Heated egg white has no effect, but lactic fermented and unheated egg white reduces abdominal fat in rats.

BACKGROUND: We previously reported the abdominal fat-reducing effect of unheated egg white proteins (EWP); however, unheated egg white is actually rarely consumed. We thus investigated the effect of heated egg white on abdominal fat in rats. METHODS: Male SD rats were divided into two groups that were allowed to consume different dietary preparations containing casein or heated egg white for 4 weeks (Trial 1). We studied whether a heated form and a lactic fermented form of egg white (FLE) are as effective as unheated egg white for reducing abdominal fat. For this, we divided male SD rats into four groups that were allowed to consume different dietary preparations containing casein, unheated egg white, heated egg white, or lactic fermented egg white for 4 weeks (Trial 2). RESULTS: Animals in the heated egg white group showed no significant difference in abdominal fat weight compared with those in the casein group (Trial 1). Animals in the unheated egg white group and the FLE group had significantly lower levels of abdominal fat weight than those in the casein group (Trial 2). Ovalbumin in heated egg white was degraded by pepsin, whereas ovalbumin in unheated egg white and lactic acid fermented egg white was not degraded appreciably by pepsin. It was reported that EWP inhibit triglyceride absorption in rat. In the present study, EWP pepsin hydrolysate inhibited the micellar solubility of fatty acids in vitro. In particular, ovalbumin inhibited the micellar solubility of fatty acids. CONCLUSIONS: These results indicate that lactic fermented egg white reduces visceral fat in rats and suggest that different levels of susceptibility of ovalbumin to pepsin digestion underlie the varying effectiveness among the egg white preparations.

Molecular profiling and genome-wide analysis based on somatic copy number alterations in advanced colorectal cancers.

To characterize somatic alterations in colorectal cancer (CRC), we conducted a genome-scale analysis of 106 CRC specimens. We assessed comprehensive somatic copy number alterations (SCNAs) in these CRC specimens. In addition, we examined microsatellite instability (MSI; low and high), genetic mutations (KRAS, BRAF, TP53, and PIK3CA), and DNA methylation status (classified into low, intermediate, and high type). We stratified molecular alterations in the CRCs using a hierarchical cluster analysis. The examined CRCs could be categorized into three subgroups using hierarchical cluster analysis. Tumors in subgroup 1 were characterized by a low frequency of SCNAs and a high frequency of MSI-high status, whereas tumors in subgroups 2 and 3 were closely associated with a high frequency of SCNAs. Tumors in subgroup 1 were preferentially present in the right-sided colon and showed frequent MSI-high status. Subgroup 3 was distinguished by specific alterations, including gains at 1q23-44, 1p11-36, 10q11-26, 10p11-13, 12q24-24, and 13q33-33. In contrast, tumors in subgroup 2 were characterized by copy-neutral LOH at 12p12-13, 1q24-25, and 10q22. In addition, KRAS mutations were more frequently found in subgroup 3 than in subgroup 1. TP53 mutations and intermediate levels of DNA methylation were common alterations in the three subgroups. SCNAs contributed to sporadic CRC, and there were three subgroups based on SCNAs that played a different role in driving the development of this disease.

Molecular subtypes of colorectal cancers determined by PCR-based analysis.

Tumor tissue consists of a heterogeneous cell population. The allelic imbalance (AI) ratio, determined in isolated tumor glands, is a good index of tumor heterogeneity. However, associations of the patterns of AI and microsatellite instability (MSI) development, observed in most cases of colorectal cancer (CRC), with tumor progression have not been reported previously. In this study, we examined whether CRC genetic profiles stratified by a combination of the AI ratio and MSI facilitate categorization of CRC, and whether these genetic profiles are associated with specific molecular alterations in CRC. A crypt isolation method was used to isolate DNA from tumors and normal glands obtained from 147 sporadic CRCs. AI and MSI statuses were determined using PCR-based microsatellite analysis and stratified based on AI ratio and MSI status. DNA methylation status (high methylation, intermediate methylation and low methylation status and mutations in KRAS, BRAF, and TP53 were examined. In addition, mucin markers were immunostained. Based on this analysis, four subgroups were categorized. Subgroup 1 was characterized by a high MSI status and BRAF mutation; subgroup 2 was closely associated with a high AI ratio, which accumulated during the early phases of colorectal carcinogenesis, and TP53 mutation; subgroup 3 was associated with a low AI ratio, seen during the later phases of colorectal carcinogenesis, and KRAS mutation; and subgroup 4 was defined as a minor subgroup. These results confirmed that classification of distinct molecular profiles provides important insights into colorectal carcinogenesis.

