation of CXCR4-positive Ewing sarcoma cell lines, which could be abrogated by AMD3100. CXCR4 expression was not correlated with occurrence of metastatic disease. Also, therapy-naïve tumors demonstrated higher CXCR4 expression as compared to metastases (p?=?0.027). Evaluation of in vivo hypoxia-inducible factor-1α expression and culture of cells under hypoxic conditions revealed no role for hypoxia in CXCR4 expression.

Conclusions

Together, our results imply a crucial role for the CXCR4-CXCL12 axis in auto- and/or paracrine growth stimulation. Integration of CXCR4-targeting strategies into first- and/or second-line treatment regimens may represent a promising treatment option for Ewing sarcoma.

     

Promoter methylation of the Wnt/β‐catenin signaling antagonist Dkk‐3 is associated with poor survival in gastric cancer

BACKGROUND:

Abnormal activation of the Wnt/β‐catenin signaling pathway is common and critical in the pathogenesis of digestive cancers. In this study, the authors investigated the promoter methylation of the dickkopf homolog 3 gene Dkk‐3 in these cancers and its prognostic significance in gastric cancer.

METHODS:

Dkk‐3 methylation was assessed in 173 patients with gastric cancers (including 104 patients who were followed for up to 4090 days) and in 128 patients with colorectal cancer. Cell growth was evaluated by using a colony‐formation assay. For survival analyses, the authors used Kaplan‐Meier plots, the log‐rank test, and Cox proportional regression.

RESULTS:

Dkk‐3 was silenced or down‐regulated in 12 of 17 gastric cancer cell lines (70.6%) and in 3 of 9 colon cancer cell lines (33.3%). The loss of gene expression was associated with promoter methylation, which could be restored by demethylating agents. Ectopic expression of Dkk‐3 suppressed colony formation. Moreover, methylation of Dkk‐3 was detected in 117 of 173 primary gastric tumors (67.6%) and in 67 of 128 colorectal tumors (52.3%). The clinical significance and the prognostic value of Dkk‐3 methylation also were examined in 104 gastric cancers and in 84 colorectal cancers. Multivariate analysis indicated that Dkk‐3 methylation was associated significantly and independently with poor disease survival (relative risk, 2.534; 95% confidence interval, 1.54–4.17; P = .002) in gastric cancer, but not in colorectal cancer. Kaplan‐Meier survival curves revealed that patients who had Dkk‐3 methylated gastric cancers had a significantly shorter survival (median, 0.76 years) compared with patients who did not have Dkk‐3 methylation (median, 2.68 years; P < .0001; log‐rank test).

CONCLUSIONS:

Epigenetic silencing of the Dkk‐3 gene by promoter methylation was a common event in gastric cancer and was associated with a poor outcome in such patients. Cancer 2009. © 2008 American Cancer Society.     

Combined mobilization and stimulation of tumor‐infiltrating dendritic cells and natural killer cells with Flt3 ligand and IL‐18 in vivo induces systemic antitumor immunity

It was hypothesized that if dendritic cells (DC) could be efficiently manipulated in vivo, this might enable functional maturation and retention of their potent functions and might represent a more promising approach in DC immunotherapy. The present study focused on the modulation of DC in tumor microenvironment using Fms‐like thyrosine kinase 3 ligand (Flt3L) combined with interferon‐γ‐inducing factor (IL‐18). Tumor‐inoculated mice were treated with in vivo electroporation (IVE) of expression plasmids carrying complementary DNA of Flt3L. As a combination therapy, mice in the other group were treated with intra‐tumoral injection of adenoviral vector carrying IL‐18 gene (Ad.IL‐18). Significant antitumor effect was observed in mice treated with Ad.IL‐18 alone when compared with that of control (P < 0.01). Complete eradication was observed more frequently (100%versus 33%: P < 0.05) in the mice treated with Flt3L and Ad.IL‐18 when compared with the mice treated with Ad.IL‐18 alone. In un‐injected distant tumor, significant antitumor responses were observed only in the mice treated with combination therapy. Lymphoid cells in lymph nodes of mice treated with combination therapy showed significant cytolytic activity against inoculated tumor cells and YAC‐1 cells when compared with the lymphoid cells in other groups. In the tumor microenvironment, combination therapy resulted in the recruitment of mobilized DC into the tumor bed, although Flt3L–IVE alone had an effect in the peri‐tumoral area. Tumor‐infiltrating DC in mice treated with combination therapy showed higher CD86 expression and more potent allogeneic T‐cell stimulatory capacity. These results may suggest that local expression of IL‐18 combined with in vivo DC mobilization with Flt3L is clinically applicable as a new strategy of DC immunotherapy. (Cancer Sci 2008; 99: 2028–2036)