Real-time visualization of intratumoral necrosis using split-luciferase reconstitution by protein trans-splicing.

Necrosis, a form of cell death, occurs not only with the development of various diseases but also with a tumor tissue response to cancer treatment. Therefore, pursuing progress for cancer therapy through induction of necrosis may be one of the most effective approaches for cancer eradication. We herein describe the development of a real-time imaging system to visualize intratumoral necrosis. The system is composed of two types of cells expressing either one of two necrosis imaging reporters that consist of a DnaE intein sequence linking to one of two split-luciferase fragments. When necrosis occurs in a tumor composed of both of the cells, the two types of leaked reporters can reconstitute the enzymatic activity as a result of protein trans-splicing and thereby emit bioluminescence in the presence of the substrate. This system, which was constructed with shrimp-derived luciferase, allowed in vitro imaging of necrosis. We further confirmed real-time imaging of intratumoral necrosis caused by physical or chemical tissue disruption, validating its application in in vivo necrosis imaging. Thus, the constructed imaging system could be a powerful tool for the optimization of the therapeutic condition for cancer therapy and for the evaluation of novel anticancer drugs targeting necrosis.

Reduction of fatty liver in rats by nicotinamide via the regeneration of the methionine cycle and the inhibition of aldehyde oxidase.

Nonalcoholic fatty liver disease, which has been rapidly increasing in the world in recent years, is roughly classified into nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis. This study was based on our previous reports that stated that the combination treatment of N1-methylnicotinamide (MNA) and hydralazine (HYD) improves fatty liver in NAFL model rats. This finding was attributed to the MNA metabolism inhibition by HYD, which is a strong inhibitor of aldehyde oxidase (AO); this results in an increase in hepatic MNA and improved fatty liver. We hypothesized that orally administered nicotinamide (NAM), which is the precursor of MNA and is a form of niacin, would be efficiently metabolized by nicotinamide N-methyltransferase in the presence of exogenous S-adenosylmethionine (SAM) in NAFL rats. To address this issue, NAFL model rats were orally administered with NAM, SAM, and/or HYD. As a result, liver triglyceride (TG) and lipid droplet levels were barely altered by the administration of NAM, SAM, NAM+SAM, or NAM+HYD. By contrast, the triple combination of NAM+SAM+HYD significantly reduced hepatic TG and lipid droplet levels and significantly increased hepatic MNA levels. These findings indicated that the combination of exogenous SAM with AO inhibitors, such as HYD, has beneficial effects for improving fatty liver with NAM.

Comparative image analysis of conventional and thin-layer preparations in endometrial cytology.

We evaluated the differences in cytologic findings between conventional and thin-layer preparations in endometrial cytology to introduce the thin-layer method into routine cytology. Eighty patients who had undergone endometrial cytology and biopsy on the same day were selected and we compared the cytological findings between conventional- and thin-layer preparations (TLP) in endometrial cytology. The numbers of neutrophils and cell clusters in the thin-layer method were lower than those in the conventional smear (CSS) method. The average number of neutrophils in endometrioid adenocarcinoma was significantly higher than that in normal morphology endometrium and endometrial hyperplasia. Regarding the shape of the cell clusters, ball-like patterns and round-edged cell clusters were not identified in CSS. The average number of clusters in CSS was significantly greater than that using the TLP. The average of the nuclear area in CSS was significantly larger than that using the TLP, indicating that the nuclear areas in CSS were more uneven than that using the TLP. In the future, it is expected that liquid-based cytology will be applied to the cytological diagnosis of a variety of lesions. The influence on cells due to fixation is considerable in liquid-based preparations. Therefore, if we strive to pick up the differences between CSS and TLP of endometrial samples, the diagnostic accuracy of the latter could be improved.

Cytological significance of abnormal squamous cells in urinary cytology.