Galectin-9 as a Predictive Marker for the Onset of Immune-Related Adverse Effects Associated with Anti-CCR4 MoAb Therapy in Patients with Adult T Cell Leukemia.

Adult T-cell leukemia/lymphoma (ATL/ATLL) is one of the most malignant lymphomas with poor prognosis. ATL/ATLL cells express CC chemokine receptor 4, and mogamulizumab (anti-CCR4 monoclonal antibody) exhibits strong cytotoxicity for ATL/ATLL cells. We analyzed plasma samples of 6 patients with ATL/ATLL treated with chemotherapy followed by mogamulizumab therapy (mogatherapy) for changes in the levels of biomarkers in relation to immune-related adverse effects. As treatment is often associated with skin eruptions, we investigated the profiles of inflammatory cytokines, including galectin-9 (Gal-9), which becomes increased in various infectious diseases and allergic patients. Gal-9, soluble interleukin (IL)-2 receptor, tumor necrosis factor-α, and IL-10 levels were increased before chemotherapy, and Gal-9 levels were associated with the sIL-2 receptor, which reflects tumor burden. Inflammatory levels decreased after chemotherapy. After mogatherapy, 5 of 6 patients attained complete remission (CR), whereas 1 patient showed no response (NR) and died. Among 5 patients with CR, the biomarkers remained low during mogatherapy, except for a 3-5-fold increment in Gal-9 (associated with skin eruptions). A skin biopsy showed infiltration by inflammatory cells and Gal-9 synthesis in areas with CD8 cell infiltration. In the patient with NR, increased levels of Gal-9 and the aforementioned biomarkers were noted 3 days after mogatherapy, followed by opportunistic infections resembling immune reconstitution inflammatory syndrome. Therefore, an increased Gal-9 plasma level in ATL/ATLL indicates tumor burden and reflects immune activation by mogatherapy. These findings may indicate that an increase in the Gal-9 level, a novel immune checkpoint molecule, can reflect immune-related adverse effects of various biotherapies.

Heating Has No Effect on the Net Protein Utilisation from Egg Whites in Rats.

Egg whites (EW) are a good source of protein; however, they are typically heated prior to consumption. Therefore, we investigated the effects of different heating conditions on the protein utilisation rate of EW. Male Sprague-Dawley rats (n = 36, 198 ± 1 g) were divided into six groups and fed American Institute of Nutrition-76 chow containing unheated EW, soft-boiled EW, boiled EW, milk whey protein, soybean protein, or no protein over a 10-day period using pair-feeding. Urine and faeces were sampled daily beginning on day 5 to measure nitrogen content and the net protein utilisation (NPU) rate. The soybean protein group had a significantly lower level of food intake and was thus excluded from subsequent analyses. The NPU value was similar among the unheated, soft-boiled, and boiled EW groups (97.5 ± 0.4, 96.5 ± 0.1, and 96.5 ± 0.7, resp.). The EW group values were significantly higher than the whey group values (90.5 ± 1.0). These results show that EW serve as a good source of protein, irrespective of heating.

Secretion of IFN-γ Associated with Galectin-9 Production by Pleural Fluid Cells from a Patient with Extrapulmonary Tuberculosis.

