Urinary WT1-positive cells as a non-invasive biomarker of crescent formation.

OBJECTIVE: The purpose of this study was to assess the relationship between urinary WT1-positive cells (podocytes and active parietal epithelial cells) and WT1-positive cells in renal biopsy to investigate whether urinary WT1-positive cells are useful for detection of crescent formation. METHODS: Fifty-two patients with kidney disease were investigated (15 cases with crescentic lesions and 37 cases with non-crescentic lesions) for immunoenzyme staining using anti-WT1 antibody for urine cytology and renal biopsy. Numbers of WT1-positive cells in urine and renal biopsy were counted. RESULTS: There was no correlation between urinary WT1-positive cells and WT1-positive cells in renal biopsy. However, the number of urinary WT1-positive cells in patients with crescentic lesions was significantly higher than in patients with non-crescentic lesions. In addition, the best cut-off value to detect patients with crescentic lesions using urinary was 5 cells/10-mL (area under the concentration-time curve=0.735). CONCLUSIONS: The results of our study suggest urinary WT1-positive cells can be used to detect patients with crescent formation using 5 cells/10-mL cutoff value. WT1-positive glomerular podocytes and parietal epithelial cells may be shed into urine in active glomerular disease. This study, investigating the relationship between WT1-positive cells in urine and in the renal biopsy found no correlation; however, the results do suggest that, using a cutoff value of 5 cells/10 mL, WT1 positive urinary cells can be used to detect patients with crescent formation.

WT1 immunoenzyme staining using SurePath(™) processed urine cytology helps to detect kidney disease.

OBJECTIVES: Damage and detachment of podocytes and loss into the urine have been implicated in the progression of kidney diseases. The purpose of this study was to investigate the potential role of urine cytology based on SurePath(™) combined with immunoenzyme staining using Wilms' tumour 1 (WT1) antibody as a podocyte marker in the discrimination of normality and non-renal urinary tract disease from kidney disease. METHODS: Sixty-six patients with kidney disease, 45 patients with lower urinary tract disease and 30 healthy volunteers were examined. Urine cytology slides were prepared using the SurePath method and immunoenzyme stained with WT1 antibody, and the number of WT1-positive cells was counted. RESULTS: In kidney disease, WT1-positive cells were found in 33 (50%) of 66 samples. No WT1-positive cells were found in 45 patients with lower urinary tract disease or in 30 healthy volunteers. The positive rates for WT1 varied with disease type, but not significantly: immunoglobulin A (IgA) nephropathy, (14/23); membranous glomerulonephritis, (4/10); Henoch-Schönlein purpura nephritis, (3/5); diabetic glomerulopathy, (5/5); minor glomerular abnormality/minimal change nephrotic syndrome (0/4). CONCLUSIONS: The results suggest that WT1 immunoenzyme staining of urine cytology can be used to detect some types of kidney disease.

Local steroid injection into the artificial ulcer created by endoscopic submucosal dissection for gastric cancer: prevention of gastric deformity.

BACKGROUND AND STUDY AIMS: Endoscopic submucosal dissection (ESD) of large gastric lesions results in an extensive artificial ulcer that can lead to marked gastric deformity. The aim of the current study was to evaluate therapeutic efficacy in the prevention of gastric deformity of local triamcinolone acetonide (TCA) injection into the extensive artificial ulcer following ESD. PATIENTS AND METHODS: A total of 45 patients who were diagnosed with early gastric cancer were enrolled. Patients were randomly assigned by the sealed-envelope randomization method to either local TCA injections (n = 21) or sham-control (n = 20) groups. Two clips were placed at the two maximum outer edges of the artificial ulcer after the lesion had been resected (Day 0). Local TCA injections were performed on postoperative Day 5 and Day 12. The distance between the two clips was measured by endoscopic measuring forceps on Days 5, 12, 30, and 60. Granulation formation and gastric deformity were evaluated by visual analog scale (VAS) on Days 30 and 60. RESULTS: Local TCA injection did not alter clip-to-clip distance on postoperative Day 60, and formation of flat granulation tissue over the ulcer was followed by regenerative mucosa without any gastric deformity. The sham-control group showed significant shortening of clip-to-clip distance compared with the local steroid-injected group and protruded forms of granulation tissue with mucosal convergence. Histological evaluation revealed prominent growth of neovessels, swelling, and marked increases in endothelial cells in the local steroid-injected group compared with the sham-control group. CONCLUSIONS: Local steroid injection into the floor of a post-ESD artificial ulcer promotes the formation of granulation tissue at an early stage of the healing process leading to regeneration of gastric mucosa without mucosal convergence or gastric deformity.