The aim of this study was to evaluate the significance of abnormal squamous cells (ASCs) in urinary cytology to clarify whether finding of ASCs could improve diagnostic accuracy. A total of 3,812 urine specimens were reviewed. We focused on three parameters of ASCs, necrotic debris, and ASC clusters, and linked them to histological diagnosis and clinical information. ASCs were identified in 34 (0.9%) specimens from 21 different patients. The incidence of ASCs was higher in females than in males. The 34 urine specimens were categorized as voided urine (16 cases), bladder-catheterized urine (17 cases), and bladder-washed fluid (1 case). Six (28.6%) of 21 patients were histologically diagnosed as having combined urothelial carcinoma and squamous cell carcinoma (SCC). Eight patients (38.1%) were histologically diagnosed as having SCC originating from sites other than the urinary tract; those urine specimens showed ASCs that were likely to have been exfoliated from malignant lesions. Necrotic debris and ASC clusters were identified in 12 specimens (35.3%) from 11 patients and 4 specimens (11.8%) from 4 patients, respectively, from a total of 34 specimens. Our results indicate that a great amount of care is needed for cytological diagnosis when attempting to recognize ASCs in urine specimens because ASCs were identified in not only SCC of the bladder but also in carcinoma or nonmalignant lesions of nonurinary tracts. Necrotic debris was found not only in patients who had malignant bladder tumors but also in those who had malignant lesions in locations other than the bladder.

Immunohistochemical, molecular, and clinicopathological analyses of urothelial carcinoma, micropapillary variant.

The prognosis of urothelial carcinoma, micropapillary variant (MPV), of the bladder has been shown to be worse than that of the conventional urothelial carcinoma (UC). However, it remains to be clarified why the MPV is more aggressive. We therefore here focused on the correlation between clinical features and histological, immunohistochemical and molecular findings for eight MPV and 35 UC, evaluating expression of MUC1, Ki-67, p53, CD147, CD34, D2-40, and extracellular matrix proteins. The Ki-67 labeling index was significantly higher in UC than in MPV but densities of venous and lymphatic tumor emboli were significantly higher in the MPV cases and lymph node metastasis was more frequent, with a poorer prognosis. Tenascin-C and fibronectin also showed significantly greater expression in MPV than in UC at the epithelial-mesenchymal interfaces. Direct sequencing showed point mutations of KRAS exon 1 in three MPV with significantly more frequency compared to UC. Occupation rate of the MPV area in the tumor showed significant inverse correlation with overall survival. Thus our histopathological findings provide clues to explaining why prognosis is poorer in the MPV than UC.

Clinical significance of human papillomavirus genotype by linear array assay in Japanese women with uterine cervical lesions and type 16 physical status by in situ hybridization.

The aim of this study was to clarify the relation of human papillomavirus (HPV) genotypes and physical status in the cervical neoplasm of Japanese patients with the grade of the disease. Human papillomavirus genotype was detected using a linear array genotyping assay. Human papillomavirus status, diffuse or punctate signal pattern, was studied by biotynyl-tyramide-based in situ hybridization for positive cases of HPV-16. Human papillomavirus types 16, 52, 58, and 31, in descending order of frequency, were prevalent. The rates of HPV infection in patients with cervical intraepithelial neoplasia (CIN) or squamous cell carcinoma (SCC) were significantly higher than those in patients without cervical lesions. The frequency of HPV single infection in SCC was higher than that in CIN1 or CIN2. In an unspecified-risk HPV, types 66 and 70 were found in SCC and 62, 71, and 82 were detected in CIN3. The diffuse pattern was more frequent in CIN, and the punctate pattern was more frequent in SCC. Human papillomavirus types 16, 52, 58, and 31 were frequently detected in Japanese women with cervical neoplasias, and several unspecified-risk HPVs might be high-risk types. A single infection of HPV and a punctate signal pattern seemed to be closely correlated with cervical carcinogenesis.

Liquid-based preparation for endometrial cytology--usefulness for predicting the prognosis of endometrial carcinoma preoperatively.

