Independent histological risk factors for lymph node metastasis of superficial esophageal squamous cell carcinoma; implication of claudin-5 immunohistochemistry for expanding the indications of endoscopic resection.

Endoscopic resection is curative for superficial esophageal squamous cell carcinoma (ESCC) limited to the lamina propria. Endoscopic resection is not recommended for superficial ESCC invading muscularis mucosa or submucosa, however, because of the high frequency of lymph node metastasis (LNM) in such patients. Methods to more accurately predict LNM by analysis of endoscopically resected specimens are needed. Patients with superficial ESCC who underwent surgery without prior chemoradiotherapy (n= 110) were retrospectively examined to determine whether LNM correlated with immunohistochemical parameters and conventional histological parameters, including depth of invasion and vascular permeation. Cancer cell expression of claudins-1, 5, and 7, E-cadherin, beta-catenin, and matrix metalloproteinase 7 was evaluated. Univariate analysis revealed that LNM correlated with claudin-5 expression, but not any other immunohistochemical parameter examined. Multivariate analysis revealed three independent risk factors for LNM: aberrant claudin-5 expression in cancer cells (odds ratio; OR [95% confidence interval]= 4.61[1.44-14.77]), depth of submucosal invasion greater than 200 microm (3.55 [1.02-13.17]), and positive lymphatic permeation (3.34 [1.22-9.15]). LNM was found in one of 29 (3.4%) patients with none of these three risk factors, and in 32 of 81 (39.5%) patients with one or more of these risk factors. In superficial ESCC, routine analysis of claudin-5 expression in cancer cells together with depth of invasion and lymphatic permeation may be useful for predicting LNM and thereby reducing the number of patients undergoing additional surgery after successful endoscopic resection.

Aurora kinase B is a predictive factor for the aggressive recurrence of hepatocellular carcinoma after curative hepatectomy.

BACKGROUND: Patterns of cancer recurrence hold the key to prognosis after curative resection. This retrospective study aimed to identify a predictor and therapeutic candidate for aggressive recurrence of hepatocellular carcinoma (HCC). METHODS: Primary HCC tissues from 107 patients who had curative resection were analysed. Genome-wide gene expression profiles were investigated using a microarray technique, and clustering analysis was carried out based on the first diagnosis of recurrence according to the Milan criteria. Immunohistochemical expression and array-based comparative genomic hybridization (array-CGH) were also assessed. RESULTS: Microarray analysis revealed overexpression of Aurora kinase B, a chromosome passenger protein kinase, as the most significant predictor of the aggressive recurrence of HCC. Aurora kinase B protein expression was significantly associated with aggressive recurrence (P < 0.001) and prognosis (P < 0.001). Multivariable analysis identified Aurora kinase B as the only independent predictor of aggressive recurrence of HCC (P = 0.031). Array-CGH analysis showed that genomic instability was closely related to Aurora kinase B expression (P = 0.011). CONCLUSION: Aurora kinase B is an effective predictor of aggressive HCC recurrence, in relation to the genomic instability. It might be worth considering as a molecular target for the adjuvant therapy of HCC.

Basic fibroblast growth factor (bFGF) receptors decrease with luteal age in rat ovarian luteal cells: colocalization of bFGF receptors and bFGF in luteal cells.

