Differences in the frequencies of K-ras c12-13 genotypes by gender and pathologic phenotypes in colorectal tumors measured using the allele discrimination method.

The frequencies of different genotypes of the K-ras oncogene in colorectal cancer (CRC) reveal complex relationships among gender, age, and tumor aggression, however, differences among these studies could also be attributed to a lack of standardization of the detection methods used. We developed the allele discrimination assay, which uses dual-color real-time polymerase chain reaction (qPCR) as a fast K-ras genotyping method, and demonstrated higher sensitivity and specificity than DNA sequencing with formalin-fixed paraffin tissues. The assay detected K-ras mutations among 83 of 204 patients with CRC (40.7%); 20.6% of these mutations were G12D (GAT) mutations, 7.4% were G13D (GAC) and G12V (GTT), and 5.3% were other types. A higher proportion of females was observed overall in tumors with K-ras mutations (60.2%, P = 0.01), codon 12 mutations (63.2%, P = 0.005), and transversions (69.6%, P = 0.02), which reflected the higher prevalence of females among the well- to moderately differentiated tumors (29% in males vs. 53% in females; interaction P = 0.03). The opposite was observed for poorly differentiated tumors (47% in males vs. 35% in females). No significant influence of age was found on the prevalence of K-ras mutation. Males with pathological changes and females with poorly differentiated tumors displayed GAT as a less common genotype compared with most other prevalence studies. In conclusion, allele discrimination, with no additional amplification step, is a fast and reliable genotyping method for detecting K-ras c12-13 mutations. Using this method, we demonstrate differences in the frequencies of K-ras genotypes by gender and pathologic phenotypes of CRC.

Lipopolysaccharide preferentially induces 4-1BB ligand expression on human monocyte-derived dendritic cells.

Dendritic cells (DCs) represent a promising tool for immunotherapy. A key feature in their action is to provide co-stimulatory signals for full activation of T cells. In view of recent studies demonstrating the critical role of 4-1BB co-stimulation in T cell response, it is of importance to optimize 4-1BB ligand (4-1BBL) expression on human monocyte-derived DCs (MDDCs), the DC source of many clinical studies. In this study, two types of MDDCs, generated in granulocyte-macrophage colony-stimulating factor and interleukin-4 (GM-CSF/IL-4-DCs) or in interferon-beta and IL-3 (IFN-beta/IL-3-DCs), were analyzed for 4-1BBL expression in response to several known DC activators. Immature MDDCs expressed 4-1BBLs at very low levels. Lipopolysaccharide (LPS) was the only activator that preferentially triggered 4-1BBL expression on either MDDCs, but 4-1BBL-positive cells were significantly more frequently observed on LPS-activated GM-CSF/IL-4-DCs (30.2+/-2.6% versus 14.3+/-1.2%). Combinations of multiple activating signals did not bring about enhanced 4-1BBL stimulatory capacity. In addition, plasmid DNA transfection and necrotic cell pulsing of GM-CSF/IL-4-DCs for antigen loading also resulted in 4-1BBL up-regulation. However, in all circumstances, the induced 4-1BBL levels were low in comparison with CD80 co-stimulatory molecule. Finally, by demonstrating LPS-matured GM-CSF/IL-4-DCs from sorted 4-1BBL(high) population augmented T cell expansion and survival, we propose that efforts are required to increase 4-1BBL levels on MDDCs achieved by current activation schemes.

Activated human CD4+ T cells induced by dendritic cell stimulation are most sensitive to transforming growth factor-beta: implications for dendritic cell immunization against cancer.

The secretion of immunosuppressive factors like transforming growth factor-beta (TGF-beta) by tumor cells has been recognized as one of the mechanisms involved in tumor immunological escape. This study aimed to examine whether dendritic cell (DC) immunization could reverse TGF-beta-induced immunosuppression by simulating the in vivo interaction among infused DCs, host T cells, and tumor-secreted TGF-beta in an in vitro study. We found that both immature and mature DCs were relatively resistant to TGF-beta. The addition of TGF-beta to naive human CD4+ T cells, which are required by genetically modified DC to elicit antitumor immunity, resulted in their hyporesponsiveness to DC stimulation in a dose-dependent manner. When activated by allogeneic DCs in the presence of TGF-beta, CD4+ T cells displayed a reduced capacity to proliferate. More importantly, activated CD4+ T cells induced by DC stimulation were very sensitive to TGF-beta, and this susceptibility was enhanced by their previous exposure to TGF-beta. The underlying mechanism was linked to TGF-beta-induced apoptosis of activated T cells. However, the presence of stimulation from DC or antibodies to CD3 plus CD28 could partly reverse the immunosuppressive effect of TGF-beta on activated CD4+ T cells. Taken together, our results indicate that the efficacy of DC immunization may be impaired by tumor-derived TGF-beta.