Initial Experience of Intra-Arterial Chemotherapy Using a Novel External Carotid Arterial Sheath System Combined with Radiotherapy and Systemic Chemotherapy for Locally Advanced Tongue Cancer.

We retrospectively evaluated the safety and effectiveness of an external carotid arterial sheath (ECAS) for intra-arterial chemotherapy (IACT) for locally advanced tongue cancer. Thirty-one patients with the Union for International Cancer Control's 8th TNM stage III-IV tongue cancer underwent IACT using the ECAS combined with RT and systemic chemotherapy with either cisplatin and fluorouracil (FP) or docetaxel, cisplatin, and fluorouracil (TPF) between October 2015 and February 2021. The ECAS was inserted retrogradely via the superficial temporal artery, and the tip was placed in the external carotid artery between the maxillary and facial arteries. A microcatheter was inserted into each tumor-feeding artery through the ECAS under fluoroscopy, wherein cisplatin 50 mg/m2 was administered. IACT was performed weekly with neutralization using sodium thiosulfate. Complete response of the primary lesion was achieved in 28/31 (90%) patients. The median follow-up for all patients was 39 months. The 3-year overall survival, progression-free survival, and local control rates were 81.6%, 74.2%, and 83.4%, respectively. Grade 3 and greater toxicities included oral mucositis (45%), neutropenia (39%), nausea (13%), anemia (10%), thrombocytopenia (10%), dry mouth (10%), and fever (3%). There were no severe complications associated with IACT. In conclusion, the ECAS is feasible and effective for locally advanced tongue cancer.

Epigenetic analysis of bovine parthenogenetic embryonic fibroblasts.

Although more than 100 imprinted genes have already been identified in the mouse and human genomes, little is known about genomic imprinting in cattle. For a better understanding of these genes in cattle, parthenogenetically activated bovine blastocysts were transferred to recipient cows to obtain parthenotes, and fibroblasts derived from a Day 40 (Day 0 being the day of parthenogenetic activation) parthenogenetic embryo (BpEFs) were successfully obtained. Bovine embryonic fibroblasts (BEFs) were also isolated from a normal fertilized embryo obtained from an artificially inseminated cow. The expression of imprinted genes was analyzed by RT-PCR. Paternally expressed genes (PEGs) in mouse (viz., IGF2, PEG3, ZAC1, NDN, DLK1, SGCE, and PEG10) were expressed in BEFs, but not in BpEFs, suggesting that these genes are also imprinted in cattle. However, other PEGs in mouse (viz., IMPACT, MAGEL2, SNRPN, and PEG1/MEST) were expressed in both BEFs and BpEFs. These genes may not be imprinted in BEFs. The expression of seven maternally expressed genes in mouse was also analyzed, and only CDKN1C was not expressed in BpEFs. The DNA methylation patterns of repetitive elements (Satellite I, Satellite II, alpha-satellite, and Art2) were not different between the BEFs and BpEFs; however, the differentially methylated region (DMR) of paternally methylated H19 was hypomethylated, whereas those of maternally methylated PEG3 and PEG10 were hypermethylated in BpEFs, as expected. The methylation of the SNRPN DMR was not different between the BEFs and BpEFs, in accordance with the SNRPN expression levels in both cell types. The XIST gene, which is essential for X chromosome inactivation in females, was expressed in BpEFs, whereas its DMR was half-methylated, suggesting that X chromosome inactivation is normal in these cells. Microarray analysis was also applied to identify novel PEGs that should be expressed only in BEFs but not in BpEFs. More than 300 PEG candidate genes, including IGF2, PEG3, and PEG10, were obtained. These results illustrate the epigenetic characteristic of bovine parthenogenetic embryos and contribute to the identification of novel imprinted genes in cattle.

Classification of patients with non-alcoholic fatty liver disease using rapid immunoassay of serum type IV collagen compared with liver histology and other fibrosis markers.