Ovarian growth factors have been implicated in the development and differentiation of corpus luteum. We have characterized both high and low affinity receptors for basic fibroblast growth factor (bFGF) in luteal cells and tissue throughout the life span of corpus luteum using gonadotropin-treated rat luteinized ovaries. Additionally, we determined bFGF location in luteal tissue. High affinity (Kd, approximately 0.2 nM) and low capacity (approximately 500-6000 sites/cell, depending on luteal ages) [125I] bFGF-binding sites (mol wt, 140 kilodaltons, determined by affinity labeling) were found on luteal cells. [125I]bFGF binding to luteal cells and corpus luteum membranes progressively decreased in binding capacity without affecting binding affinity as the age of corpus luteum advanced. bFGF receptor (flg) mRNA in luteinized ovaries decreased with the luteal age similar to the [125I]bFGF binding. In contrast, low affinity binding sites, identified as heparan sulfate proteoglycans, which were immunohistochemically visualized around luteal cells, did not appear to change with luteal age. The signals for heparan sulfate proteoglycans that were found only in granulosa, but not theca-interstitial, cells of follicles became intense during folliculogenesis. The functionality of the bFGF receptors was shown by tyrosine phosphorylation of 16.5- and 18-kilodalton proteins in luteal cells with the antiphosphotyrosine antibody. Immunohistochemistry localized bFGF to steroidogenic luteal cells, and immunoblotting displayed larger molecular forms of bFGF in luteal cells. These results suggest that the bFGF receptors may be associated with the development and differentiation of corpus luteum in an autocrine manner.

Glycogen storage disease confined to the heart with deficient activity of cardiac phosphorylase kinase: a new type of glycogen storage disease.

The case of a male infant with marked deposition of glycogen, confined to the heart, is presented. Clinically, prominent cardiomegaly had been evident from immediately after birth until the infant's death due to heart failure. There were no significant clinical manifestations in other organs, including liver and skeletal muscle, during the clinical course. Autopsy revealed abnormal deposition of normally structured glycogen in the heart, but no deposition in the liver, skeletal muscle, or other systemic organs. This unusual pattern of glycogen deposition was also confirmed by measurement of the glycogen content of each organ. This is the first report of glycogen storage disease confined to the heart. Enzymatic analysis revealed no decrease in the activities of acid maltase, amylo-1,6-glucosidase, and phosphorylase in the heart or in the liver or skeletal muscle. However, phosphorylase kinase activity was not detectable in the heart, although high activity levels were observed in the liver and skeletal muscle. In this case the inborn error of metabolism responsible for the isolated deposition of glycogen in heart muscle may have been due to a deficiency of cardiac phosphorylase kinase.

Production of monoclonal antibodies directed to Hanganutziu-Deicher active gangliosides, N-glycolylneuraminic acid-containing gangliosides.

We have established three kinds of monoclonal antibodies against gangliosides containing N-glycolylneuraminic acid (NeuGc) by immunization of BALB/c mice with the purified gangliosides inserted into liposomes comprising Salmonella minnesota R595 lipopolysaccharides, and fusion of spleen cells with a mouse myeloma cell line. One monoclonal antibody, SHS-1, which was generated by immunizing mice with purified i-active ganglioside(NeuGc), reacted specifically with the i-active ganglioside(NeuGc) used as an immunogen. Structurally related gangliosides, such as GM3(NeuGc), sialosylparagloboside (SPG) (NeuGc), or I-active ganglioside(NeuGc), corresponding gangliosides [GM3 containing N-acetylneuraminic acid (NeuAc), SPG(NeuAc), i-active ganglioside(NeuAc), and I-active ganglioside(NeuAc)], other gangliosides, or neutral glycosphingolipid (GSL) were not recognized by the monoclonal antibody. These findings indicate that the SHS-1 monoclonal antibody may be specific for NeuGc-containing i-active ganglioside. On the other hand, the other two monoclonal antibodies, MSG-1 and SPS-20, which were generated by immunizing mice with purified ganglioside GM3(NeuGc) and SPG(NeuGc), respectively, showed cross-reactivity to structurally related gangliosides. The MSG-1 monoclonal antibody exhibited reactivity to ganglioside GM3(NeuAc). The SPS-20 monoclonal antibody also cross-reacted with SPG(NeuAc), i-active ganglioside(NeuGc), and i-active ganglioside(NeuAc). Neither MSG-1 nor SPS-20 reacted with corresponding gangliosides, other gangliosides, or neutral GSLs tested. Using the SHS-1 antibody specific for i-active ganglioside(NeuGc), we studied the expression of NeuGc-containing antigen in human colon cancer tissue. An NeuGc-containing glycoconjugate was detected in the colon cancer tissue.

