Comparison between Ki67 labeling index determined using image analysis software with virtual slide system and that determined visually in breast cancer.

BACKGROUND: In recent papers, Ki67 labeling index (LI) has been used to classify breast cancer patients into the low and high Ki67LI groups for comparison studies, which showed significant differences in many prognostic factors. It has not been clarified whether image analysis software can be used for calculating LI in breast cancer. In our study, we examined whether Ki67LI in breast cancer calculated using image analysis software correlates with that measured on the basis of visual. METHODS: Fifty patients were randomly selected among breast cancer patients who underwent surgical operation from March, 2010 to May, 2010 in our hospital without preoperative chemotherapy. In this study, for the virtual slide system (VSS: VS120-L100, Olympus, Tokyo, Japan), the high-resolution VSs of all the 50 patients were prepared as samples. The image analysis software use for calculating LI was Tissuemorph Digital Pathology (Tissuemorph DP: Visiopharm, Hoersholm, Denmark). The calculated LI was extracted from 3 to 5 views containing hot spots. The LI calculated using Tissuemorph DP was designed as LI/image/T. The digital image of 3 to 5 LI/image/T views was printed out, and on the digital photograph, we counted visually the number of Ki67-immunopositive cells in exactly the same area, and the percentage of Ki67-immunopositive cells was designed as LI/direct. Moreover, a pathologist's assistant (PA) determined the tumor area in the same specimen using VSS and calculated LI using Tissuemorph DP, which was designed as LI/image/PA. The chief pathologist (CP) similarly calculated LI which was designed as LI/image/CP. We evaluated the degree of agreement between different data sets "LI/image/T and LI/direct" and "LI/image/T, LI/image/CP, and LI/image/PA" by using interclass correlation coefficient (ICC). RESULTS: The average counts of cells were as follows: LI/direct, 3209.7 ± 1970.4 (SD); LI/image/T, 2601.6 ± 1697.1; LI/image/PA, 2886.5 ± 2027.5; LI/image/CP, 18805.5 ± 22293.4. The values of LI/direct and LI/image/T showed almost perfect agreement as showed by an ICC of 0.885 (95 % CI, 0.806-0.933; p < 0.001). The agreement among three investigators was almost perfect. The obtained ICC was 0.825 (95 % CI, 0.739-0.890; p < 0.001) among the data of LI/image/T, LI/image/CP and LI/image/PA. There were five cases that immunopositivity for Ki67 showed a more than 10 % disagreement between LI/direct and LI/image/T. CONCLUSION: The merits of calculating Ki67 LI using Tissuemorph DP are as follows. First, the staining intensity of the cells to be counted can be adjusted. Second, the portion of a tumor including "hot spots" for counting can be chosen. Third, many cancer cells can be counted more rapidly using Tissuemorph DP than by visual observation. However, it is important that pathologist should check and carry out the final decision of the data, when Ki67 LI using Tissuemorph DP is calculated.

Characteristics of the Japanese histological classification for breast cancer: correlations with imaging and cytology.

The Japanese histological classification of breast cancer is a unique classification that was published as the 2nd edition of the General Rules for Clinical and Pathological Recording of Breast Cancer in 1971. According to this classification, breast cancers are divided into three subgroups: noninvasive, invasive, and Paget's disease. Invasive carcinomas are further divided into two groups: invasive ductal carcinomas (IDCs) and special types. IDCs are the most common types, representing 70-80 % of all breast cancers, and most of them correspond to invasive carcinoma-not special type-according to the latest WHO classification (4th edition). In particular, IDCs are subdivided into three subgroups as follows: (1) papillotubular carcinoma, which is characterized by the projection of papillae into spaces, and includes cribriform and comedo patterns; (2) solid-tubular carcinoma, which is a solid cluster of cancer cells with expansive growths that form relatively sharp borders; and (3) scirrhous carcinoma, which grows in a scirrhous manner characterized by cancer nests or cells accompanied by marked fibrosis. The concept of subclassification originated in Japan. The Japanese histological classification has taken on important roles since its publication, in particular for comparisons between histology and imaging or cytology.

