Survival analysis of platinum-refractory patients with advanced esophageal cancer treated with docetaxel or best supportive care alone: a retrospective study.

The survival benefit of second-line chemotherapy with docetaxel in platinum-refractory patients with advanced esophageal cancer (AEC) remains unclear. A retrospective analysis of AEC patients with Eastern Cooperative Oncology Group performance status (PS)≤2 was performed, and major organ functions were preserved, who determined to receive docetaxel or best supportive care (BSC) alone after failure of platinum-based chemotherapy. The post-progression survival (PPS), defined as survival time after disease progression following platinum-based chemotherapy, was analyzed by multivariate Cox regression analysis using factors identified as significant in univariate analysis of various 20 characteristics (age, sex, PS, primary tumor location, etc) including Glasgow prognostic score (GPS), which is a well-known prognostic factor in many malignant tumors. Sixty-six and 45 patients were determined to receive docetaxel and BSC between January 2007 and December 2011, respectively. The median PPS was 5.4 months (95% confidence interval [CI] 4.8-6.0) in the docetaxel group and 3.3 months (95% CI 2.5-4.0) in the BSC group (hazard ratio [HR] 0.56, 95% CI 0.38-0.84, P=0.005). Univariate analysis revealed six significant factors: treatment, PS, GPS, number of metastatic organs, liver metastasis, and bone metastasis. Multivariate analysis including these significant factors revealed three independent prognostic factors: docetaxel treatment (HR 0.62, 95% CI 0.39-0.99, P=0.043), better GPS (HR 0.61, 95% CI 0.46-0.81, P=0.001), and no bone metastasis (HR 0.31, 95% CI 0.15-0.68, P=0.003). There was a trend for PPS in favor of the docetaxel group compared with patients who refused docetaxel treatment in the BSC group (adjusted HR 0.61, 95% CI 0.29-1.29, P=0.20). Docetaxel treatment may have prolonged survival in platinum-refractory patients with AEC.

Hepatic differentiation of mouse embryonic stem cells in a bioreactor using polyurethane/spheroid culture.

BACKGROUND: We have previously developed a hybrid artificial liver (HAL) using polyurethane foam (PUF)/hepatocyte spheroid culture. The PUF-HAL has been successfully scaled up to a clinical level. However, one of the most difficult problems for clinical application of HALs is obtaining a cell source. We now focused our attention on embryonic stem (ES) cells as a potential source for HAL. In this study, we investigated the differentiation of mouse ES (mES) cells into functional hepatocytes in the PUF-HAL module. METHODS: The PUF-HAL module included a cylindrical PUF block having many capillaries for medium flow. mES cells were immobilized in the module. To induce hepatic differentiation, growth factors were added to the culture medium. We evaluated cell density, gene expression analysis, and liver-specific functions. RESULTS: mES cells spontaneously formed spherical multicellular aggregates (spheroids) in the pores of PUF. mES cells proliferated by 20 days, achieving a high cell density (about 1 x 10(8) cells/cm3 PUF). Differentiating ES cells expressed endodermal-specific genes such as alpha-fetoprotein, albumin, and tryptophan 2, 3-deoxygenase. The activity of ammonia removal of mES cells per unit volume of the module was detectable by 15 days and increased with culture time. Maximal expression levels were comparable to those of primary (porcine and human) hepatocytes. SUMMARY: mES cells immobilized in the PUF module expressed liver-specific functions at high level, because of high cell density in culture and hepatic differentiation. These results indicated that PUF module-immobilized mES cells may be useful as a biocomponent of HALs.

Hepatic differentiation of embryonic stem cells in HF/organoid culture.