AIM: Non-alcoholic fatty liver disease (NAFLD) can progress to non-alcoholic fatty liver (NAFL) or non-alcoholic steatohepatitis (NASH). We investigated the association among serum type IV collagen level, liver histology, and other fibrosis markers in NAFLD progression. METHODS: We evaluated 184 patients diagnosed with NAFLD following biopsy, including 89 males and 95 females with an average age of 52.6 and 62.6 years, respectively. Non-alcoholic fatty liver disease was classified as NAFL or NASH using Matteoni's classification, and the grade and stage of NASH were assessed using Brunt's classification. Serum type IV collagen was measured by a rapid and sensitive latex particle-enhanced turbidimetric immunoassay. RESULTS: Forty-two patients with NAFL and 142 patients with NASH were included in this study. Compared with patients with NAFL, patients with NASH showed more significant liver function disorder and increased expression of fibrosis markers including type IV collagen, collagen 7S, Mac2-binding protein (M2BP), and hyaluronic acid (HA). Expression of type IV collagen and collagen 7S, but not M2BP and HA, was more significantly elevated in patients with stage 1 NASH than in patients with NAFL, indicating that type IV collagen and collagen 7S may be better discriminators of NASH and NAFL than M2BP and HA at an early stage of fibrosis. When patients were stratified by NAFLD activity score, type IV collagen and collagen 7S were significantly elevated as NAFLD activity score progressed, whereas M2BP and HA expression were not significantly elevated. CONCLUSION: Type IV collagen may be a useful measure of NASH severity as latex particle-enhanced turbidimetric immunoassay-based rapid type IV collagen assay can be carried out routinely.

[Primary small intestinal Burkitt lymphoma treated with rituximab-combined CODOX-M/IVAC therapy without surgical resection].

A 38-year-old man complaining of abdominal pain was admitted to our hospital and diagnosed with small bowel obstruction. Whole body computed tomography(CT)scan showed moderate right pleural effusion with pleural and anterior chest wall thickening, with a mass approximately 100 mm in diameter at the terminal ileum. Histopathological and immunohistorical analysis of the endoscopic biopsy from the terminal ileum mass revealed diffuse infiltration of medium-sized, monotonous, atypical B lymphocytes with scanty basophilic cytoplasms carrying the so-called "starry sky" appearance, and primary small intestinal Burkitt lymphoma(BL)was diagnosed. Because of his advanced clinical stage(stage IV with Ann Arbor staging system)and the need for immediate treatment, he was promptly treated with cyclophosphamide and doxorubicin without surgical resection. Fortunately this initial therapy was received without the occurrence of bowel perforation. After initiating additional rituximab combined high-dose chemotherapy consisting of alternating courses of rituximab, cyclophosphamide, vincristine, doxorubicin, and methotrexate(R-CODOX-M)and rituximab, ifosfamide, etoposide, and cytarabine(R-IVAC), he achieved complete remission and remains without signs of disease now more than seven years after his last treatment. While further investigation will of course be needed, if possible, immediate chemotherapy without surgical resection is a treatment worth considering for improving the prognosis of those with small intestinal BL.

[Clinicopathological evaluation of preoperative chemotherapy with doxorubicin plus cyclophosphamide followed by paclitaxel for locally advanced breast cancer].

While the optimum regimen and therapeutic duration of preoperative chemotherapy have not been established for advanced breast cancer, the combination regimen of anthracycline series and taxane series, which has been well received as a postoperative chemotherapy, is also recommended for preoperative chemotherapy. We here present a clinicopathological evaluation of the safety and efficacy of the regimen and the predictors of the effectiveness of preoperative chemotherapy conducted retrospectively in patients with advanced breast cancer who were treated preoperatively with the combination of doxorubicin and cyclophosphamide followed by sequential weekly paclitaxel during the period between January 2004 and June 2007 at Yamada Red Cross Hospital. Eleven patients were treated preoperatively with the regimen during the study period. Tumors shrank in all patients following the preoperative chemotherapy with a mean reduction rate of 64.1%. Adverse reactions to the preoperative chemotherapy included leukopenia; grade 3 in 1 patient and grade 2 or less in the remaining patients. Response rates evaluated in the primary tumors and lymph nodes were high. As for the predictors of the effectiveness of the regimen as preoperative chemotherapy, estrogen receptor(ER)and the presence of HER2 were investigated, and antitumor effects were high in patients with negative ER and positive HRE2.