BACKGROUND: The authors evaluated the applicability and usefulness of immunocytochemical staining for cyclin A, p53, estrogen receptor alpha (ER-alpha), and progesterone receptor B (PR-B) as a preoperative prognostic indicators for endometrial carcinoma using endometrial cytology with the liquid-based cytology (LBC) method. METHODS: Cytologic specimens from 44 patients who had endometrial carcinoma were prepared with the LBC method. The results of immunocytochemical and immunohistochemical staining for cyclin A, p53, ER-alpha, and PR-B were compared with clinicopathologic parameters and prognosis. RESULTS: Patients who had positive results for cyclin A and p53 and negative results for ER-alpha and PR-B appeared to have unfavorable clinicopathologic characteristics, such as high-grade histology, advanced clinical stage, lymphovascular space involvement (LVSI), and deeper myometrial invasion (MI), and had a poor prognosis. In contrast, patients who had positive results for ER-alpha and PR-B, and negative results for cyclin A and p53 had favorable characteristics, such well differentiated tumor, early clinical stage, negative LVSI, and less MI, and had a good prognosis. Immunostaining results from cytologic specimens obtained in the clinic and at surgery and from histologic specimens obtained at surgery were correlated positively. CONCLUSIONS: Consistent specimens that were prepared using the LBC method facilitated multiple immunocytochemical analyses. Endometrial cytology with the LBC method was useful for predicting the prognosis of patients with endometrial carcinoma before therapy.

[Educational program in the Medical Science Course, Kitasato University School of Allied Health Sciences].

The aim of education in the Medical Laboratory Science course, Kitasato University School of Allied Health Sciences, is to bring up train students who have Kitasato spirit, for careers in laboratory medicine of hospital or scientific staff of medical companies or as researchers. General and enlightening education concerning "Kitasato spirit" and professional education composed of major subjects was carried out in the first and during the 2nd and two third of 3rd grade, respectively. Medical practice and research training were alternatively carried out for 6 months between November of the 3rd year and November of the 4th year, in order to gain practical experience. Two problem-based learning (PBL) tutorial courses, "Infectious Diseases Course" and "Team Medical Care--Interprofessional Collaborations" were also carried out at the end of the 3rd and beginning of the 4th years, respectively, in order to convert a memory to knowledge. Team medical care course enrolls 1000 students at the School of Allied Health Sciences, Medicine, Nursing, Pharmacy and Kitasato College Applied Clinical Dietetics Course, is now one of special courses available at our university. This attempt is thought to result in a way of thinking that recognizes the importance of co-operation as a team member and personal contributions to actual team medical care.

Basement membrane-like substance in cytologic diagnosis in clear cell adenocarcinoma of the minor salivary gland of the palate. A case report.

BACKGROUND: Clear cell adenocarcinoma (CCA) of the minor salivary gland accounts for < 1% of all tumors of the salivary gland. CASE: A 32-year-old woman with a history of papillary carcinoma of the thyroid 1 year earlier complained of pain on the left side of the neck. After a detailed examination, the patient underwent the resection of a tumor located at the palate. Imprint cytology of the tumor revealed cohesive tumor cells of uniform size containing an abundant clear cytoplasm and round nuclei with extra but fine granular chromatin and conspicuous nucleoli. A basement membrane-like substance (BMS) was stained in light green with Papanicolaou staining and was positive for laminin with immunohistochemical staining. Histopathologic analysis confirmed the trabecular or nest-like arrangement of the cells with the clear cytoplasm and BMS substance surrounded by tumor cells, which were positive for laminin and AE1 immunohistochemically. CONCLUSION: Although CCA of the palate is extremely rare, an accurate cytologic diagnosis can be made if the characteristic findings of CCA, including BMS, are imaged.

Intractable wounds caused by calcific uremic arteriolopathy treated with bisphosphonates.