OBJECTIVE: The use of embryonic stem cells (ES cells) has recently received much attention as a novel cell source for various hybrid artificial organs. To use ES cells, it is necessary to be able to produce functional mature cells from ES cells in large quantities. We applied HF/organoid culture, where cultured cells formed cylindrical multicellular aggregates (organoids) in the lumen of hollow fibers, to mouse and cynomolgus monkey ES cells for hepatic differentiation. MATERIALS AND METHODS: ES cells were injected into hollow fibers. The hollow fibers were centrifuged to induce organoid formation and cultured in medium including factors for hepatic differentiation. To determine the characteristics of cells in the bundle, we evaluated gene expression and liver-specific functions. RESULTS: ES cells immobilized inside hollow fibers proliferated and formed cylindrical organoids. In mouse ES cell cultures, the expression of mRNAs of hepatocyte-specific genes increased with culture time. Ammonia removal activity detected at 15 days increased with culture time. Albumin secretion activity detected at 12 days increased by 21 days. In cynomolgus monkey ES cell cultures, ES cells showed spontaneous ammonia removal functions. The maximum levels of these functions per unit volume of the hollow fibers were roughly comparable to those of primary hepatocyte-organoids. CONCLUSIONS: ES cells differentiated into hepatocyte-like cells using the organoid culture technique. The results indicated that the combination of ES cells and an organoid culture technique was useful to obtain mature hepatocytes.

Impacts of excision repair cross-complementing gene 1 (ERCC1), dihydropyrimidine dehydrogenase, and epidermal growth factor receptor on the outcomes of patients with advanced gastric cancer.

Using laser-captured microdissection and a real-time RT-PCR assay, we quantitatively evaluated mRNA levels of the following biomarkers in paraffin-embedded gastric cancer (GC) specimens obtained by surgical resection or biopsy: excision repair cross-complementing gene 1 (ERCC1), dihydropyrimidine dehydrogenase (DPD), methylenetetrahydrofolate reductase (MTHFR), epidermal growth factor receptor (EGFR), and five other biomarkers related to anticancer drug sensitivity. The study group comprised 140 patients who received first-line chemotherapy for advanced GC. All cancer specimens were obtained before chemotherapy. In patients who received first-line S-1 monotherapy (69 patients), low MTHFR expression correlated with a higher response rate (low: 44.9% vs high: 6.3%; P=0.006). In patients given first-line cisplatin-based regimens (combined with S-1 or irinotecan) (43 patients), low ERCC1 correlated with a higher response rate (low: 55.6% vs high: 18.8%; P=0.008). Multivariate survival analysis of all patients demonstrated that high ERCC1 (hazard ratio (HR): 2.38 (95% CI: 1.55-3.67)), high DPD (HR: 2.04 (1.37-3.02)), low EGFR (HR: 0.34 (0.20-0.56)), and an elevated serum alkaline phosphatase level (HR: 1.00 (1.001-1.002)) were significant predictors of poor survival. Our results suggest that these biomarkers are useful predictors of clinical outcomes in patients with advanced GC.

Application of chimerism-based drug-induced tolerance to rat into mouse xenotransplantation.

The current critical shortage of human donor organs has stimulated the feasibility of the xenogenic transplantation, such as swine to primate. We have previously reported the induction of donor-specific tolerance in MHC-disparated recipient mice by using our cyclophosphamide (CP)-induced tolerance conditioning. In this study, we examined the efficacy of our CP-induced tolerance conditioning in xenogenic transplantation model. F344 rats and B10 mice were used as donors and recipients. Recipient mice were treated with donor spleen cells, CP, Busulfan and bone marrow cells, with or without prior NK-cell depletion. Donor mixed chimerism, and the presence of donor reactive T-cell population were analysed by flow cytometry. The survival of the donor skin grafts were observed after the conditioning. Donor mixed chimerism was temporary induced but terminated at 10 weeks after treatments. Donor-specific prolongation of the skin graft survival was observed after the treatments, however, grafts were rejected in the long term. NK-cell depletion, prior to the treatments, did not affect the levels of the mixed chimerism or graft prolongation. The donor-reactive recipient T-cell population was remained the same level as the untreated mice, suggesting the failure of the induction of the central T-cell tolerance. Thus, partial efficacy of our CP-induced tolerance treatments in the rat to mice xenotransplantation was observed. Our results suggested that the additional treatments were required to establish the stable xenogenic tolerance.

Differentiation effects by the combination of spheroid formation and sodium butyrate treatment in human hepatoblastoma cell line (Hep G2): a possible cell source for hybrid artificial liver.