In this study, we investigated the role of a matricellular protein galectin-9 (Gal-9) in pleural effusion related to tuberculosis (TB). Plasma and pleural fluid of a patient with extrapulmonary TB were analyzed for cytokine content by ELISA and Luminex. Peripheral blood mononuclear cells (PBMCs) and pleural fluid cells (PFCs) were examined for interferon-γ (IFN-γ) secretion by the enzyme-linked immunospot (ELISPOT) assay or IFN-γ ELISA, for apoptosis and necrosis by Cell Death Detection ELISA, and also underwent cell sorting. The results indicate that compared to plasma, pleural fluid had increased levels of IFN-γ (1.6 vs. 55.5 pg/mL), IL-10, IL-12p40, vascular endothelial growth factor (VEGF), and Gal-9 (3.0 vs. 936.0 pg/mL), respectively. PFCs culture supernatant exhibited higher concentration of Gal-9 compared to PBMCs in culture, consistent with enriched Gal-9 staining in the granuloma that is in closer vicinity to PFCs compared to PBMCs. PFCS displayed higher IFN-γ secretion after stimulation with TB antigens ESAT-6/CFP-10. Furthermore, in PFCs, Gal-9 alone could stimulate IFN-γ synthesis in culture or ELISPOT, which was inhibited by a Gal-9 antagonist lactose, and which may promote apoptosis and necrosis. These findings suggest that Gal-9 could modulate immune responses and participate in immunopathology of pleural effusion during TB.

Molecular differences in the microsatellite stable phenotype between left-sided and right-sided colorectal cancer.

Differences in the pathogenesis of microsatellite stable (MSS) sporadic colorectal cancers (CRCs) between left-sided CRC (LC) and right-sided CRC (RC) have not been clarified. To identify pathogenesis-related genomic differences between MSS CRCs within the two locations, we performed a comprehensive molecular analysis using crypt isolation with samples from 92 sporadic CRCs. Microsatellite instability (MSI; high and low/negative) and DNA methylation status (low methylation epigenome; intermediate methylation epigenome [IME] or high methylation epigenome [HME]) were determined using polymerase chain reaction (PCR) microsatellite analysis and PCR-bisulfite pyrosequencing, respectively. Additionally, mutations in the TP53, KRAS, BRAF and PIK3CA genes were examined using PCR-bisulfite pyrosequencing (for KRAS and BRAF mutations) or PCR-single conformation polymorphism (for TP53 and PIK3CA mutations), followed by sequencing of aberrant bands. Finally, a genome-wide study using a copy number alteration (CNA)-targeted single nucleotide polymorphism array was performed. Ninety-two CRCs were classified into 71 MSS and 21 MSI phenotypes. We examined 71 CRCs with the MSS phenotype (LC, 56; RC, 15). Mutations in KRAS were associated with RC with the MSS phenotype, whereas mutations in TP53 were more frequently found in LC with the MSS phenotype. There were significant differences in the frequencies of KRAS and TP53 mutations in the IME between LC and RC with the MSS phenotype. Although CNA gains were associated with LC with the MSS phenotype, CNA losses were not major alterations associated with the MSS phenotype. These findings suggested that the molecular pathogenesis of the MSS phenotype in LC was different from that in RC.

Tumor microenvironment in invasive lobular carcinoma: possible therapeutic targets.

Invasive ductal and lobular carcinomas (IDC and ILC) are the two most common histological types of breast cancer, and have been considered to develop from terminal duct lobular unit but their molecular, pathological, and clinical features are markedly different between them. These differences could be due to different mechanisms of carcinogenesis and tumor microenvironment, especially cancer-associated fibroblasts (CAFs) but little has been explored in this aspect. Therefore, in this study, we evaluated the status of angiogenesis, maturation of intratumoral microvessels, and proliferation of CAFs using immunohistochemistry and PCR array analysis to explore the differences of tumor microenvironment between ILC and IDC. We studied grade- and age-matched, luminal-like ILC and IDC. We immunolocalized CD34 and αSMA for an evaluation of CAFs and CD31, Vasohibin-1, a specific marker of proliferative endothelial cells and nestin, a marker of pericytes for studying the status of proliferation and maturation of intratumoral microvessel. We also performed PCR array analysis to evaluate angiogenic factors in tumor stromal components. The number of CAFs, microvessel density, and vasohibin-1/CD31 positive ratio were all significantly higher in ILC than IDC but nestin immunoreactivity in intratumoral microvessel was significantly lower in ILC. These results did indicate that proliferation of CAFs and endothelial cells was more pronounced in ILC than IDC but newly formed microvessels were less mature than those in IDC. PCR array analysis also revealed that IGF-1 expression was higher in ILC than IDC. This is the first study to demonstrate the differences of tumor microenvironment including CAFs and proliferation and maturation of intratumoral vessels between ILC and IDC.