Exendin-4 regulates the expression of the ATP-binding cassette transporter A1 via transcriptional factor PREB in the pancreatic ß cell line.

BACKGROUND: PRL regulatory element-binding (PREB) protein is a transcription factor that regulates insulin promoter activity in the rat anterior pituitary. The PREB protein is expressed not only in the anterior pituitary but also in pancreatic ß cells. Previously, we have reported that PREB plays an important role in glucose-mediated insulin gene expression in pancreatic ß cells. The ATP-binding cassette transporter A1 (ABCA1) in pancreatic ß cells influences insulin secretion and glucose homeostasis. Exendin-4 (Ex-4), a longacting agonist of the glucagon-like peptide 1, stimulates ABCA1 expression in pancreatic ß cells. AIMS: In this study, we examined the role played by PREB in Ex-4-induced ABCA1 expression in pancreatic ß cells. MATERIAL/SUBJECTS AND METHODS: PREB mRNA and protein expression were evaluated in pancreatic ß cell line (INS-1 cells) treated with Ex-4 (10 nM). RESULTS: Ex-4 stimulated PREB protein and mRNA expression in INS-1 cells. PREB stimulated the activity of the luciferase reporter protein that was under the control of the ABCA1 promoter. Chromatin immunoprecipitation assay showed that PREB mediates its transcriptional activity by directly binding to the ABCA1 promoter region. Finally, we used small interfering RNA to inhibit PREB expression in the cells and demonstrated that the knockdown of PREB expression attenuated the effects of Ex-4 on ABCA1 expression. CONCLUSION: PREB mediates Ex-4-stimulated transcription of the ABCA1 gene in pancreatic ß cells.

Expression of vimentin and high-molecular-weight cytokeratin (clone 34ßE12) in differentiating reactive renal tubular cells from low-grade urothelial carcinoma cells in voided urine.

OBJECTIVE: Reactive renal tubular cells show features of an atypical repair reaction. Differentiation between reactive renal tubular cells and low-grade urothelial carcinoma (LG-UC) cells can therefore be a diagnostic challenge based on morphology alone. In this study, we evaluated the diagnostic utility of vimentin and a high-molecular-weight cytokeratin antibody (clone 34ßE12) in differentiating reactive renal tubular cells from LG-UC. METHODS: We evaluated voided urine cytology and surgical specimens from 40 patients with renal disease, and 17 patients with LG-UC. All slides were stained with vimentin and 34ßE12. RESULTS: In the reactive renal tubular cells in voided urine cytology, vimentin showed strong cytoplasmic staining in 39/40 (97.5%) cases, but all were negative for 34ßE12. LG-UC cells showed positive staining for 34ßE12 in 3/17 (17.6%) cases, whereas none were positivity for vimentin. The reactive renal tubular cells of histological specimens in the renal disease group demonstrated positive for vimentin in all 40 cases and all were negative for 34ßE12. The LG-UC group showed abnormal staining for 34ßE12 in 4/17 (23.5%) cases, whereas none were positive for vimentin. CONCLUSIONS: Vimentin expression in urine cytology can help to distinguish reactive renal tubular cells from LG-UC. However, 34ßE12 does not appear to be a useful adjunct to distinguish these two groups in voided urine cytology.

Value of computer-assisted quantitative nuclear morphometry for differentiation of reactive renal tubular cells from low-grade urothelial carcinoma.

OBJECTIVE: To assess whether computer-assisted quantitative morphological parameters can be an effective tool for objectively distinguishing reactive renal tubular cells from low-grade urothelial carcinoma cells (LG-UCs) in voided urine. METHODS: Nuclear morphometry was performed by a computer-assisted image analyser system on Papanicolaou-stained cytological specimens. The circumference of reactive renal tubular cells (n = 40) or LG-UC (n = 20) nuclei were manually traced, and the following nuclear morphometric parameters were analysed: (i) area, (ii) perimeter, (iii) roundness factor, (iv) maximum length, and (v) linear factor. For each nuclear measurement, we calculated the maximum, minimum, mean and standard deviation. RESULTS: The mean nuclear area and nuclear perimeter were higher in reactive renal tubular cells compared to the LG-UCs. The mean of roundness and linear factors (reflecting a tendency for the nuclear outline to be regular and oval, respectively) were higher in LG-UCs compared with reactive renal tubular cells. Among nuclear areas, the nuclear perimeter, roundness factors and maximum length did not show any significant differences between reactive renal tubular cells and LG-UCs. On the other hand, the linear factor showed a mean higher value among LG-UCs than reactive renal tubular cells (P = 0.023). CONCLUSIONS: Of five quantitative nuclear morphological parameters, only linear factor was statistically significant in differentiating reactive renal tubular cells in renal disease from LG-UCs.