The aim of this study was to investigate the feasibility of human hepatoblastoma cell line (Hep G2), which differentiates by spheroid formation, and treatment with sodium butyrate (SB) as a cell source for hybrid artificial liver (HAL). Hep G2 spontaneously formed spheroids in polyurethane foam (PUF) within 3 days of culture and restored weak ammonia removal activity. Treatment with SB, which is a histone deacetylase inhibitor, further increased the ammonia removal activity of Hep G2 spheroids in a concentration-dependent manner. The activation of ornithine transcarbamylase--a urea cycle enzyme--was significantly related to the upregulation of ammonia removal by spheroid formation, but scarcely contributed to the further upregulation following SB treatment. In contrast with ammonia removal, treatment with SB reduced the albumin secretion of Hep G2 spheroids in a concentration-dependent manner. In the PUF-HAL module in a circulation culture, the ammonia removal rate and albumin secretion rate (per unit volume of the module) of Hep G2 spheroids treated with 5 mM SB were almost the same as those of primary porcine hepatocyte spheroids. These results suggest that simultaneous use of spheroid formation and SB treatment in Hep G2 is beneficial in enhancing the functions of human hepatocytes with potential applications in regenerative medicine and drug screening.

Effects of N-alpha-methyl-histamine on human H(2) receptors expressed in CHO cells.

BACKGROUND: Production of N-alpha-methyl-histamine (NAMH), a histamine H(3) receptor (H3R) agonist, is reportedly promoted in Helicobacter pylori infected human gastric mucosa. NAMH was suggested to act directly on histamine H(2) receptors (H2Rs) in animals to stimulate acid secretion and to be a H2R agonist. As H2Rs and H3Rs play different roles in gastric acid secretion, it is very important to verify that NAMH is a H2R agonist. AIMS: To determine whether NAMH is a H2R agonist, as well as a H3R agonist. METHODS: We used a Chinese hamster ovary (CHO) cell line expressing human H2Rs (CHO-H2R) and control CHO cells. Expression of human H2Rs was confirmed by tiotidine binding. cAMP production in CHO-H2R and control cells in response to histamine or NAMH was measured. cAMP production in response to 10(-7) M NAMH was also measured in the presence or absence of the H2R antagonist famotidine and the H3R antagonist thioperamide. RESULTS: NAMH dose dependently stimulated cAMP productions in CHO-H2R cells. This production was inhibited by famotidine but not by thioperamide. Control CHO cells were unresponsive to either histamine or NAMH. In addition, the effect of NAMH, in terms of cAMP production in CHO-H2R cells, was more potent than that of histamine-that is, with a lower EC(50) concentration and higher maximal cAMP production. Both NAMH and histamine, but not R-alpha-methyl-histamine, effectively inhibited [(3)H] tiotidine binding to CHO-H2R cells. CONCLUSIONS: NAMH, which is produced in the gastric mucosa by H pylori, is a potent H2R agonist as well as a H3R agonist.

Association of allelic losses at 3p25.1, 13q12, or 17p13.3 with poor prognosis in breast cancers with lymph node metastasis.

To identify specific allelic losses that might correlate with postoperative mortality of patients with node-positive breast carcinomas, we examined tumors from a cohort of 263 such patients, who were followed clinically for 5 years postoperatively, for allelic losses among 18 microsatellite markers. Patients whose tumors had lost an allele at 3p25.1, 13q12, or 17p13.3 had significantly higher risks of mortality than those whose tumors retained both alleles at those loci. At 3p25.1, the 5-year mortality rate was 33.8% among patients with losses vs. 16.8% with retention (P = 0.0154); at 13q12, 30.3% vs. 13.0% (P = 0.0241); and at 17p13.3, 30.4% vs. 16.2% (P = 0.0243). Combined losses at 3p25.1 and 17p13.3 increased the predicted postoperative mortality risk by a factor of 4.9 (5-year mortality rate of 38.2% vs. 8.0%, P = 0.0006), and combined losses at 3p25.1 and 13q12 raised the predicted postoperative mortality risks by a factor of 2.9 (34.7% vs. 12.7%, P = 0.0441). These data indicate that loss of heterozygosity (LOH) at any one or a pair of loci at 3p25.1, 13q12, or 17p13.3 is a significant predictor of postoperative mortality for breast-cancer patients.

Allelic losses as prognostic markers for breast cancers.