Aberrant activation of Notch signaling in extrahepatic cholangiocarcinoma: clinicopathological features and therapeutic potential for cancer stem cell-like properties.

BACKGROUND: Little is known about the roles of Notch signaling in cholangiocarcinoma (CC). The expression of hairy and enhancer of split 1 (Hes-1) has not been investigated yet in resected specimens of CC. Notch signaling has been reported to be related to cancer stem cell (CSC) like properties in some malignancies. Our aim is to investigate the participation of Notch signaling in resected specimens of extrahepatic CC (EHCC) and to evaluate the efficacy of CC cells with CSC-like properties by Notch signaling blockade. METHODS: First, the expression of Notch1, 2, 3, 4 and Hes-1 was examined by immunohistochemistry in 132 resected EHCC specimens. The clinicopathological characteristics in the expression of Notch receptors and Hes-1 were investigated. Second, GSI IX, which is a γ-secretase-inhibitor, was used for Notch signaling blockade in the following experiment. Alterations of the subpopulation of CD24+CD44+ cells, which are surface markers of CSCs in EHCC, after exposure with GSI IX, gemcitabine (GEM), and the combination of GSI IX plus GEM were assessed by flow cytometry using the human CC cell lines, RBE, HuCCT1 and TFK-1. Also, anchorage-independent growth and mice tumorigenicity in the cells recovered by regular culture media after GSI IX exposure were assessed. RESULTS: Notch1, 2, 3, 4 and Hes-1 in the resected EHCC specimens were expressed in 50.0, 56.1, 42.4, 6.1, and 81.8 % of the total cohort, respectively. Notch1 and 3 expressions were associated with poorer histological differentiation (P = 0.008 and 0.053). The patients with the expression of at least any one of Notch1-3 receptors, who were in 80.3 % of the total, exhibited poorer survival (P = 0.050). Similarly, the expression of Hes-1 tended to show poor survival (P = 0.093). In all of the examined CC cell lines, GSI IX treatment significantly diminished the subpopulation of CD24+CD44+ cells. Although GEM monotherapy relatively increased the subpopulation of CD24+CD44+ cells in all lines, GSI IX plus GEM attenuated it. Anchorage-independent growth and mice tumorigenicity were inhibited in GSI IX-pretreated cells in RBE and TFK-1 (P < 0.05). CONCLUSION: Aberrant Notch signaling is involved with EHCC. Inhibition of Notch signaling is a novel therapeutic strategy for targeting cells with CSC-like properties.

Immunohistochemical analysis of steroidogenic enzymes in ovarian-type stroma of pancreatic mucinous cystic neoplasms: Comparative study of subepithelial stromal cells in intraductal papillary mucinous neoplasms of the pancreas.

Mucinous cystic neoplasms (MCNs) are generally defined as cyst-forming epithelial neoplasms that arise in the pancreas and harbor characteristic ovarian-type stroma beneath the epithelium. In this study, we compared the immunoreactivity of steroid-related factors in these subepithelial stromal cells in MCNs to those in intraductal papillary mucinous neoplasms (IPMNs) to further characterize this unique MCN ovarian-type stroma through evaluation of sex steroid biosynthesis. Twenty MCNs and twenty IPMNs were examined. Immunoreactivity of steroid hormone receptors, including estrogen receptor (ERα and ERß), progesterone receptor (PR, PR-A, and PR-B), and androgen receptor (AR), was more frequently detected in MCN ovarian-type stromal cells than in IPMN stromal cells (P < 0.01). The H-scores (mean ± SD) of steroidogenic factor (SF)-1 were also significantly higher in MCNs (112.3 ± 33.1) than in IPMNs (0.9 ± 1.2) (P < 0.01). The steroidogenic enzymes cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), cytochrome P450 17 alpha-hydroxylase (P450c17) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) showed immunoreactivity in 9/20 (45.0 %), 15/20 (75.0 %) and 13/20 (65.0 %), respectively, of ovarian-type stroma from MCN cases. These results demonstrate that the ovarian-type stroma of MCNs can express steroidogenic enzymes. Thus, the ovarian-type stroma of MCNs can produce sex steroids that may also act on these cells.

