Recent studies have revealed that microsatellite instability due to deficiency of DNA mismatch repair systems could be correlated to both hereditary and sporadic cancer, and involve a mechanism of acquired resistance to anti neoplastic agents. We report the current understanding of the correlation between DNA mismatch repair functions and resistance to platinum agents in epithelial ovarian cancer based on our previous report using chromosome transfer, microsatellite assay for surgically resected specimens, and a review of the English literature.
The TMA is an intensive and efficient method, and the technique should be used by all means in the experiment of the analytical cytometry. The good points of TMA are to save time and cost greatly for cancer research. A sample tissue for TMA is cut from a common paraffin-embedded tissue block, it is inserted in a new paraffin block (recipient-paraffin-block). Finally, a recipient-paraffin-block for TMA including many (may be more than 100) tissue samples is completed. It is thinly cut, and can be stained using antigen-antibody reaction or hybridization technique. TMA samples suit virtual microscopy system well because it has high density tissue information. To make better use of TMA, this brief review introduces outline of TMA system based on published reports and informs the limits, problems, and notes.
We have recently found that intra-bone marrow - bone marrow transplantation (IBM-BMT) can be used to prevent graft-versus-host disease (GvHD), even when intensive donor lymphocyte infusion (DLI) is carried out. In the present study, in conjunction with IBM-BMT, allogeneic splenic T cells as DLI were also injected into the bone marrow cavity of lethally irradiated (8.5Gy) recipients. The extent of GvHD was compared with that of recipients that had received allogeneic IBM-BMT plus intravenous injection of allogeneic T cells (IV-DLI). GvHD in recipients treated with allogeneic IBM-BMT plus IBM-DLI was far milder than in those treated with allogeneic IBM-BMT plus IV-DLI. This was confirmed macroscopically and histopathologically. The frequency of regulatory T cells (Tregs) detected as CD4+ CD25+ or CD4+ Foxp3+ cells was significantly higher in recipients treated with IBM-BMT plus IBM-DLI than in those treated with IBM-BMT plus IV-DLI. Donor-derived helper T (Th) cells polarized to Th2 type in recipients treated with IBM-BMT plus IBM-DLI, while Th1 cells were dominant in recipients treated with IBM-BMT plus IV-DLI. Furthermore, the production of TGF-ß and HGF from bone marrow stromal cells (BMSCs) was enhanced after IBM-DLI. Thus, IBM-BMT plus IBM-DLI seem to preferentially induce Tregs and Th2, and also induce the production of TGF-ß and HGF from BMSCs, thereby resulting in the prevention of GvHD. These findings indicate that IBM-BMT is an excellent way of BMT not only for facilitating donor engraftment but also for preventing GvHD even when T cells are contained in BMCs or intensive donor lymphocyte infusion is carried out.
Using comparative genomic hybridization (CGH)-based lineage analyses, we have recently demonstrated that there are two genetic lineages in poorly differentiated adenocarcinoma (POR) with tubular component (TC) in the stomach: one derived form signet ring cell carcinoma and the other from tubular adenocarcinoma (TUB). It still remains open whether the POR with TC that derives from TUB is genetically continuous to the TUB that is detected as intramucosal carcinoma. We have applied CGH and TP53 mutation and LOH analyses to this problem. The CGH profile indicated genetic continuity between POR component and TC and discontinuity between the TC and intramucosal TUB. PORs with TC were characterized by high incidence of wild-type p53 inactivation by mutation and LOH of TP53 or genomic gain of MDM2 that may downregulate p53 not only in POR component but also in TC. The pattern of TP53 mutation was common between the POR components and TC. These findings suggest that wild-type p53 inactivation precedes dedifferentiation of TC to POR. The precursors of POR with TC may thus include early TUBs with this inactivation.