Oncologic outcomes and technical considerations of nipple-sparing mastectomies in breast cancer: experience of 425 cases from a single institution.

BACKGROUND: Nipple-sparing mastectomy (NSM) is an advantageous treatment option, providing a complete cure and good cosmetic results. We tested whether NSM is a surgically and oncologically safe technique. METHODS: We evaluated the oncological outcome of 425 breasts in 413 patients who underwent NSM between January 2000 and March 2013. We retrospectively reviewed patient data and analyzed all patient characteristics as potential risk factors of recurrence at the nipple-areola complex (NAC). To confirm the oncological safety of NSM, we compared outcomes of NSM and conventional total mastectomy. RESULTS: The median follow-up time after surgery was 46.8 months (range 6-158 months). Nipple necrosis was observed in 6 cases (1.4 %). The cumulative local recurrence rate after NSM was 5.8 % (25/425 cases), similar to that of conventional total mastectomy in the same period (5.6 %, 49/878 cases). Furthermore, the cumulative local recurrence rate at the NAC was 2.3 % (10 cases). HER2-enriched tumors and young age (<40 years) were significant risk factors for recurrence at the NAC. In patients with recurrence, the site of recurrence was easily excised, and good cosmetic results were achieved in breast reconstruction cases. CONCLUSION: NSM is safe with a low complication rate. No significant difference was observed in cumulative local recurrence rate, cumulative distant disease recurrence rate, and overall survival between patients who underwent NSM or conventional total mastectomy, confirming that NSM was surgically and oncologically safe.

Effects of 5-aminolevulinic acid on a murine model of diet-induced obesity.

The effects of 5-aminolevulinic acid (5-ALA) on obesity were investigated using a murine model (diet-induced obese mice). Diet-induced obese mice were divided into 4 groups: a control group (C group), which was fed a high-fat diet; a low-5-ALA dose (10 mg/kg/day) group (10A group); a moderate-5-ALA dose (30 mg/kg/day) group (30A group); and a high-5-ALA dose (100 mg/kg/day) group (100A group). 5-ALA was administered by mixing the high fat diet for 8 weeks. Body weight increases in the 30A and 100A groups were significantly smaller compared with those of the C group. Body fat measurements by X-ray computed tomography indicated that the 100A group showed a tendency toward low visceral fat quantities during the final week of the study. Visceral fat weights in the 30A and 100A groups were slightly low. The levels of serum alanine aminotransferase (ALT) and total cholesterol (TC) in the 10A group was slightly low, whereas the 30A and 100A groups showed significantly lower ALT and TC values. Liver lipid concentration showed a dose-dependent decrease with ALA. Thus, in this diet-induced obese murine model, administration of 5-ALA had a significantly beneficial impact on the visceral fat, serum ALT and TC, and liver lipid concentration.

Positive predictive value for malignancy of pure flat epithelial atypia diagnosis by percutaneous needle biopsy of the breast: management of FEA in ultrasonography.

BACKGROUND: Some reports suggest that the rate of definitive diagnosis of malignant tumors, namely, the final diagnosis being revised to a higher stage, in patients diagnosed as having flat epithelial atypia (FEA) by percutaneous needle biopsy of the breast (PNB) is as low as 0-3 %. However, other reports suggest that the rate is as high as 10 % or more, bringing confusion on this issue. We examined the positive predictive value for malignancy in the patients diagnosed as having pure FEA and the patients' radiolopathological characteristics observed in our hospital. METHODS: Of the patients who underwent PNB in our facility, those who were diagnosed as having pure FEA were recruited as the subjects of this study. RESULTS: Of the 4,197 consecutive patients who underwent PNB, 44 (1.0 %) were diagnosed as having pure FEA following a re-examination. Among 44 cases, 39 cases were selected as the subjects of this study. Among the 39 patients, six patients were diagnosed as having malignant lesions, two of whom had invasive carcinoma of no special type (papillotubular type), one had tubular carcinoma, one had ductal carcinoma in situ (DCIS) of high nuclear grade, one had DCIS of intermediate nuclear grade, and one had DCIS of low nuclear grade. The diameters of 6 malignant lesions were 10-30 mm at ultrasonography (US) examination. Five of the 39 patients had contralateral breast cancer. CONCLUSION: The positive predictive value for malignancy of pure FEA was 15.7 %. The patients with pure FEA may make a follow up without an excisional biopsy when the lesion sizes less than 10 mm on US examination.