Prognostic significance of tumor-infiltrating CD8+ and FOXP3+ lymphocytes in residual tumors and alterations in these parameters after neoadjuvant chemotherapy in triple-negative breast cancer: a retrospective multicenter study.

INTRODUCTION: The status of tumor-infiltrating lymphocytes (TILs) has been recently proposed to predict clinical outcome of patients with breast cancer. We therefore studied the prognostic significance of CD8(+) TILs and FOXP3(+) TILs in residual tumors after neoadjuvant chemotherapy (NAC) and the alterations in these parameters before and after NAC in patients with triple-negative breast cancer (TNBC). METHODS: One hundred thirty-one TNBC patients who received NAC at three institutions were examined. CD8(+) TIL and FOXP3(+) TIL in residual tumors and biopsy specimens were evaluated by double-staining immunohistochemistry. The CD8(+) TIL and FOXP3(+) TIL status of the residual tumors was assessed, and the rates of their changes before and after NAC were calculated. RESULTS: TNBC patients with high CD8(+) TIL levels or a high CD8/FOXP3 ratio in residual tumors had significantly better recurrence-free survival (RFS) and breast cancer-specific survival (BCSS) than patients with low values of these parameters. In multivariate analyses, CD8(+) TIL exhibited strong prognostic significance for RFS, with a hazard ratio (HR) of 3.09 (95 % confidence interval (CI) 1.537-6.614, P=0.0013). The CD8/FOXP3 ratio was also significantly correlated with RFS (HR=2.07, 95 % CI 1.029-4.436, P=0.0412). TNBC with larger residual tumor size and positive lymph node status, which are known prognostic factors, was independently associated with worse RFS (P=0.0064 and P=0.0015, respectively). High CD8(+) TIL levels were a markedly powerful indicator of improved BCSS, with an HR of 3.59 (95 % CI 1.499-9.581, P=0.0036). Nodal status was also associated with BCSS (P=0.0024). TNBC with a high rate of CD8(+) TIL changes was associated with significantly better RFS compared with the low group (P=0.011). Higher rates of changes in the CD8/FOXP3 ratio were significantly correlated with both better RFS and BCSS compared with lower rates (P=0.011 and P=0.023, respectively). CONCLUSIONS: This is the first study to demonstrate that high CD8(+) TIL and a high CD8/FOXP3 ratio in residual tumors and increment of these parameters following NAC and accurately predict improved prognosis in TNBC patients with non-pathological complete response following NAC. These parameters could serve as a surrogate one for adjuvant treatment in patients with residual disease in the neoadjuvant setting.

Osteopontin-integrin interaction as a novel molecular target for antibody-mediated immunotherapy in adult T-cell leukemia.

BACKGROUND: Adult T-cell leukemia (ATL) is a CD4(+) T-cell neoplasm with a poor prognosis. A previous study has shown that there is a strong correlation between the secreted matricellular protein osteopontin (OPN) level and disease severity in ATL patients. Here, we investigated the role of OPN in ATL pathogenesis and the possible application of anti-OPN monoclonal antibody (mAb) for ATL immunotherapy in NOD/Shi-scid,IL-2Rg (null) (NOG) mice. RESULTS: Subcutaneous inoculation of ATL cell lines into NOG mice increased the plasma level of OPN, which significantly correlated with metastasis of the inoculated cells and survival time. Administration of an SVVYGLR motif-recognizing anti-OPN mAb resulted in inhibition not only of tumor growth but also of tumor invasion and metastasis. The number of fibroblast activating protein-positive fibroblasts was also reduced by this mAb. We then co-inoculated mouse embryonic fibroblasts (MEFs) isolated from wild-type (WT) or OPN knockout mice together with ATL-derived TL-OmI cells into the NOG mice. The mice co-inoculated with WT MEFs displayed a significant decrease in survival relative to those injected with TL-OmI cells alone and the absence of OPN in MEFs markedly improved the survival rate of TL-OmI-inoculated mice. In addition, tumor volume and metastasis were also reduced in the absence of OPN. CONCLUSION: We showed that the xenograft NOG mice model can be a useful system for assessment of the physiological role of OPN in ATL pathogenesis. Using this xenograft model, we found that fibroblast-derived OPN was involved in tumor growth and metastasis, and that this tumor growth and metastasis was significantly suppressed by administration of the anti-OPN mAbs. Our findings will lead to a novel mAb-mediated immunotherapeutic strategy targeting against the interaction of OPN with integrins on the tumor of ATL patients.

