Detection of Serum Free DNA Hypermethylation in Surgically Resected Adenocarcinoma of the Lung

PURPOSE : Aberrant DNA methylation patterns have been commonly associated with human cancers. We have investigated the frequency of DNA hypermethylation in promoter regions from adenocarcinomas of the lung and then attempted to detect the same epigenetic changes from patient serum samples. MATERIALS AND METHODS : We collected tissues from 72 cases of lung adenocarcinomas. The cancer and normal lung tissues were tested for DNA hypermethylation using methylation-specific PCR (MSP). The genes investigated were DAPK, RARbetaP2 and p16. We selected 12 patients where promoter hypermethylation was present for all three genes and four patients where hypermethylation was not seen for any of the three genes. Serum-free DNA was extracted and was tested for promoter hypermethylation. The status of serum-free DNA methylation was analyzed; the hypermethylation status was compared to clinical variables and cancer outcomes. RESULTS : DNA hypermethylation was observed in 32% of samples for DAPK, 63% of samples for RARbetaP2 and 83% of samples for p16 from the cancer tissues. Among the 12 matched serum samples where the primary tumor showed hypermethylation in all three gene promoter regions, we were able to detect five incidences of serum DNA hypermethylation in four patients. The four patients had TNM stage II or higher disease. None of the patients with stage I disease showed serum-free DNA hypermethylation. CONCLUSION : Aberrant promoter hypermethylation was frequently observed in surgically resected adenocarcinoma of the lung. Concurrent serum-free DNA hypermethylation was detected in 34% of patients where the primary tumor showed hypermethylation in all three gene promoter regions. The findings suggest that the serum-free DNA methylation status might be used as a potential target for the diagnosis of lung cancer. However, the low sensitivity should be improved for use in a clinical application

Effect of Antibody Titer on Xenograft Survival in Pig-To-Dog Heterotopic Cardiac Xenotransplantation: Opening of Xenotransplantation Era / 대한흉부외과학회지

BACKGROUND: Xenotransplantation in discordant species results in immediate and irreversible hyperacute rejection due to natural antibodies, IgM. With this, antibody depletion is one option to reduce hyperacute rejection, we investigated the effect of PCPP (postcentrifugal plasmapheresis) on the depletion of natural antibodies and the effect of antibody titer on xenograft survival. MATERIAL ANDMETHOD: Outbred swines (n=4) weighing 10~20 kg were used as donors and mongrel dogs (n=4) weighing 25~30 kg were used as recipients. Recipient canines underwent plasmapheresis (COBE TPE Laboratories, Lakewood. CO, USA). Pre-transplantation PCPP was performed on day - 2 and day 0. There were three groups (Group 0: no PCPP, Group 1: 1 pla sma-volume (PV) at day 2 and 2 PV at day 0, Group 2: 2 PV at day - 2 and 2 PV at day 0). A swine heart was heterotopically transplanted into a recipient's abdominal infrarenal aorta and inferior vena cava. Mean percent depletion of total IgM and IgG in plasma of the recipients was calculated. Serum albumin, elecctrolyte, complement activity and coagualtion factors were measured. Histopathologic examination of heart specimens was performed. RESULT: Mean percent depletion of IgM and IgG were 95.7+/-1.2%, 80.5+/-2.4% in the group 2 at the end of PCPP. The percent depletion of serum albumin concentration was decreased from 2.8 to 1.4 g/dL in the group 1 and 3.0 to 1.5 g/dL in the group 2. Complement hemolytic activity was decreased in group 1 and 2, but returned to normal level within 24 hours. Complement hemolytic activity was reduced to 10% of pre-PCPP level in group 2. Serum fibrinogen decreased to 20% or less and was recovered within 24 hours in group 2. Antithrombin III decreased but less than fibrinogen. PT and aPTT were sometimes but not always prolonged during plasmapheresis. After plasmapheresis, PT and aPTT were prolonged beyond the measurable level. D-dimer was not found during PCPP, but appeared and maintained from 10 minutes after transplantation. Graft survival time was 5 min in group 0, and it was 90+/-0 min in the group 2. Histopathologic changes were more typically characterized by edema, hemorrhages, thrombosis in all groups at the end of experiment. CONCLUSION: PCPP effectively removed immuoglobulins and reduced the titer of natural antibodies, as a result, significantly prololonged swine heart xenograft survival.

A Xenoreactive Complement Hemolytic Assay in Xenotransplantation / 대한진단검사의학회지

BACKGROUND: Xenotrasplantation is a possible alternative for organ shortage in clinical transplantation, but hyperacute xenograft rejection has been a big huddle. Pre-existing natural xenoreactive antibodies and consequent activation of the complement system are thought to play major roles in hyperacute rejection. To set a monitorig test for the hyperacute rejection in xenotransplantation, we optimised a complement hemolytic assay and evaluated its in-vitro precisions and clinical implications. METHODS: Complement hemolytic activities of normal human sera on rabbit or porcine red blood cells (RBCs) in each gelatin veronal buffer with or without dextrose were compared to retrieve optimal conditions for assay. The precision and activity range of normal human sera were evaluated at a given optimum condition. And we also assayed complement hemolytic activities of the sera obtained from various models of xenotransplantated animal, and assessed its association with other clinical parameters. RESULTS: The assay with rabbit RBCs in gelatin veronal buffer containing dextrose showed linear hemolytic reactions in the broadest range of serum dilutions with the least background hemolysis. Its intra- and inter-assay coefficient variation was 1.3% and 8.1%, respectively. The complement hemolytic activity was dependent on the serum levels of C3 and IgM. Severe hyperacute rejection in lung xenotransplantation was accompanied with a rapid decline of serum complement hemolytic activities compared to the basal level. CONCLUSIONS: The complement hemolytic assay using rabbit red cells has a clinically acceptable range of precision, and seems to be useful for the evaluation of hyperacute rejection in clinical xenotransplantation.