BACKGROUND: Calcific uremic arteriolopathy (calciphylaxis) is a calcification syndrome that predominantly affects relatively small vessels and is a life-threatening entity usually seen in patients with end-stage renal disease. Intractable skin necrosis sometimes causes lethal sepsis because it progresses rapidly as a result of mechanical stress. OBJECTIVE: We sought to investigate the efficacy of etidronate disodium (bisphosphonates) in treating intractable ulcers occurred in a patient on hemodialysis accompanied with calcific uremic arteriolopathy. METHODS AND RESULTS: A 53-year-old patient receiving hemodialysis with chronic renal failure accompanied with calciphylaxis had bilateral leg ulcers caused by minor trauma. The aggressive debridement worsened his skin condition as is usually seen in pyoderma gangrenosum. It eventually healed by lowering calcium-phosphorus levels with the administration of bisphosphonates and with the continuous use of sevelamer hydrochloride. LIMITATIONS: This study reporting a single case limits the interpretation of results. CONCLUSION: Bisphosphonates may be effective in treating calciphylaxis and arteriosclerosis obliterans by reducing the formation of ectopic calcification around blood vessels.

Significance of matrix metalloproteinase-7 [correction of matrix metalloproteinase-2], -11 and tissue inhibitor of metalloproteinase-1 expression in normal, hyperplastic and neoplastic endometrium.

BACKGROUND: Matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) are key factors in the degradation of extracellular matrix and basement membranes. This study aimed to examine the expressions of MMP-7 and -11 and TIMP-1 in normal, hyperplastic and neoplastic endometrium and their correlation to clinicopathologic factors. PATIENTS AND METHODS: Tissue samples of 40 normal endometria, 20 endometrial hyperplasias and 120 endometrial endometrioid adenocarcinomas were used for the study. Immunohistochemical staining for MMP-7 and -11 and TIMP-1 protein was performed on formalin-fixed and paraffin-embedded tissue samples. These expressions were represented as incidence of expression. RESULTS: MMP-7 was highly expressed in the glands of the basal and functional layers during the proliferative and menstrual phases. MMP-11 expression in the gland of the basal layer and the stroma of the functional layer fluctuated during the menstrual cycle. TIMP-1 was highly expressed in the late secretory and menstrual phases. MMP-7 was expressed at significantly higher levels in endometrial hyperplasia than normal endometrium, whereas MMP-11 was expressed at lower levels. In endometrial adenocarcinoma, MMP-7, MMP-11 and TIMP-1 were expressed at the same levels as in hyperplasia. MMP-7 expression in endometrial carcinoma was correlated with myometrial invasion and estrogen receptor expression. The expression of MMP-7 in the adjacent stroma was associated with a poor prognosis. CONCLUSION: MMP-7, MMP-11 and TIMP-1 expression may be regulated by the menstrual cycle, and related to the degradation and remodeling of the normal endometrium. MMP-7 expression might be a prognostic factor in endometrial carcinoma.

High expression of skp2 correlates with poor prognosis in endometrial endometrioid adenocarcinoma.

PURPOSE: Skp2 interacts with the degradation of cyclin-dependent kinase inhibitor p27. This study aimed to investigate the correlation of skp2 expression with the expression of p27 and other cell cycle regulators, and clinicopathological parameters in endometrial endometrioid adenocarcinoma. METHODS: Tissue samples of 136 endometrioid adenocarcinomas, in addition to 20 endometrial hyperplasias and 20 normal endometria, were immunohistochemically stained for skp2. The expression was represented as a labeling index (LI), which indicates the percentage of positive nuclei. RESULTS: Skp2 staining was localized in the nuclei of the glandular cells of the proliferative phase endometrium, and endometrial hyperplasia and carcinoma cells. Skp2 expression was increased significantly in those of higher histological grade. The high level of skp2 expression was significantly correlated with the presence of lymph node metastasis and lymph-vascular space involvement. The LI of skp2 in endometrial carcinoma was significantly correlated with that of p27, Ki-67, cdk2, cyclin A, cyclin D1, cyclin E, p53 and PTEN. The high level of skp2 expression (LI> or =20%) was significantly correlated with the patients' poor survival. CONCLUSIONS: The skp2 level might have increased due to p27 accumulation and may be a good indicator of proliferative activity and poor prognosis.

Cytologic scoring of endometrioid adenocarcinoma of the endometrium.