Evaluation of matricellular proteins in systemic and local immune response to Mycobacterium tuberculosis infection.

Matricellular proteins such as osteopontin (OPN), galectin-9 (Gal-9), and tenascin-C (TN-C) are expressed not only under normal physiological conditions, but also during infection, inflammation and tumorigenesis. Plasma concentrations of matricellular proteins were studied to determine their diagnostic value as potential markers of tuberculosis (TB) activity. It was found that concentrations of OPN and TN-C were higher in patients with active TB than in healthy controls and individuals with latent infection. Moreover, LTBI patients had higher concentrations of OPN than did healthy controls. Gal-9 concentrations did not differ significantly between groups. Concentrations of matricellular proteins were higher in pleural fluid than in the plasma of patients with TB. Expression of matricellular proteins was also investigated in TB granulomas and other granulomatous diseases. Positive OPN and Gal-9 staining was observed in TB and sarcoidosis granulomas, but not in Crohn disease granulomas. The fibrotic ring around granulomas stained positive for TN-C in TB and sarcoidosis, but not in Crohn disease. Of the three matricellular proteins studied, OPN and TN-C may serve as reliable plasma markers for monitoring TB activity, whereas Gal-9 seems to be expressed more at the site of infection than in the systemic circulation.

Simultaneous improvement of specificity and affinity of aptamers against Streptococcus mutans by in silico maturation for biosensor development.

In silico evolution with an in vitro system can facilitate the development of functional aptamers with high specificity and affinity. Although a general technique known as systematic evolution of ligand by exponential enrichment (SELEX) is an efficient method for aptamer selection, it sometimes fails to identify aptamers with sufficient binding properties. We have previously developed in silico maturation (ISM) to improve functions of aptamers based on genetic algorithms. ISM represents an intelligent exploitation of a random search within a defined sequence space to optimize aptamer sequences and improve their function of interest. Here we demonstrated a successful application of ISM of aptamers to simultaneously improve specificity and affinity for Streptococcus mutans with discovery of a core sequence, which was required to form a polymerized guanine quadruplex structure for target binding. We applied ISM to aptamers selected by whole-cell SELEX and identified an aptamer with up to 16-fold improvement in affinity compared to its parent aptamers, and specificity was increased to show 12-fold more binding to S. mutans than to Lactobacillus acidophilus. Furthermore, we demonstrated a specific flow-through detection of S. mutans at a concentration range of 1 × 10(5) -10(8) CFU/mL using the evolved aptamer immobilized on gold colloids.

Oncocytic adrenocortical neoplasm arising from adrenal rest in the broad ligament of the uterus.

Oncocytic adrenocortical neoplasm is characterized by abundant eosinophilic cytoplasm containing mitochondria, occasional nuclear atypia and diffuse growth pattern. Oncocytic adrenocortical neoplasm arising in adrenal rest is, however, extremely rare. We report a case of oncocytic adrenocortical neoplasm arising in adrenal rest of the broad ligament with associated marked lipomatous metaplasia. A well circumscribed tumor was accidentally detected in the pelvic cavity of a 29 year old Japanese woman, adjacent to the broad ligament of the uterus. The tumor was composed of large eosinophilic cells associated with diffuse growth pattern and abundant mature adipose tissue admixed with foci of clear cells. Both steroidgenic factor 1 (SF-1) and alpha-inhibin were immunohistochemically positive in tumor cells. Abundant mitochondria detected by immunohistochemical and electron microscopic examination confirmed the diagnosis of oncocytic adrenocortical neoplasm. The absence of necrosis, capsular and vascular invasion as well as the low mitotic index indicated the benign potential of this tumor. The tumor cells were also positive for dehydroepiandrosteron-sulfotransferase (DHEA-ST), 17ß-hydroxysteroid dehydrogenase type 5 (17ß-HSD5), 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and steroid 17α-hydroxylase (P450-c17), suggesting a possible production of testosterone of this tumor. This is the first reported case of oncocytic adrenocortical adenoma arising in adrenal rest of the broad ligament.