Can cytological features differentiate reactive renal tubular cells from low-grade urothelial carcinoma cells?

OBJECTIVE: To compare the cytomorphological and immunocytochemical features of reactive renal tubular cells and low-grade urothelial carcinoma cells (LG-UCs). METHODS: We examined 15 cytological parameters in 38 cases with reactive renal tubular cells in renal disease and 20 cases of LG-UCs from bladder cancer that had been diagnosed by histological examination. Voided urine cytological parameters evaluated were as follows: (i) maximum cell numbers of clusters, (ii) cannibalism, (iii) rosette-like arrangement, (iv) hobnail-shaped cells, (v) vacuolated cytoplasm, (vi) intracytoplasmic haemosiderin, (vii) irregular nuclear contours, (viii) chromatin pattern, (ix) prominent nucleoli, (x) cast encasement, (xi) casts, (xii) dysmorphic erythrocytes, (xiii) isomorphic erythrocytes, (xiv) necrosis, and (xv) vimentin reactivity. The above parameters were determined using Mann-Whitney U-test and chi-square test, with differences considered significant at P < 0.05. RESULTS: In reactive renal tubular cells, low to moderate cell numbers of clusters (fewer than 50 cells), rosette-like arrangement, hobnail-shaped cells, vacuolated cytoplasm, intracytoplasmic haemosiderin, euchromatin pattern, prominent nucleoli, dysmorphic erythrocytes and vimentin reactivity were present in significantly higher proportions compared with those in LG-UCs. In LG-UCs, high cell numbers of clusters (50 cells or more), cannibalism, heterochromatin pattern, isomorphic erythrocytes and necrosis were seen in significantly higher proportions. No significant differences were observed in irregular nuclear contours, cast encasement or casts. CONCLUSIONS: Based on results of the present study, maximum cell numbers of clusters, cannibalism, rosette-like arrangement, hobnail-shaped cells, vacuolated cytoplasm, intracytoplasmic haemosiderin, chromatin pattern, prominent nucleoli, dysmorphic erythrocytes, isomorphic erythrocytes, necrosis, and vimentin reactivity were capable of distinguishing reactive renal tubular cells from LG-UCs.

'Cannibalism' (cell phagocytosis) does not differentiate reactive renal tubular cells from urothelial carcinoma cells.

OBJECTIVE: Cannibalism of one cell by another in voided urine cytology has been considered a cytological feature for differentiating urothelial carcinoma (UC) from benign lesions. Recently, however, we observed cannibalism in voided urine obtained from patients with renal glomerular disease (RGD). The purpose of this study was to determine the cytomorphological and immunocytochemical characteristics of cannibalism in voided urine from RGD. METHODS: Seventy cytology specimens of voided urine were examined and the findings were compared with the histological findings. In addition, we compared the cytomorphological and immunocytochemical differences in cannibalism found in RGD and cases of UC selected as showing cannabilism. RESULTS: Cannibalism in voided urine was found in three (5.5%) of 55 RGD cases. The finding was measured as (1+) < 5 cells, (2+) 5-20 cells, and (3+) > 20 cells and was (1+) in all three RGD cases, compared with 6.7%, 60% and 33.3% respectively in 15 UC cases. Differences in low cellularity cases (1+) and moderate to high cellularity cases (2+ or 3+) were statistically significant between RGD (3 and 0) and UC (1 and 14) (P=0.005). The maximum diameter of cannibalized cells in RGD was 24.3-33.0 microm (mean 29.8 microm) versus 18.0-30.4 microm (mean 23.3 microm) in UC (P=0.004). Necrosis and isomorphic erythrocytes were absent in RGD, but were found in 46.7% and 86.7%, respectively, of UC cases (P=0.245 and P=0.012). Dysmorphic erythrocytes were identified in all three cases with RGD and 13.3% of UC (P=0.012). Vimentin reactivity was found in all cases with cannibalism in RGD, but never in UC (P=0.001). CONCLUSIONS: Our results demonstrated that cannibalism in voided urine is present not only in UC but also in RGD. Furthermore, we showed that cellularity of cannibalism, vimentin reactivity and background differed significantly and can be used for differential diagnosis between the two groups.