BACKGROUND: Endometrial carcinoma is one of the most frequent malignancies in the female genital tract, and its incidence has been increasing in Japan. Histologic grade is an important factor for organizing treatment strategies, including hormone therapy, and for predicting the prognosis of the patient. The objective of this study was to evaluate the applicability and usefulness of cytologic scoring in assessing the morphologic differentiation of endometrioid adenocarcinomas of the endometrium using endometrial smears. METHODS: Sixty-four endometrial cytologic samples of endometrioid adenocarcinomas of the endometrium were used in this study. All patients underwent endometrial cytology before hysterectomy, and the diagnosis was confirmed by histologic examination of the extirpated uterus. Each cytologic specimen was scored according to a scoring system established by the authors. The cytologic grade based on those estimated scores was compared with the histologic grade and clinicopathologic parameters, respectively. RESULTS: The cytologic grade (CG) was correlated positively with the histologic grade. A high cytologic score was correlated with p53 mutation and myometrial invasion and was correlated negatively with estrogen receptor and progesterone receptor status. The concordance rates of cytologic grade with well differentiated (Grade 1), moderately differentiated (Grade 2), and poorly differentiated (Grade 3) histologic grades were 83.3% (35 of 42 tumors), 9.1% (1 of 11 tumors), and 100% (11 of 11 tumors), respectively. The total concordance rate was 73.4% (47 of 64 tumors). The best cut-off value for distinguishing histologic Grade 1 from the others was a cytologic score of 17, representing a sensitivity of 83% and a specificity of 81%. For distinguishing histologic Grade 3 from the others, the best cut-off value was a cytologic score of 20, representing a sensitivity of 100% and a specificity of 83%. CONCLUSIONS: The cytologic scoring system studied for endometrioid adenocarcinoma was useful for predicting histologic grade and tumor malignant potential.

Expression of tumor suppressor gene product p14ARF in endometrioid adenocarcinoma of the uterine corpus.

p14 activates p53 by inhibiting MDM2 expression and arrests the cell cycle in G1 and G2/M. Abnormal p14 expression has been reported in various human cancers. This study investigated p14 expression in endometrioid adenocarcinoma of the uterine corpus in an attempt to clarify its correlation with other cell cycle-regulators and clinicopathologic parameters. The specimen studied consisted of 124 endometrioid adenocarcinomas, 20 normal endometria, and 20 endometrial hyperplasias. Immunohistochemical staining of formalin-fixed and paraffin-embedded tissues was performed using a Catalyzed Signal Amplification System. Cells with >5% positive staining were classified as positive for p14. A staining score of 1 was adopted when the percentage of positive nuclei was <5%, a score of 2 when it was 5 to 50%, and a score of 3 when it was >50%. In normal endometrium, the frequency of positive staining in the proliferative phase and secretory phase was 50% (4/8) and 58.3% (7/12), with staining scores of 1.8+/-0.9 and 1.6+/-0.5, respectively. The frequency of staining in simple hyperplasia (SH), complex hyperplasia (CH), and complex atypical hyperplasia (CAH) was 88.9% (8/9), 25% (1/4), and 42.9% (3/7), respectively; the staining scores were 1.9+/-0.3, 1.3+/-0.5, and 1.4+/-0.5, respectively. Among endometrioid adenocarcinomas, the frequency of staining of well-differentiated (G1), moderately differentiated (G2), and poorly differentiated (G3) adenocarcinomas was 69% (49/71), 64% (16/25), and 42.9% (12/28) respectively, with staining scores of 2.1+/-0.8, 2+/-0.9, and 1.8+/-1, respectively. Thus expression levels of p14 were higher in G1 tumors than in normal endometria or endometrial hyperplasias, and the frequency of its staining in endometrioid carcinomas was inversely correlated with histologic grade. The staining score for endometrioid adenocarcinomas also was inversely correlated with the labeling index (LI) of Ki-67, but not with that of cyclin A, cyclin D1, cyclin E, cdk2, p27, p53, or other clinicopathologic parameters. In conclusion, p14 expression correlated with histologic grade and Ki-67, but not other prognostic factors in endometrioid adenocarcinoma. Long-term follow-up studies are needed to analyze the significance of p14 expression in these tumors.