Expression of the cyclin dependent kinase inhibitor p21WAF1/CIP1 in oesophageal squamous cell carcinomas.

To elucidate the role of CDK inhibitor p21WAF1/CIP1 in human oesophageal squamous cell carcinomas, we examined its expression immunohistochemically using surgically resected tissues from 25 patients, and have analyzed the relationship with alteration of p53 gene (F-SSCP analysis), proliferative activity (Ki-67 labelling index), frequency of apoptosis (in situ DNA nick end labelling), and degree of differentiation. P21 expression was observed in 11 cases (44%) with a percentage of positive cells ranging between 1% and 10%. Of the 25 cases, 4 cases showed > 5% of positive cells. As for the relationship with p53 gene, all 7 p53-mutation positive cases were negative for p21 expression, whereas 11 out of 18 mutation negative cases showed positive for p21 expression. As for the relationship with degree of tumour differentiation, 6 out of 8 well differentiated type cases showed positive for p21 expression. By contrast, all 8 cases of poorly differentiated type were negative for p21 expression. Frequency of apoptotic cells was significantly higher in p21 positive cases than negative cases although Ki-67 labelling index was almost the same regardless of the expression of p21. P21 expressing cells were distributed mainly in the middle layers of the invading nests, especially around the keratinization, which was almost similar to the distribution of apoptotic cells. Our results suggest that expression of p21 in human oesophageal squamous cell carcinomas is induced by a p53-dependent pathway and affects apoptosis and differentiation of carcinoma cells.

Proliferative activity and p53 protein accumulation correlate with early invasive trend, and apoptosis correlates with differentiation grade in oesophageal squamous cell carcinomas.

Using oesophageal squamous cell carcinoma samples of both intramucosal and advanced types, proliferative activity (Ki-67 labelling index), p53 protein accumulation and apoptosis (in situ DNA nick end labelling) were assessed, and the relation of these values to progression or differentiation grade of tumours was analysed. In terms of proliferative activity and the proportion of positive cases with p53 accumulation, a statistically significant difference was demonstrated between intraepithelial carcinomas and intramucosal carcinomas with stromal invasion (17.2% vs 31.7% for the Ki-67 labelling index, and 23.5% vs 67.4% for the proportion of positive cases of p53 accumulation). Values for the latter were almost comparable to those of advanced carcinomas. Immunohistologically, Ki-67 positive, proliferating cells were distributed preferentially in the peripheral fronts of invading nests. Apoptotic cells were observed in the inner areas of the invading nests of the intramucosal carcinomas with stromal invasion and in more advanced lesions, but were rarely observed in the normal epithelium or intraepithelial carcinomas. Apoptotic cells were seen mainly around areas of keratinization, and the apoptotic cell index was higher in well and moderately differentiated types of advanced carcinomas than in the poorly differentiated type (2.59% vs 1.09%). An increase in proliferative activity and an accumulation of p53 protein are associated with the onset of early carcinomatous invasion, while apoptosis is closely linked with the differentiation grade of carcinoma cells.

Gastric mucosal density of Helicobacter pylori estimated by real-time PCR compared with results of urea breath test and histological grading.