Type 2 diabetic conditions in Otsuka Long-Evans Tokushima Fatty rats are ameliorated by 5-aminolevulinic acid.

A precursor of protoporphyrin IX, 5-aminolevulinic acid (5-ALA) is used as a prodrug for photodiagnosis and photodynamic therapy. Recently, it has been shown that 5-ALA reduces glucose levels during fasting and after glucose loading in prediabetic subjects. We hypothesized that 5-ALA ameliorates diabetic conditions through mitochondrial changes in visceral adipose tissue. In order to explore the metabolic effects on the type 2 diabetic state, we administered ALA hydrochloride in combination with sodium ferrous citrate to Otsuka Long-Evans Tokushima Fatty (OLETF) rats at intragastric doses of 20 and 300 mg kg(-1) d(-1) for 6 weeks. The administration of 300 mg kg(-1) d(-1) of 5-ALA improved glucose intolerance, hypertriglyceridemia, and hyperleptinemia in OLETF rats more effectively than the administration of an equivalent dose of metformin, in accordance with reductions in food intake and body weight. Furthermore, the weight of the retroperitoneal fat tended to decrease and cellular mitochondrial content of the fat was markedly reduced by the 5-ALA administration, showing a positive correlation. These results suggest that 5-ALA ameliorates diabetic abnormalities in OLETF rats by reducing the visceral fat mass and mitochondrial content of adipocytes in a site-specific manner.

Aberrant DNA methylation status of DNA repair genes in breast cancer treated with neoadjuvant chemotherapy.

Dysregulation of homologous recombination (HR) DNA repair has been implicated in breast carcinogenesis and chemosensitivity. Here, we investigated the methylation status of sixteen HR genes and analyzed their association with tumor subtypes and responses to neoadjuvant chemotherapy. Core specimens were obtained before neoadjuvant chemotherapy from sixty cases of primary breast cancer of the following four subgroups: luminal breast cancer (LBC) with pathological complete response (pCR), LBC with stable disease, triple-negative breast cancer (TNBC) with pCR and TNBC with poor response. The aberrant DNA methylation status of the following HR related-genes was analyzed using bisulfite-pyrosequencing: BRCA1, BRCA2, BARD1, MDC1, RNF8, RNF168, UBC13, ABRA1, PALB2, RAD50, RAD51, RAD51C, MRE11, NBS1, CtIP and ATM. Among the genes analyzed, only the incidence of BRCA1 and RNF8 methylation was significantly higher in TNBC than that in LBC. Whereas the incidence of BRCA1 methylation was tended to be higher in pCR cases than in poor-response cases in TNBC, that of RNF8 was significantly lower in pCR cases than in poor-response cases. Our results indicate that the methylation status of HR genes was not generally associated with TNBC subtype or chemosensitivity although hypermethylation of BRCA1 is associated with TNBC subtype and may impact chemosensitivity.

The effect of 5-aminolevulinic acid on cytochrome c oxidase activity in mouse liver.

BACKGROUND: 5-Aminolevulinic acid (ALA) is a precursor of heme that is fundamentally important in aerobic energy metabolism. Among the enzymes involved in aerobic energy metabolism, cytochrome c oxidase (COX) is crucial. In this study, the effect of ALA on cytochrome c oxidase activity was measured. FINDINGS: c57BL/6N species of mice were administered ALA orally for 15 weeks. After ALA administration, mice were sacrificed and livers were obtained. COX activity in mitochondria from ALA-administered mouse livers was 1.5-fold higher than that in mitochondria from PBS-administered mouse livers (P < 0.05). Furthermore, ATP levels in ALA-administered mouse livers were much higher than those in PBS-administered mouse livers. These data suggest that oral administration of ALA promotes aerobic energy metabolism, especially COX activity. CONCLUSIONS: This is the first report of a drug that functions in aerobic energy metabolism directly. Since COX activity is decreased in various diseases and aging, the pharmacological effects of ALA will be expanding.