The clinical significance of splice variants and subcellular localisation of survivin in non-small cell lung cancers.

Survivin is a member of the inhibitor of apoptosis protein family. Survivin has splice variants with different biological functions associated with tumorigenesis. We investigated 134 non-small cell lung cancers (NSCLCs) to study the clinical significance of wild-type survivin, survivin-2B, and survivin-deltaEx3. Real-time PCR analyses were performed for their gene expressions. The subcellular localisation of survivin proteins was evaluated by immunohistochemistry. The Ki-67 proliferation index and the apoptotic index were also evaluated. The survivin-deltaEx3 gene expression was significantly higher in stage II-III than in stage I (P=0.0174), and significantly correlated with the nuclear pan-survivin expression (P<0.0001). The Ki-67 index was significantly higher in wild-type survivin-positive tumours (P<0.0001), survivin-deltaEx3-positive tumours (P<0.0001), and tumours with positive expression of the nuclear pan-survivin (P=0.0047). In contrast, the apoptotic index was significantly lower only in wild-type survivin-positive tumours (P<0.0001). Thus, the wild-type survivin gene expression was associated with apoptotic inhibition and tumour proliferation. Furthermore, the survivin-deltaEx3 gene expression was strongly associated with tumour proliferation, especially in advanced stage NSCLCs. In contrast, the survivin-2B gene expression did not correlate with tumour proliferation or tumour apoptosis.

Clinical application of biological markers for treatments of resectable non-small-cell lung cancers.

We performed a clinical study to identify biological markers useful for the treatment of resectable non-small-cell lung cancers (NSCLCs). In all, 173 patients were studied. By immunohistochemistry, we evaluated the Ki-67 proliferation index, tumour vascularity, thymidylate synthase (TS), vascular endothelial growth factor (VEGF)-A, VEGF-C, and E (epithelial)-cadherin. Concerning the survival of NSCLC patients, tumour vascularity (P<0.01), VEGF-A status (P=0.03), VEGF-C status (P=0.03), and E-cadherin status (P=0.03) were significant prognostic factors in patients with stage I NSCLCs. The Ki-67 proliferation index (P=0.02) and TS status (P<0.01) were significant prognostic factors in patients with stage II-III NSCLCs. In patients with stage II-III NSCLCs, furthermore, the survival of UFT (a combination of tegafur and uracil)-treated patients with TS-negative tumours was significantly better than those of any other patients. Biological markers associated with tumour angiogenesis or metastasis are useful for the detection of aggressive tumours among early-stage NSCLCs. Postoperative chemotherapy might be necessary in such tumours even in stage I. In contrast, tumour proliferation rate and TS status are useful markers for identifying less aggressive tumours in locally advanced NSCLCs. Thymidylate synthase expression is also a useful marker to evaluate responsiveness of UFT-based chemotherapy for these tumours.

The tumour-stromal interaction between intratumoral c-Met and stromal hepatocyte growth factor associated with tumour growth and prognosis in non-small-cell lung cancer patients.

Immunohistochemical analyses of the effects of hepatocyte growth factor (HGF) and c-Met expression on tumour growth and angiogenesis were performed on 88 patients with non-small-cell lung cancers (NSCLCs). In all, 22 carcinomas (25.0%) were intratumoral HGF-positive, 14 carcinomas (15.9%) were stromal HGF-positive, and 36 carcinomas (40.9%) were intratumoral c-Met-positive. None of the carcinomas were stromal c-Met-positive. Examination of tumour growth revealed that the frequency of tumours with a high Ki-67 index was significantly greater for stromal HGF-positive tumours than for stromal HGF-negative tumours (P=0.0197). The frequency of tumours with a high Ki-67 index was also significantly greater for intratumoral c-Met-positive tumours than for intratumoral c-Met-negative tumours (P=0.0301). However, there was no significant difference in tumour vascularity with relation to intratumoral HGF status, stromal HGF status, and intratumoral c-Met status. The survival rate of patients with intratumoral c-Met-positive tumours was significantly lower than for patients with c-Met-negative tumours (P=0.0095). Furthermore, the survival rate of patients with both intratumoral c-Met-positive and stromal HGF-positive tumours was significantly lower than for patients with either positive tumours, and that of patients with both negative tumours (P=0.0183 and P=0.0011, respectively). A univariate analysis revealed that intratumoral c-Met expression was a significant prognostic factor of NSCLC patients (relative risk=2.642, P=0.0029). The present study demonstrates that tumour-stromal interaction between tumour cell-derived c-Met and stromal cell-derived HGF affects tumour growth and the prognosis of NSCLC patients.