Renal epithelioid angiomyolipoma with malignant features: Histological evaluation and novel immunohistochemical findings.

Renal epithelioid angiomyolipoma (EAML) is a potentially malignant tumor type whose characteristics and biomarkers predictive of malignant behavior have not been elucidated. Here, we report three cases of renal EAML with malignant features but without histories of tuberous sclerosis complex. Case 1 involved a 29-year-old man with a 12-cm solid mass in the right kidney who underwent radical right nephrectomy. Case 2 involved a 22-year-old woman with a retroperitoneal mass who underwent radical right nephrectomy and retroperitoneal tumorectomy. Local recurrence was detected 7 years post-surgery. Case 3 involved a 23-year-old man with a 14-cm solid mass in the left kidney who underwent radical left nephrectomy. Microscopically, the tumors in all cases demonstrated proliferation of epithelioid cells with atypia, mitotic activity, necrosis, hemorrhage, and vascular invasion. Epithelioid cells in all cases were immunohistochemically positive for melanocytic and myoid markers and weakly positive for E-cadherin and ß-catenin. Immunohistochemistry revealed activation of the mammalian target of rapamycin pathway. Here, we report the morphological and immunohistochemical features of clinically or histologically malignant renal EAML.

Significance of CD133 expression in esophageal squamous cell carcinoma.

BACKGROUND: CD133 was recently reported to be a cancer stem cell marker and a prognostic marker for several tumors. However, few studies have investigated CD133 expression in esophageal squamous cell carcinoma (ESCC). Therefore, we examined whether CD133 could serve as a prognostic marker of ESCC and investigated the correlation between CD133 expression and the clinicopathological findings of ESCC patients and several markers. METHODS: We studied 86 ESCC patients who underwent curative surgery without neoadjuvant treatment at Tohoku University Hospital (Sendai, Japan) between January 2000 and December 2005. We analyzed tissue specimens by immunohistochemical staining for CD133, p53, p16, p27, murine double minute 2 (MDM2), Ki-67, and epidermal growth factor receptor (EGFR). RESULTS: Pathological tumor depth and tumor stage were significantly more advanced among CD133-negative patients than among CD133-positive patients. A log-rank test showed that CD133 immunoreactivity was significantly correlated with the overall survival of the patients (P = 0.049). However, multivariate analysis showed that it was not significantly correlated (P = 0.078). Moreover, CD133 was significantly positively correlated with p27 immunoreactivity (P = 0.0013) and tended to be positively correlated with p16 immunoreactivity (P = 0.057). In addition, p16 immunoreactivity was correlated with smoking history (P = 0.018), pathological lymph node status (P = 0.033), and lymphatic invasion (P = 0.018). CONCLUSIONS: This study indicated that CD133 immunoreactivity is a good predictor of prognosis in ESCC patients. In addition, CD133 may play a role in the regulation of tumor cell cycle through p27 and p16 in ESCC. At present, it thus remains controversial whether CD133 expression is a valid prognostic marker for ESCC. To elucidate this relationship, further investigations are required.

Development of a quantitative diagnostic method of estrogen receptor expression levels by immunohistochemistry using organic fluorescent material-assembled nanoparticles.