The accuracy of the urea breath test (UBT) and histological grading for estimation of the density of Helicobacter pylori in gastric mucosa is not known. Real-time (TaqMan) PCR was used to estimate the total number of H. pylori genomes in biopsy samples. These values were compared with those obtained by the UBT and the histological grade obtained by the Sydney system. The UBT and endoscopy with antral and corporal biopsies were performed in 88 consecutive untreated patients with dyspepsia. Bacterial culture and the rapid urease test were done with fresh biopsy materials. TaqMan PCR and histological examination were done on serial paraffin sections of the biopsy samples. Of the five methods tested, TaqMan PCR had the highest sensitivity and specificity (both 100%) in the diagnosis of H. pylori infection. The mean density of H. pylori genomes for pairs of biopsy samples from individual patients was compared with the individual values obtained by the UBT; correlation between the results was significant. The density of H. pylori genomes was higher in histological grades 1, 2 and 3 than in grade 0, without significant differences between adjacent grades from 1 to 3. These results suggest that the severity of H. pylori infection of the stomach can be estimated by the UBT and that histopathologists might state whether the organism is present or absent, rather than making a quantitative statement as recommended in the Sydney system.

Peripheral neuropathy with predominantly motor manifestations in a patient with carcinoma of the uterus.

An autopsy case of a 56-year-old woman who had carcinoma of the corpus uteri and peripheral neuropathy with predominantly motor manifestations is described. The neurological abnormalities included subacute weakness of the limbs and loss of deep reflexes, which improved after the surgical removal of the uterine carcinoma. Neuropathologically, peripheral nerves mainly presented features of axonal degeneration with a mild loss of myelinated fibres. Anterior horns of the spinal cord showed central chromatolysis of the motor nerve cells and many spheroids without neuronal loss. Axonopathy of peripheral nerves was considered to be the main pathological process in this paraneoplastic syndrome.

Proliferative response of peripheral blood mononuclear cells and levels of antibody to recombinant protein from Propionibacterium acnes DNA expression library in Japanese patients with sarcoidosis.

BACKGROUND AND AIM OF THE WORK: The causes of sarcoidosis are unknown. Propionibacterium acnes has been isolated from sarcoid lesions, and many genomes of P. acnes or P. granulosum have been detected in all biopsy samples tested from Japanese patients with sarcoidosis. We searched for protein antigens from propionibacteria that caused immune responses in patients with sarcoidosis but not in subjects without sarcoidosis. METHODS: A lambda gt11 genomic DNA expression library of P. acnes was screened with sera from patients with sarcoidosis. Antibodies to a recombinant protein from the insert recovered by the screening were measured in serum and bronchoalveolar lavage (BAL) fluid from patients with or without sarcoidosis by an immunofluorescence-based method. Peripheral blood mononuclear cells from patients with and without sarcoidosis were used to examine the lymphoproliferative response to the protein. RESULTS: Of 180,000 plaques screened, two clones coded for an identical recombinant protein, termed RP35, were recognized by sera. RP35 was the C-terminal region of P. acnes trigger factor. RP35 caused sarcoidosis specific proliferation of the mononuclear cells from 9 (18%) of the 50 patients with sarcoidosis; in a similar way, purified protein derived from Mycobacterium tuberculosis evoked specific responses in 8 (38%) of 21 patients with tuberculosis. Serum levels of IgG and IgA antibodies to RP35 were high in patients with sarcoidosis and other lung diseases. In BAL fluid levels IgG or IgA antibodies were high in 7 (18%) and 15 (39%), respectively, of 38 patients with sarcoidosis, and in 2 (3%) and 2 (3%), respectively, of 63 patients with other lung diseases. CONCLUSIONS: The RP35 protein from P. acnes causes a cellular immune response in some patients with sarcoidosis but not in subjects without sarcoidosis.

Transferrin receptor in oral tumors.

The transferrin receptor (TfR) appears in vigorously proliferating cells. We did an immunohistochemical study of TfR in oral tissues and a quantitative analysis by flow cytometry of TfR in a cancer cell line after an anticancer drug treatment. TfR was found in the parabasal and basal layers of the normal epithelium, but rarely in benign tumors. Generally, in the malignant tumors, the poor prognostic cases showed strong staining regardless of the differentiation of the tumor. In the flow cytometric analysis, the amount of TfR decreased according to the reduction of the proliferative ability of cancer cells. These results suggest that TfR expression may be useful as a prognostic marker.