Comparative gene expression analysis of bovine nuclear-transferred embryos with different developmental potential by cDNA microarray and real-time PCR to determine genes that might reflect calf normality.

Placental abnormalities are the main factor in the high incidence of somatic cell clone abnormalities. The expression of several trophoblast cell-specific molecules is enhanced during gestational days 7 to 14. To determine the possible genes whose expression patterns might reflect calf normality, we first compared the gene expression profiles on day 15 between in vitro-fertilized (IVF) embryos and two types of somatic cell nuclear-transferred embryos with either a high (FNT) or low (CNT) incidence of neonatal abnormalities using a cDNA microarray containing 16 of 21 placenta-specific genes developed from tissues collected across gestation. To identify significant genes from the screening of day 15 embryos, genes with a less than two-fold difference in expression between IVF and CNT embryos, and those with a greater than two-fold difference between IVF and FNT and between CNT and FNT were considered to contribute to clone abnormalities. These two comparisons revealed 18 down-regulated and 18 upregulated genes of the 1722 genes examined. We then examined the expression levels of 10 genes with known functions in eight-cell and blastocyst-stage embryos by real-time PCR. The mRNA expression pattern of interferon (IFN)-tau, a trophectoderm-related gene, differed between IVF, CNT, and FNT eight-cell embryos; few or none of the IVF or CNT eight-cell embryos expressed IFN-tau mRNA, but all eight-cell FNT embryos expressed IFN-tau. IFN-tau mRNA expression was significantly higher in IVF blastocysts, however, than in nuclear-transferred blastocysts. Average IFN-tau mRNA expression in FNT blastocysts was not different from that in CNT blastocysts, due to one CNT blastocyst with high expression. The precise relation between early expression of IFN-tau mRNA and inferior developmental potential in cloned embryos should be examined further.

[Splenic marginal zone lymphoma associated with autoimmune hemolytic anemia treated with splenectomy and rituximab].

A 51-year-old woman, who presented with dyspnea on effort and was diagnosed with autoimmune hemolytic anemia in July 2004, was suspected of having splenic marginal zone lymphoma (SMZL) because of clonality of B cell on bone marrow and splenomegaly. She underwent splenectomy, and histopathological examination of the resected specimen confirmed the diagnosis of SMZL. The patient was treated with rituximab, and complete remission was achieved. Up to the present, three years after diagnosis, the patient has shown no evidence of progression.

CD21S antigen expression in tumour cells of diffuse large B-cell lymphomas is an independent prognostic factor indicating better overall survival.

To evaluate the clinical significance of CD21S expression of diffuse large B-cell lymphoma (DLBCL) tumour cells, we compared their clinical features, immunophenotype, response to therapy and outcome in relation to CD21S expression. Between 1987 and 1999, frozen sections from 240 DLBCL cases were examined for CD21S expression by immunohistochemical methods. CD21S expression was detected on the tumour cells of 87 (36%) cases. The median age of the CD21S(+) DLBCL cases was 65 years (range: 17-84 years), the male-female ratio was 42:45, and they showed the following clinical features: Eastern Cooperative Oncology Group score >1 in 14%, lactate dehydrogenase greater than normal levels in 38%, extranodal sites >1 in 14%, stages III/IV disease at diagnosis in 29%, B symptoms in 17%, and a high/high-intermediate International Prognostic Index (IPI) in 23%. They also showed a better overall survival (P = 0.00001, log-rank test) and a better complete remission rate (P = 0.00004, chi-square test) than CD21S(-) DLBCL. Moreover, CD21S(+) DLBCL showed a better survival than CD21S(-) DLBCL for both low/low-intermediate and high/high-intermediate risk categories of IPI (P = 0.045 and P = 0.0016 respectively). Multivariate analysis identified CD21S expression as an independent factor for survival when compared with the five IPI factors. These findings indicate that CD21S expression of DLBCL tumour cells is a useful prognostic factor for survival.

mRNA expression of leukemia-associated antigens in patients with acute myeloid leukemia for the development of specific immunotherapies.

