We attempted to develop a highly sensitive and specific method for the detection of circulating tumor DNA (ctDNA) using a digital PCR (dPCR) assay for PIK3CA mutations (i.e., H1047R, E545K, and E542K) in primary breast cancer patients. The sensitivity of the dPCR assay for the mutant alleles was examined using cell lines with PIK3CA mutations and proved to be 0.01 %. Serum samples were collected pre-operatively from 313 stage I-III breast cancer patients, of whom 110 were found to have PIK3CA mutant tumors. The serum samples from these patients with PIK3CA mutant tumors were subjected to the dPCR assay, and 25 (22.7 %) were found to be positive. No PIK3CA mutant ctDNA was detected in the serum samples of 50 healthy women and 30 breast cancer patients with PIK3CA non-mutant tumors. The patients with PIK3CA mutant ctDNA were dichotomized into mutant ctDNA-high (ctDNA(high)) and ctDNA-low (ctDNA(low)) groups based on the median. The ctDNA(high) patients exhibited significantly shorter recurrence-free survival (RFS; P = 0.0002) and overall survival rates (OS; P = 0.0048) compared to those exhibited by the combined ctDNA(low) patient and ctDNA-free patient group. Multivariate analysis revealed that ctDNA(high) status significantly predicted poor RFS and OS and did so independently of conventional histological parameters. These results suggest that dPCR is a highly sensitive and specific method for the detection of PIK3CA mutant ctDNA and that ctDNA(high) but not ctDNA(low) status is a significant and independent prognostic factor for primary breast cancer patients.

Background/Aims Hepatocellular carcinoma (HCC) has a poor survival rate due to recurrent intrahepatic metastases and lack of effective adjuvant therapy. Aspartate-β-hydroxylase (ASPH) is an attractive cellular target since it is a highly conserved transmembrane protein overexpressed on both murine and human HCC tumors, and promotes a malignant phenotype as characterized by enhanced tumor cell migration and invasion. Methods Dendritic cells (DCs), expanded and isolated from the spleen, were incubated with a cytokine cocktail to optimize IL-12 secretion and co-stimulatory molecule expression, then subsequently loaded with ASPH protein for immunization. Mice were injected with syngeneic BNL HCC tumor cells followed by subcutaneous inoculation with 5–10×105 ASPH loaded DCs using a prophylactic and therapeutic experimental approach. Tumor infiltrating lymphocytes (TILs) were characterized, and their role in producing anti-tumor effects determined. The immunogenicity of ASPH protein with respect to activating antigen specific CD4+ T cells derived from human peripheral blood mononuclear cells (PBMCs) was also explored. Methods We found that immunotherapy with ASPH-loaded DCs suppressed and delayed established HCC and tumor growth when administered prophylactically. Ex-vivo re-stimulation experiments and in vivo depletion studies demonstrate that both CD4+ and CD8+ cells contributed to anti-tumor effects. Using PBMCs derived from healthy volunteers and HCC patients, we showed that ASPH stimulation led to significant development of antigen-specific CD4+ T-cells. Conclusion Immunization with ASPH-loaded DCs has substantial anti-tumor effects which could reduce the risk of HCC recurrence.

Sox17 is a Sry-related HMG-box transcription factor developmentally expressed in both the definitive endoderm and extraembryonic endoderm (ExE). Although Sox17–/– mouse embryos have a defective definitive gut endoderm, their developing ExE is morphologically intact. Here, we aimed to investigate the role of Sox17 in ExE development by using an in vitro differentiation system of embryonic stem cells (ESCs). Although forced Sox17 expression in ESCs did not affect ExE commitment, it facilitated the differentiation of ESC-derived primitive endoderm cells into visceral and parietal endoderm cells. This event was inhibited by the forced expression of Nanog, a negative regulator of differentiation of ESCs into the ExE. Although Sox17–/– ESCs could differentiate into primitive endoderm cells, further differentiation was severely impaired. These results indicate a substantial involvement of Sox17 in the late stage of ExE differentiation in vitro. Furthermore, the expression of Sox7 – another Sox factor, concomitantly expressed with Sox17 in the developing ExE – was suppressed during the in vitro differentiation of Sox17–/– ESCs, but it was maintained at a high level in the extraembryonic tissues of Sox17–/– embryos. These findings possibly explain the discrepancy between the ExE phenotype derived from Sox17–/– ESCs and that of Sox17–/– embryos.

Systematic screening of differentially expressed genes among androgenetic, parthenogenetic, and normal embryos by means of fluorescent differential display revealed five imprinted genes. One of them, named Rian, was expressed exclusively from the maternal allele and was closely linked to an imprinted gene, Meg3(Gtl2), mapped to the distal end of chromosome 12. The Rian transcript did not have any apparent open reading frame, and its transcript was exclusively localized to the nucleus.