[Influence of the supporting mechanism of removable partial denture on the stomatognathic system].

The connecting rigidity of the retainer and the denture supporting area may influence the distribution of the occlusal load. Three types of retainers (wrought wire clasp, Akers cast clasp and conical crown telescopic retainer) and three outline forms of denture bases designed for distal extension removable partial dentures were assessed in six patients with Kennedy Class II situations. To evaluate the influence of oral function, mandibular displacement, abutment tooth displacement, and denture base exerted pressure were measured simultaneously during maximum voluntary clenching. The mandibular displacement increased when the connecting rigidity decreased or the size of the denture base was reduced. The abutment tooth displacement increased as the denture base was reduced with the Akers clasp. The denture base pressure increased as the denture base reduced with the wrought wire clasp. The conical crown telescopic retainer did not cause overloading to the supporting tissues even when the denture base was reduced. It was also revealed that the abutment tooth tended to displace distally when a distal occlusal rest was prepared on the abutment tooth.

Point-of-care Helicobacter pylori urine antibody detection in a multi-ethnic adult population in the United States.

A need exists for accurate point-of-care tests for diagnosis of Helicobacter pylori (H. pylori) infection to evaluate a rapid urine-H. pylori antibody test device for detection of H. pylori infection in a point-of-care setting in the United States. A multi-center study in a multi-ethnic population compared the RAPIRUN urine antibody test with the (13)C-urea breath test (C-UBT) and a traditional serologic test, the high-molecular-weight cell-associated protein enzyme immunoassay (HM-CAP EIA). The primary comparator was with "definite positive" and "definite negative" patients defined as a concordance of combined results of the UBT and the HM-CAP IgG EIA. Overall, 188 eligible patients were enrolled (61 men, age range: 18-73 years, including 84 Hispanics, 73 Asian-Pacific Americans, 22 Black African-Americans, 6 non-Hispanic Caucasians, and 3 of "other" ethnicity). Compared with "definite positive" and "definite negative" results, the sensitivity and specificity of the urine antibody test were 0.9 and 1.0, respectively. The urine antibody test proved suitable for point-of-care rapid diagnosis of anti-H. pylori antibodies indicative of active or past H. pylori infection.

An ex vivo method for rapid generation of monoclonal antibodies (ADLib system).

Here, we describe a protocol for using the ADLib (Autonomously Diversifying Library) system to rapidly generate specific monoclonal antibodies using DT40, a chicken B-cell line that undergoes constitutive gene conversion at both light- and heavy-chain immunoglobulin loci. We previously developed the ADLib system on the basis of our finding that gene conversion in DT40 cells was enhanced by treatment of the cells with a histone deacetylase inhibitor, trichostatin A (TSA). TSA treatment evolves a diversified library of DT40 cells (ADLib), in which each cell has different surface IgM specificity. Antigen-specific DT40 cells are selected from ADLib using antigen-conjugated magnetic beads, and their specificity can be examined by various immunological assays, using culture supernatant containing secreted IgM. The whole process from selection to screening can be completed in about 1 week. Thus, the ADLib system will accelerate biological studies, including drug discovery and design.

Improved infrared spectrophotometer for point-of-care patient 13C-urea breath testing in the primary care setting.

UNLABELLED: The 13C-urea breath test provides non-invasive testing for Helicobacter pylori infection with the possibility of analysis at the point of care. Point of care tests require accurate and efficient desktop instrumentation. OBJECTIVE: To compare results obtained from a new infrared spectrophotometer (10 kg, 2 min sample measurement) to the previously approved UBiT-IR300 (22.5 kg, 5-6 min sample measurement). DESIGN AND METHODS: Adults underwent urea breath testing; breath samples were analyzed using the new and the predicate instruments. RESULTS: There were 220 adults enrolled (age range 18-74 years; M/F = 35:65); with 86 UBT positive and 134 negative cases. The overall agreement between instruments was 99.6% (95% C.I. = 97.6 to 99.9); the positive agreement was 100%. CONCLUSION: Technical performance of the new instrument was excellent.