Specific allelic losses in the DNA of tumor cells are potential indicators of postoperative prognosis. Patients whose tumors showed allelic losses at 1p34, 3p25, 8p22, 13q12, 17p13.3, or 17q21.1 had a significantly higher risk of postoperative mortality than women whose tumors retained both alleles at those loci (the 5-year mortality rates in patients with loss vs those with retention were: at 1p34, 23% vs 10%, P = 0.0100; at 3p25, 22% vs 9%, P = 0.0014; at 8p22, 24% vs 7%, P = 0.0177; at 13q12, 19% vs 8%, P = 0.0093; at 17p13.3, 19% vs 9%, P = 0.0078; and at 17q21.1, 17% vs 10%, P = 0.0475). Allelic losses at these loci may serve as negative prognostic indicators to guide postoperative management, especially in the selection of patients who should be offered intensive adjuvant therapy.

Study on endoscopic esophageal mucosal resection with ligating device. I--Clinical study.

BACKGROUND/AIMS: EEMRL (endoscopic esophageal mucosal resection with a ligating device) has become increasingly popular. In this article, we review 13 clinical cases of EEMRL. METHODOLOGY: Since 1993, we have performed EEMRL to treat 15 lesions in 13 patients. Twelve squamous cell carcinomas (mucosal cancer in 10 and submucosal cancer in 2) were included among the 15 lesions. RESULTS: EEMRL failed to achieve complete resection of the 2 submucosal lesions (3.0 and 2.8 cm in maximum diameter). However, esophageal lesions could be removed successfully when 2.5 cm or less in maximum diameter. The procedure was not associated with any complication. CONCLUSIONS: Our clinical study showed that this technique may be indicated for esophageal cancer with a maximum diameter < or = 2.5 cm and confined to the mucosa. EEMRL is a technically easy and minimally invasive therapy which could be useful for the treatment of early esophageal cancer.

[Effect of G-CSF (nartograstim) on neutropenia (leukopenia) induced by taxane in metastatic breast cancer--time-course changes in neutrophil and leukocyte counts].

Since 1997, we have used docetaxel and paclitaxel as the second-line and third-line chemotherapies against anthracycline-resistant metastatic breast cancer. However, these taxane compounds induced neutropenia and leukopenia, which may be reversed by G-CSF (Nartograstim). We thus examined the therapeutic efficacy of nartograstim for time-course changes in neutrophil and leukocyte counts in these patients. No difference was observed in neutrophil or leukocyte count whether the patient was treated with docetaxel or paclitaxel. Neutrophil and leukocyte counts reached a nadir on days 7 to 8 after administration. With a 5-6 day administration of nartograstim, neutrophil or leukocyte counts recovered by the second or third day after the nadir, indicating that the chemotherapy was given safely with nartograstim. In these same patients receiving a given treatment cycle, the number of days until reaching the nadir were almost identical for neutrophils and leukocytes; however, the duration of the nadir and the time to count recovery was significantly longer for neutrophils than for leukocytes. In the clinical setting, the parameter "leukocyte count" has been occasionally used for evaluation of the severity of myelosuppression, because the data is more readily available. However, at least during the nadir, the "neutrophil count" should be used as the parameter of choice.

Lifting of lesions during endoscopic mucosal resection (EMR) of early colorectal cancer: implications for the assessment of resectability.

BACKGROUND AND STUDY AIMS: This study assessed the indications for and limitations of endoscopic mucosal resection (EMR) for early colorectal cancer, focusing on the way in which the lesion lifts after submucosal injection. PATIENTS AND METHODS: The study included 94 patients with early colorectal cancer who received EMR treatment. The lifting of the lesion after submucosal injection was analyzed (classified as completely lifted/soft; completely lifted/hard; incompletely lifted; and non-lifted) along with the endoscopic findings, pathological findings, and clinical course. RESULTS: Almost all completely lifted/soft lesions were mucosal cancers. Some of the completely lifted/hard lesions were staged as sm2. The incompletely lifted lesions included stages sm1 to sm3. Non-lifting lesions were almost always deeper than sm3. The lifting condition was significantly associated with the depth of invasion, and the lesion type was related to the extent of lifting but not to tumor size or recurrent disease. Recurrent disease was noted in three patients who underwent piecemeal EMR. CONCLUSIONS: The indication for EMR is easily assessed on the basis of the lifting characteristics of the tumor after submucosal injection, which was found to be significantly related to the depth of invasion. The factor limiting the indication for EMR is not the size of a tumor, but its lifting condition.