Accumulation of germanium in the tissues of a long-term user of germanium preparation died of acute renal failure.

Acute renal failure developed in a patient accompanied by systemic manifestations such as myopathy and skin rash. The patient, a middle aged house wife, had been taking 600 mg of germanium (Ge) preparation daily for 18 months as an elixir. The main component of the preparation was GeO2 and some organic compound was also present. Histological study of the kidney post mortem showed foamy cell transformation of glomerular epithelia, degeneration of tubular epithelia with red blood cell casts and urate crystals, and a mild proliferation of mesangial matrix. Analysis of the tissue content of Ge, prompted by her history, revealed an increased accumulation of the metal. As compared to a non-user died of liver cirrhosis, the concentration of the metal was higher particularly in the spleen (183X), thyroid gland (175X), psoas muscle (93X), jejunum (76X), and renal cortex (69X). So far, neither accumulation of Ge in humal tissue nor systemic toxicity of the Ge in human has been reported. The relevance of massive accumulation of Ge to the renal failure as well as to other systemic manifestations the patient presented remains to be clarified.

Development of the immune system in severe combined immunodeficiency mice reconstituted with transferred fetal liver cells.

Severe combined immunodeficiency (SCID) mice were immunologically reconstituted by the transfer of fetal liver cells (FLC) from BALB/c (SCID-isoFLC mice) or C57BL/6 mice (SCID-alloFLC mice). The developmental process of the immune system in the peripheral blood (PB) was almost comparable between SCID-isoFLC and SCID-isoFLC mice. Analysis of the lymphoid organs and the PB from SCID-alloFLC mice indicated that slg+ B cells appeared and were distributed to the periphery within 2 weeks after the transfer of FLC. It was also suggested that precursor T cells entered the thymus before 4 weeks after the transfer, and were differentiated into mature CD4+ or CD8+ T cells which then migrated to the periphery by 6 weeks. All of mature lymphocytes in the periphery of the SCID-alloFLC mice were shown to express donor-type H-2 antigens. Additionally, in the SCID-isoFLC mice, cell-mediated immunity such as rejection against alloantigens was functioning from 6 weeks, and humoral immune function was suggested by the detection of cells generating antigen-specific antibodies. We discuss that development of the immune system in SCID mice receiving transferred FLC was comparable to that normally seen in the fetal and neonatal stages.

Histological differentiation and basement membrane distribution in gastric adenocarcinomas.

To analyse the histological distribution of basement membranes (BM) in gastric adenocarcinomas, we produced a monoclonal antibody, BM909 (IgG 2b, kappa), which has been found useful for identifying the BM in routinely processed specimens. The BM 909 antibody was shown by ELISA to be negative against the three major BM components (type IV collagen, laminin and fibronectin). Gastric carcinomas from 78 patients including both differentiated and undifferentiated adenocarcinomas were classified into three types (tubular, trabecular, and isolated) based on the histological arrangement of the cancer cells. Histological distributions of the BM were also classified into four patterns (peritubular, peritrabecular, intratrabecular, and pericellular) based on the immunostaining results with the BM 909 antibody. Our results demonstrated a close relationship between these two parameters as follows: 1) the tubular type of gastric carcinomas showed a peritubular pattern of BM distribution in the differentiated adenocarcinomas; 2) The isolated type of gastric carcinomas showed a pericellular pattern of BM distribution in the undifferentiated adenocarcinomas; and 3) The trabecular type of gastric carcinomas showed either peritrabecular or intratrabecular patterns of BM distribution, in both differentiated and undifferentiated adenocarcinomas. In conclusion, we suggest that all gastric adenocarcinomas are accompanied by BM deposition regardless of the degree of histological differentiation.

Roles of mucosal bacteria and succinic acid in colitis caused by dextran sulfate sodium in mice.