Specific immunotherapies for patients with acute myeloid leukemia (AML) using leukemia-associated antigens (LAA) as target structures might be a therapeutic option to enhance the graft-vs.-leukemia effect observed after allogeneic stem cell transplantation or to prolong a complete remission (CR) achieved by chemotherapy. Significant mRNA expression of LAA is a prerequisite for such immunotherapies. Here, previously characterized antigens associated with solid tumors (TAA) and newly characterized LAA were investigated for their expression in up to 60 AML patients and in leukemia cell lines. To investigate their specificity for leukemic blasts, the mRNA expression was also characterized in PBMN and CD34 positive cells of healthy volunteers and in a panel of normal tissues. The following antigens showed high mRNA expression in AML patients: MPP11 was detected in 43/50 (86%), RHAMM in 35/50 (70%), WT1 in 40/60 (67%), PRAME in 32/50 (64%), G250 in 18/35 (51%), hTERT in 7/25 (28%) and BAGE in 8/30 (27%) of AML patients. Real-time RT-PCR showed a tumor-specific expression of the antigens BAGE, G250 and hTERT, as well as highly tumor-restricted expression for RHAMM, PRAME and WT1. The antigen MPP11 was overexpressed. These antigens might be candidates for immunotherapies of leukemia patients and, because of their simultaneous expression, also for polyvalent vaccines.

Nuclear transfer of adult bone marrow mesenchymal stem cells: developmental totipotency of tissue-specific stem cells from an adult mammal.

Recent studies have demonstrated that somatic stem cells have a flexible potential greater than previously expected when they are transplanted into different tissues. On the other hand, recent studies also have revealed that these potentials might occur because of spontaneous cell fusion with recipient cells. The nuclei of somatic cells could have been reprogrammed when they were artificially or spontaneously fused with mouse embryonic stem (ES) cells. The resultant hybrid cells acquired a developmental pluripotency that the original somatic cells did not have but that ES cells did. LaBarge and Blau (Cell 2002; 111:589-601) demonstrated that adult bone marrow-derived cells contributed to muscle tissue in a stepwise biological progression. This means that bone marrow-derived cells became satellite cells of mononucleate muscle stem cells after the first irradiation-induced damage to the mouse, and after the second irradiation-induced damage, multinucleate myofibers appeared from the bone marrow-derived cells. Considered together, the differentiation potential of the somatic stem cell nucleus itself remains unclear. Although the pluripotency of somatic stem cell populations has been evaluated, the developmental totipotency of the nuclei of somatic stem cells, whether or not they fused with other cells, has not been shown, except in only one study concerning fetal neural cells (never in adult stem cells). Here, we showed the developmental totipotency of adult bovine mesenchymal stem cells by nuclear transfer.

Characterization of several leukemia-associated antigens inducing humoral immune responses in acute and chronic myeloid leukemia.

To design a specific immunotherapy for leukemia patients, the identification of leukemia-associated antigens (LAAs) is a pivotal step. Antileukemic effects after hematopoetic stem cell transplantation for myeloid leukemias are observed and might be related to the recognition of LAAs. Using the serological screening of an expression library (SEREX) of K562 cells, we identified 16 different clones encoding LAAs eliciting a humoral immune response, among them the heat shock proteins HSJ2 and HSP70, the M-phase phosphoprotein 11 (MPP11), the BRCA1-associated protein (BRAP), the Jkappa recombination binding protein (RBPJkappa) and the receptor for hyaluronic acid mediated motility (RHAMM). Serological responses to MPP11 were observed in 7/19 (37%) of patients with acute myeloid leukemia (AML) and 6/16 (38%) of patients with chronic myeloid leukemia (CML), but not in healthy volunteers (0/20). IgG antibodies directed against MPP11 were also detected in 25-50% of the sera of patients with solid tumors such as melanoma, renal cell, ovarian and breast carcinoma. mRNA expression of MPP11 was detected in 20/20 AML patients and 7/10 patients with CML. In normal tissues, strong mRNA expression of MPP11 was only detected in testis. By real-time PCR, we detected upregulation of MPP11 in leukemic blasts. Simultaneous humoral immune responses to 2 or more of the 16 LAAs identified here was observed, suggesting the feasibility of a polyvalent vaccination as an option for immunotherapies in leukemia patients.