Effect of intratumoral OK-432 administration of thymidine phosphorylase expression in human gastric carcinoma.

It is known that thymidine phosphorylase (dThdPase) is increased in various types of malignant tumors and is induced by cytokines. In this study, we have investigated the effects of OK-432, which induces multiple cytokines, on dTHdPase expression and angiogenesis in human gastric carcinomas. We examined 25 patients who underwent gastrectomy for gastric carcinoma. OK-432 was directly injected in tumors in 16 (OK group) of 25 patients via endoscopy before operation and the other 9 patients were not treated (control group). The dThdPase activity in carcinoma tissues of the OK group was significantly higher than that of the control group (P < 0.05). The amounts of IL-1 alpha, IFN-alpha, and IFN-gamma in carcinomas in the OK group were significantly higher than in the controls (P < 0.05), and these were significantly correlated with the dThdPase activity. Intratumoral OK-432 administration enhances the expression of dThdPase in gastric carcinoma cells by inducing various cytokines.

Two novel single-nucleotide polymorphisms of the Caspase-9 (CASP9) gene in the Japanese population.

We identified two single-nucleotide polymorphisms (SNPs) in the human Caspase-9 (CASP9) gene (1p36.3), which encodes an apoptosis-related cysteine protease, by screening all exons and exon-intron boundaries. These SNPs were present in coding regions. One of coding SNPs reflected an amino-acid substitution; an A to G transition at codon 221 in exon 5 would encode arginine instead of glutamine. As the gene is implicated in apoptotic cascade, the polymorphic sites will serve as useful markers for genetic studying of disorders affecting immune response and cancer susceptibility in humans.

Method to improve cosmetic outcome following craniotomy.

This technical note describes a simple method for reducing the dead space created by craniotome due to the loss of bone dust and improving the cosmetic outcome following a craniotomy. After drilling the burr holes for the craniotomy, the bone between the holes is drilled away in a standard fashion except that multiple regions of about 1 cm in length are left intact. These intact regions are broken using a periosteal elevator and fixed like a bridge when the bone is replaced. The resulting bone flap is readily returned to its original position without making the dead space created by regular craniotomy. The amount of the dead space caused by losing the bone dust is reduced and a good cosmetic recovery is obtained. This technique is useful for both craniotomy and facial bone surgery, which requires cosmetic results.

Allelic losses of loci at 3p25.1, 8p22, 13q12, 17p13.3, and 22q13 correlate with postoperative recurrence in breast cancer.

We previously defined 18 chromosomal regions in which frequent allelic losses were observed in breast cancers (T. Sato et al., Cancer RES:, 50: 7184-7189, 1990; Y. Harada et al., Cancer (PHILA:), 74: 2281-2286, 1994; I. Ito et al., BR: J. Cancer, 71: 438-441, 1995; K. Tsukamoto et al., Cancer (PHILA:), 78: 1929-1934, 1996; S. Matsumoto et al., Genes Chromosomes Cancer, 20: 268-274, 1997; T. Yokota et al., JPN: J. Cancer RES:, 88: 959-964, 1997; K. Tsukamoto et al., Cancer (PHILA:), 82: 317-322, 1998; A. Iida et al., Genes Chromosomes Cancer, 21: 108-112, 1998; K. Fukino et al., Genes Chromosomes Cancer, 24: 345-350, 1999; T. Yokota et al., Cancer (PHILA:), 85: 447-452, 1999; Y. Utada et al., JPN: J. Cancer RES:, 91: 293-300, 2000). To identify specific allelic losses that might correlate with postoperative recurrence, we examined tumors from a cohort of 504 breast cancer patients, who were followed clinically for 5 years postoperatively, for allelic losses of 18 microsatellite markers. Patients whose tumors had lost an allele at 3p25.1, 8p22, 13q12, 17p13.3, or 22q13 had significantly higher risks of recurrence than those whose tumors retained both alleles at those loci; at 3p25.1, the 5-year recurrence rate was 27% among patients with losses versus 18% with retention (P = 0.0131); at 8p22, 27% versus 14% (P = 0.0129); at 13q12, 28% versus 15% (P = 0.0109); at 17p13.3, 27% versus 20% (P = 0.0482); and at 22q13, 29% versus 20% (P = 0.0477). These data indicate that loss of heterozygosity at any one of these five specific loci is a significant predictor of postoperative recurrence among patients who have undergone surgery for breast cancer. These allelic losses can serve as negative prognostic indicators to guide postoperative management of patients.