The detection of estrogen receptors (ERs) by immunohistochemistry (IHC) using 3,3'-diaminobenzidine (DAB) is slightly weak as a prognostic marker, but it is essential to the application of endocrine therapy, such as antiestrogen tamoxifen-based therapy. IHC using DAB is a poor quantitative method because horseradish peroxidase (HRP) activity depends on reaction time, temperature and substrate concentration. However, IHC using fluorescent material provides an effective method to quantitatively use IHC because the signal intensity is proportional to the intensity of the photon excitation energy. However, the high level of autofluorescence has impeded the development of quantitative IHC using fluorescence. We developed organic fluorescent material (tetramethylrhodamine)-assembled nanoparticles for IHC. Tissue autofluorescence is comparable to the fluorescence intensity of quantum dots, which are the most representative fluorescent nanoparticles. The fluorescent intensity of our novel nanoparticles was 10.2-fold greater than quantum dots, and they did not bind non-specifically to breast cancer tissues due to the polyethylene glycol chain that coated their surfaces. Therefore, the fluorescent intensity of our nanoparticles significantly exceeded autofluorescence, which produced a significantly higher signal-to-noise ratio on IHC-imaged cancer tissues than previous methods. Moreover, immunostaining data from our nanoparticle fluorescent IHC and IHC with DAB were compared in the same region of adjacent tissues sections to quantitatively examine the two methods. The results demonstrated that our nanoparticle staining analyzed a wide range of ER expression levels with higher accuracy and quantitative sensitivity than DAB staining. This enhancement in the diagnostic accuracy and sensitivity for ERs using our immunostaining method will improve the prediction of responses to therapies that target ERs and progesterone receptors that are induced by a downstream ER signal.

Vasohibin-1 as a potential predictor of aggressive behavior of ductal carcinoma in situ of the breast.

Vasohibin-1 is a recently identified negative feedback regulator of angiogenesis induced by VEGF-A and bFGF. In this study, we first evaluated mRNA expression of vasohibin-1 and CD31 in 39 Japanese female breast carcinoma specimens including 22 invasive ductal carcinoma (IDC) and 17 ductal carcinoma in situ (DCIS) using a real-time quantitative RT-PCR (QRT-PCR) with LightCycler system. In addition, we also immunolocalized vasohibin-1 and CD31 and compared their immunoreactivity to nuclear grades and histological grades of 100 carcinoma cases (50 IDC and 50 DCIS). There were no statistically significant differences of CD31 mRNA expression and the number of CD31 positive vessels between DCIS and IDC (P = 0.250 and P = 0.191, respectively), whereas there was a statistically significant difference in vasohibin-1 mRNA expression and the number of vasohibin-1 positive vessels in DCIS and IDC (P = 0.022 and P < or = 0.001, respectively). There was a significant positive correlation between vasohibin-1 mRNA level and Ki-67 labeling index in DCIS (r(2) = 0.293, P < or = 0.001). In addition, vasohibin-1 mRNA expression was correlated with high nuclear and histological grades in DCIS cases and a significant positive correlation was detected between the number of vasohibin-1 positive vessels and Ki-67 labeling index or nuclear grade or Van Nuys classification of carcinoma cells (P < or = 0.001, respectively). These results all indicate the possible correlation between aggressive biological features in DCIS including increased tumor cell proliferation and the status of neovascularization determined by vasohibin-1 immunoreactivity.

[Performance of a portable continuous infusion pump (SUREFUSER A) in continuous infusion of 5-FU].

Therapy with mFOLFOX6/FOLFIRI used in treating colorectal cancer is typical of the regimens performed in outpatient settings. In this therapy, 46-h continuous infusion of 5-fluorouracil (5-FU) with concomitant oxaliplatin and irinotecan hydrochloride is conducted. The portable continuous infusion pump that makes continuous infusion possible has a non-electric structure, so variation in the infusion rate is seen. There are known effects of 5-FU concentration and temperature, and many studies have reported on the precision. In our hospital, we have experienced many cases of incomplete infusion and delays for the above reasons. We changed the specifications of the infusion pump to correspond to the kinematic viscosity of 5-FU and made all drug solution amounts uniform. We measured the time required to administer the drug solution from the time the infusion was started (recorded by a nurse) and the time it was completed (recorded by the patient), and confirmed the precision of the pump after the changes were made. It was found that while there was a decrease in the infusion rate at which the effect of the kinematic viscosity of 5-FU is seen, the mean infusion time was kept to within 46+/-10% hours in more than 90% of patients. There were no effects from concentration differences in 5-FU, and the completion time was reduced. The management and lifestyles of individual patients are potential factors in precision errors, and it is important to explain in advance to patients the necessity of secure fixation and infusion pump problems that might occur.