Histone H2AX phosphorylated on Ser 139, defined as γH2AX, is a reporter of DNA double strand breaks (DSBs). While induction of DNA damage by genotoxic agents lead H2AX to γH2AX, it also is phosphorylated in unterated normal and tumor cells. The latter is designated as constitutive H2AX phosphorylation. We show here flow cytometric investigation of γH2AX in relation to cell cycle phase. Even in untreated exponentially growing HL-60 cells, γH2AX was detected during the cell cycle. Some of normal endothelial cells demonstrated dot-like positivity of γH2AX in their nuclei. Oxide-releasing aspirin induced DNA damage and it was attenuated in the presence of N-acetyl-L-cysteine, a scavenger of reactive oxigen species. A decrease in a cells’metabolic activity as a result of inhibition of glycolysis by 2-deoxy-D-glucose reduced DNA damage. Exposure of HL-60 cells to camptothecin led to phosphorylation of H2AX in S-phase cells. Four hours’exposure to camptothecin induced a cell population which had dramatically increased γH2AX immunofluorescence and corresponded to apoptosis. Inhibitors of DNA replication which are used to synchronize cultured cells in G1 phase caused DNA damage in S-phase cells and led to their apoptosis, i. e., the synchronization was caused by selective kill of S-phase cells through induction of DSBs. Moreover, synchronized cells in G1 phase showed DNA damage. Thus, detection of γH2AX immunohistochemically and cytometric investigation of it in relation to cell cycle phases is advantageous to estimate the effect of many agents causing DNA damage such as oxidative stress, metabolism or chemicals, and have a possibility to contribute significantly to elucidate carcinogenesis.
Donor-derived T lymphocyte that responds to tumor antigens emerges after allogeneic hematopoietic stem cell transplantation (allo-HSCT), particularly in association with the status of immune recovery. To analyze the frequency of specific T lymphocyte against tumor antigens, such as PR1, PRAME and WT1, after alloHSCT, a tetramer-based analysis was performed in 97 samples taken from 35 patients (9 AML, 11 MDS, 2 CML, 4 ALL, 7 lymphoma and 2 renal cell carcinoma [RCC]) with the HLA-A02 phenotype. Regarding WT1, positive results were detected in 39 of 97 samples and 7 (2 CML, 1 ALL, 2 lymphoma and 2 RCC) patients, which frequency was higher than PR1 and PRAME. On the basis of those results, we performed serial analyses of WT1 specific lymphocyte during the clinical course in 2 patients with RCC who underwent alloHSCT, to examine the precise correlation between the kinetics of WT1 specific T lymphocyte, the occurrence of GVHD and the observed clinical response. A higher positive rate for WT1 specific T lymphocyte and a correlation with GVHD and the clinical response was detected. Those results might suggest the contribution of WT1 specific T lymphocyte on the antitumor effect after alloHSCT.
Although umbilical cord blood has been used successfully as an alternative donor source to treat hematological disorders, cord blood transplantation (CBT) is frequently complicated by graft failure and relapse of original diseases. Since recipient-derived hematopoietic cells persist or increase under these pathogenic conditions, analysis of donor-recipient mixed chimerism might be very useful to make early diagnosis of them. Since most CBT involves human leukocyte antigen (HLA)-mismatched transplantation, a flow cytometry-based method of chimerism analysis using anti-HLA antibodies to detect mismatched antigens has been developed in some research groups. We reviewed the features of anti-HLA antibodies used in this new developed technique.
There have been many reports of Rapid immunostaining Technique with Microwave（MW）treatment, which promote antigen-antibody reaction. There remains a problem of temperature rise and evaporation of antibody solution during MW irradiation. Here we propose a method of MW treatment using a slide seal, with which reaction solution is enclosed on a glass slide. The sealed glass slide was irradiated with MW in a chamber that is filled with water. This method was applied to immunoperoxidase staining of cytokeratin in colon cancers. A satisfactory result was obtained after MW treatment for 3 minutes with anti-cytokeratin antidody and subseguent MW treatment with Simplestain solution for 3 minutes. This method is applicable to intraoperative diagnosis.