Translocation of Bim to the endoplasmic reticulum (ER) mediates ER stress signaling for activation of caspase-12 during ER stress-induced apoptosis.

Endoplasmic reticulum (ER) stress activates caspase-12 in murine cells, triggering the ER stress-specific cascade for implementation of apoptosis. In C2C12 murine myoblast cells, activation of the cascade occurs without release of cytochrome c from mitochondria, suggesting that the cascade is independent of mitochondrial damage. Stable overexpression of Bcl-xL in C2C12 cells suppressed activation of caspase-12 and apoptosis. In ER-stressed cells, but not in normal cells, Bcl-xL was co-immunoprecipitated with Bim, a pro-apoptotic member of the Bcl-2 family, suggesting that Bcl-xL sequesters Bim, thereby inhibiting the apoptotic signaling. Fractionation of C2C12 cells revealed that ER stress led to translocation of Bim from a dynein-rich compartment to the ER, while stable overexpression of Bcl-xL suppressed accumulation of Bim on the ER. Although the toxic effect of Bim had been previously observed only at the mitochondrial outer membrane, overexpression of a Bim derivative, Bim(ER), targeted at the surface of the ER led to apoptosis. A C2C12 transfectant overexpressing the caspase-12 suppressor protein was resistant to Bim(ER), suggesting that the toxic effect of Bim on the ER is dependent on activation of caspase-12. Knockdown of Bim by RNA interference provided cells resistant to ER stress. These results suggest that translocation of Bim to the ER in response to ER stress is an important step toward activation of caspase-12 and initiation of the ER stress-specific caspase cascade.

Functional development of oligodendrocytes and open-field behavior in developing rats: an approach using monoclonal antibody to immature oligodendrocytes.

To examine the relation between functional development of oligodendrocytes and open-field behavior during the postnatal period, a mouse monoclonal antibody termed 14F7, which predominantly labels stage-specific immature oligodendrocytes, was employed. Antibody 14F7 was administered intraperitoneally into male pups on day 3 and 4 after birth. The open-field test was performed on days 12 and 18 of the postnatal period. Horizontal activity increased remarkably with the growth of pups. On day 18, horizontal activity in the group with 14F7 was significantly higher than the control, while there was no significant difference between treatments on day 12. In contrast to the horizontal activity, the frequency of hind leg rearing, vertical activity, in the group with 14F7 was significantly lower than that in the control. On day 12, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities in the cerebral cortex were similar between the groups. These activities increased with the growth of pups in both groups. In the 14F7 group on day 18, ChAT activity was the same as the control, whereas AChE activity was significantly lower compared with the control. These results suggest that neonatal exposure to 14F7 induces abnormal neurotransmission by reducing the degradation of acetylcholine and alters the spontaneous activities in developing rats.

Effects of tongue and oral mucosa cleaning on oral Candida species and production of volatile sulfur compounds in the elderly in a nursing home.

The purpose of this study was to investigate the effects of oral care using simple tools and methods on the cleanliness of the oral cavity in the elderly. Enrolled were 84 elderly subjects with a mean (+/-S.D) age of 85.1+/-7.0 years in a nursing home. They were given tongue and oral mucosa cleaning (the oral care) after lunch every day or every other day for two consecutive weeks by the authors. The effect of the oral care was studied in terms of Candida scores in tongue coating, concentration of volatile sulfur compounds (VSC) which are the main causative substance of bad breath, and change in tongue coating scores. The above parameters were measured five times; just before the oral care program, weekly during, and at the end of the oral care program. The groups of patients, who were given the oral care, especially the group of patients cared with sponge brushes every day, showed a significant reduction in Candida scores but not in VSC concentration and tongue coating scores. The present method of oral care proved effective in cleaning the tongue and oral mucosa, and the Candida scores appeared to be a reliable indicator for evaluation. It is suggested that this way of oral care is simple, easy and useful not only for the elderly at a nursing home but for the house-bound elder people who will rapidly increase in the near future in Japan.