Rapid lethality of hosts by interleukin-12 following H-2 compatible allogeneic bone marrow transplantation: Reminiscence of gut-associated acute graft-versus-host reaction.

T lymphocytes and NK cells play an important role in the graft-versus-host (GVH) disease. IL-12 is known to activate both lymphocyte subpopulations. Here, we examined the effect of IL-12 on the outcome after allogenic bone marrow transplantation (allo-BMT). Experiments were performed in a major histocompatibility complex (MHC)-matched allo-BMT model from DBA/2 to BALB/c mice. We administered IL-12 subcutaneously to recipient BALB/c mice at the day of BMT and 2 days later. At a dose of 200 ng IL-12, mice died between day 4 and day 10. When 50 ng of IL-12 was administered, 50% of the animals died, at a dose of 25 ng all mice recovered. In vivo blocking experiments with antagonistic monoclonal antibodies indicated that the early lethality was mediated by FasL, IFNgamma and TNFalpha. Recognition of third party antigens by donor, but not recipient lymphocytes caused lethality. HE staining of the large intestine of IL-12 treated animals showed a severe mucosal injury associated with infiltrating lymphocytes and apoptotic bodies. In IL-12 treated mice in contrast to untreated control animals, flow cytometry analysis revealed numerous CD3+ T cells and CD11c+ dendritic cells (DC) were found in the large intestine. The interaction of T cells and antigen presenting DC might contribute to the lethality in our system. In light of these findings, the usefulness of IL-12 application for patients after BMT is rather questionable.

Receptor for hyaluronan acid-mediated motility (RHAMM) is a new immunogenic leukemia-associated antigen in acute and chronic myeloid leukemia.

OBJECTIVE: Identification of leukemia-associated antigens (LAA) eliciting an immune response in patients is a prerequisite for specific immunotherapy of leukemias. To identify new LAA, we used the method of serologic screening of cDNA expression libraries (SEREX). MATERIALS AND METHODS: A SEREX library of the cell line K562 was subjected to allogeneic screening with sera from patients with acute myeloid leukemia (AML) or chronic myeloid leukemia (CML) vs sera from healthy volunteers. RESULTS: The receptor for hyaluronan acid-mediated motility (RHAMM) involved in cell growth and metastasis was identified as a new LAA. Serologic responses to RHAMM were observed in patients with AML (42%), CML (31%), melanoma (83%), renal cell carcinoma (40%), breast cancer (67%), and ovarian carcinoma (50%), but not in HV or patients with autoimmune diseases. RHAMM mRNA was detectable in peripheral blood mononuclear cells (PBMN) of 60% of newly diagnosed AML patients. Western blotting stained positive for RHAMM protein in 70% of AML patients. mRNA expression of RHAMM also was found in patients with CML (40%), renal cell carcinoma (73%), breast carcinoma (60%), and ovarian carcinoma (50%). In melanoma, RHAMM mRNA expression was detected in metastases (80%) but not in primary tumors. RHAMM is differentially expressed: significant mRNA expression was not found in normal tissues, except from testis, placenta, and thymus, or in PBMN- and CD34-separated cell samples of healthy volunteers. CONCLUSIONS: RHAMM is an immunogenic antigen in leukemias and solid tumors and might be a potential target structure for cellular immunotherapies and antibody therapies.