Nine novel single-nucleotide polymorphisms in the integrin beta4 (ITGB4) gene in the Japanese population.

We identified nine single-nucleotide polymorphisms (SNPs) in the human integrin beta4 (ITGB4) gene (17q24-q25), which encodes a cell-surface receptor, by screening all exons and exon-intron boundaries. Seven of these SNPs were present in coding regions and two in intronic sequences; four of the coding SNPs involved amino-acid substitutions. As the gene is implicated in the tumorigenesis of breast cancers, the polymorphic sites will serve as useful markers not only for distinguishing alleles in loss of heterozygosity (LOH) analyses but also for studying genetic susceptibility to malignancies in humans.

Silencing of the caspase-1 gene occurs in murine and human renal cancer cells and causes solid tumor growth in vivo.

Renal cell cancer is a unique solid tumor that occasionally shows spontaneous regression even at an advanced stage, of which the underlying mechanism is not well understood. To investigate a potential role of the pro-apoptotic molecule caspase-1 in the growth regulation of renal cell cancer, we created transfectants expressing exogenous caspase-1 from a murine renal cancer cell line, Renca. Overexpression of caspase-1 did not affect the growth of Renca cells in vitro at the exponential phase but induced apoptotic cell death at 50% to 75% confluence, whereas control cells underwent apoptosis only after reaching 100% confluence. When implanted into the flank of a syngeneic BALB/c mouse, caspase-1-overexpressing Renca cells did not effectively establish growth as a solid tumor, forming a measurable tumor in only 7 of 11 (64%) animals, whereas control cells formed a tumor in 6 of 6 (100%) animals. The growth of tumors from caspase-1-overexpressing cells slowed down markedly after the tumors reached 5 to 10 mm in diameter, and histological examination of such tumors revealed numerous apoptotic cells positively stained by TUNEL assay. Interestingly, endogenous caspase-1 was not detected in the tumors from control cells, which re-expressed caspase-1 when they were re-cultured and exposed to a demethylation reagent, 5-aza-2'-deoxycytidine. Furthermore, treatment of a human renal cancer cell line, ACHN, with 5-aza-2'-deoxycytidine also caused recovery of caspase-1 expression, which was not detected before treatment. These data suggest that silencing of caspase-1 through DNA methylation may be involved in the oncogenesis of some renal cell cancers growing as a solid tumor.

Potent and long-lasting action of lafutidine on the human histamine H(2) receptor.

BACKGROUND/AIM: Based on animal models, lafutidine, a novel histamine H(2) receptor (H(2)R) antagonist, is reported to show potent and long-lasting antagonisms of histamine H(2)R-mediated effects. However, no reports have been published concerning its direct interaction with the human H(2)R. This study aims at characterizing its interaction with human H(2)R. METHODS: Chinese hamster ovary cell lines stably expressing human H(2)Rs were obtained. The dose-dependent effects of lafutidine and famotidine on [(3)H]tiotidine binding and histamine-stimulated cAMP production were analyzed. The effects of preincubation with 2.78 x 10(-7) M of lafutidine or famotidine for 30 min on histamine-dependent cAMP production and [(3)H]tiotidine binding were also examined after 0, 1, 2, 4, and 12 h. This concentration is below the C(max) of lafutidine (10 mg p.o.) and above the C(max) of famotidine (20 mg p.o.). RESULTS: Lafutidine inhibited [(3)H]tiotidine binding and histamine-stimulated cAMP production as or more potently than famotidine. At higher concentrations lafutidine was more potent than famotidine. In addition, preincubation with 2.78 x 10(-7) M lafutidine, but not with 10(-5) M famotidine, had marked inhibitory effects which persisted as long as after extensive washing. CONCLUSION: Lafutidine shows a potent and long-lasting antagonism on the human H(2)R.