The intratumoral expression of vascular endothelial growth factor and interleukin-8 associated with angiogenesis in nonsmall cell lung carcinoma patients.

BACKGROUND: Angiogenesis has important effects on tumor growth and metastasis. It is regulated by a variety of angiogenic and angiostatic factors. METHODS: To evaluate the effects of tumor cell-derived angiogenic factors, we performed an immunohistochemic study to evaluate the intratumoral expression of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) in relation to intratumoral microvessel density (IMD) in tumors from 104 nonsmall cell lung carcinoma (NSCLC)patients. RESULTS: Fifty-four carcinomas were VEGF-positive, 47 carcinomas were IL-8-positive, and 53 carcinomas were hypervascular tumors. There was no significant correlation between the percentages of positive VEGF-staining and positive IL-8-staining in NSCLCs (rho = 0.174, P = 0.080). The IMD of VEGF-positive carcinomas was significantly greater than that of VEGF-negative carcinomas (P = 0.023). In addition, the IMD of IL-8-positive carcinomas was significantly greater than that of IL-8-negative carcinomas (P =0.013). The overall survival rate of patients with hypervascular tumors was significantly lower than that of patients with hypovascular tumors (41.0% versus 67.0%, P = 0.004). Cox proportional-hazards regression model also demonstrated that angiogenesis was one of the significant factors in predicting the survival of NSCLC patients (relative risk = 1.944, P = 0.041). CONCLUSIONS: Intratumoral expression of VEGF and IL-8 was associated with angiogenesis in NSCLCs. Tumor angiogenesis significantly affected the prognosis of NSCLC patients.

[Minocycline-induced pneumonitis presenting as multiple ring-shaped opacities on chest CT, pathologically diagnosed bronchiolitis obliterans organizing pneumonia (BOOP)].

A 39-year old woman was admitted to our hospital because of cough and abnormal shadows on chest radiographs. She had been treated for 5 months for acne vulgaris with minocycline hydrochloride (MINO). Chest computed tomographic (CT) scans showed multiple ring-shaped opacities in both lungs. Bronchoalveolar lavage disclosed an increase in the total number of cells and a marked increase of lymphocytes. A lung specimen obtained by transbronchial lung biopsy (TBLB) was pathologically diagnosed as bronchiolitis obliterans organizing pneumonia (BOOP). Withdrawal of minocycline led to rapid remission without treatment. The clinical course and histological findings for TBLB suggested that this case was minocycline-induced BOOP. Several cases with minocycline-induced pneumonitis have been reported. However, there are few reported cases of minocycline-induced BOOP, the present case being only the second found in the literature.

Ascitic fluid cytology of adenosarcoma of the ovary: a case report.

Extrauterine adenosarcoma is very rare and originates in the ovary, adnexa, or myometrium. Cytologic study of ascites is very important to determine clinical staging of malignant ovarian tumors and provide adequate therapy for recurrence. The cytomorphologic features of adenosarcoma have been only rarely described. A 77-yr-old woman visited a hospital with a complaint of lower abdominal pain for 1 mo. A tumor originating from the right adnexa in the pelvis, and involving the rectum, was found in surgery. In the ascitic fluid cytology, a few dispersed tumor cells with large cytoplasm and nuclei were oval-shaped, with nuclear invagination. The chromatin was finely granular; one or two nucleoli were conspicuous. To our knowledge, this is the fifteenth reported case of adenosarcoma of the ovary, and there have been no prior reports describing the cytological features of ascitic fluid cells in adenosarcoma of the ovary.

Intrathyroidal branchial cleft-like cyst in chronic thyroiditis.

An extremely rare case of intrathyroidal branchial cleft-like cyst is reported. A 71-year-old man complained of a growing mass in the right lateral neck. A cystic mass in the upper lobe of the right thyroid was demonstrated by ultrasonography and computed tomography. The surgical specimen revealed a cystic mass with dense fibrous capsule, 22 x 20 x 10 mm in size. Microscopically, the cyst walls and the surrounding thyroid tissue contained severe lymphoid cell infiltration with lymphoid follicle. Squamous epithelium lined the cyst wall. Immunohistochemically, squamous epithelium was positive for keratin, cytokeratin 19, carcinoembryonic antigen, and epithelial membrane antigen, but negative for calcitonin and chromogranin A. The patient is currently well with no evidence or recurrence for 43 months.