Intestines of mice with colitis caused by dextran sulfate sodium (DSS) contain more Bacteroidaceae cells than untreated controls. We investigated the roles of intestinal bacteria and succinic acid, a by-product of Bacteroidaceae metabolism, in this model of colitis. CBA/J mice were given 3% DSS in water for 14 days. After mice were anesthetized and killed, concentrations of organic acids in stools from the cecum and colon were measured. The resected rectum and colon were washed with sterile saline; some specimens were incubated with imipenem in saline for 1 h to kill bacteria on the surfaces and others were not. Their homogenates were cultured anaerobically and aerobically. Separately, 1 mL of 20 mM succinic acid was infused into the rectum of mice, whose anal verge was glued. Animals were anesthetized and killed the next day. The rectum and colon were examined histologically. Concentrations of succinate were higher everywhere in the colon of mice with colitis than in controls. Mice with colitis had more Bacteroidaceae cells, especially B. caccae, than controls. Mice given succinate enemas had focal erosions of the mucosa and edema of the submucosa. Succinic acid, produced abundantly by members of the family Bacteroidaceae, especially B. caccae, may be the ulcerogenic agent in DSS colitis.

[Small cell carcinoma of the urinary bladder: a case report and review of the Japanese literature].

A 50-year-old man presented with asymptomatic gross hematuria which he had first noticed 3 months earlier. Clinical examinations revealed a non-papillary, broad-based tumor on the left lateral wall of the urinary bladder with a clinical stage of T3N0M0. The pathological diagnosis of a transurethral biopsy tissue specimen was small cell carcinoma. Neoadjuvant intraarterial infusion chemotherapy using cisplatin and adriamycin was initially administered but proved to be ineffective. Thus, we performed a radical cystectomy. The tumor tissue was apparently homogenous and composed of small cells arranged in sheets and solid patterns, and was staged to be pT3bR1L2V0N0. An electron microscopic study confirmed small cell carcinoma with neurosecretory granules. Postoperatively, 4 courses of adjuvant chemotherapy consisting of cisplatin, etoposide and ifosfamide were administered. The patient is alive without any evidence of tumor recurrence 26 months after the operation.

[A study of reduction of NF 2 protein, mRNA and loss of 22 q in sporadic meningiomas].

Neurofibromatosis 2(NF 2) is one of the tumor suppressor genes, and its disorder has been reported in sporadic meningiomas. We investigated some reduction of expression of NF 2 protein and mRNA in 33 sporadic meningiomas, using immunohistochemistry and in situ hybridization, respectively. Seventeen of 33 (51.5%) meningiomas demonstrated significantly reduced or absent NF 2 protein expression. Its mRNA was also reduced or absent in 11 of 24(45.8%) meningiomas, and some statistically significant correlation was shown between reductions of NF 2 protein and its mRNA. Furthermore, we studied about loss of 22 q in 5 meningiomas with fluorescence in situ hybridization. In the cases with reduction of NF 2 protein and/or mRNA, 22 q telomere signals were observed as loss of heterozygosity. On the other hand, in the cases with expression of NF 2 protein and mRNA, 22 q telomere signals were normal. In conclusion, reduction or absence of NF 2 protein and mRNA may play an important role in the tumorigenesis of about half number of sporadic meningiomas.

[Seeking a causative agent of sarcoidosis].

Bacterial components (MDP and IT27 antigen), demonstrated in sarcoid granulomas, strongly suggest the causative agent of sarcoidosis to be derived from some bacteria but many kinds of bacteria are known to have these bacterial components. Our PCR detection of mycobacterial DNA could not demonstrate a significant contribution of M. tuberculosis to the pathogenesis. We then developed a monoclonal antibody to the causative agent by immunizing mice with a crude homogenate of the affected lymph nodes. Hybridized spleen cells were checked with ELISA and all the Ig-producing cell colonies were examined by immunostaining on paraffin sections of lymph nodes with sarcoidosis or tuberculosis. The established antibody (SG5) recognized some extrinsic antigens sequestrated in sarcoid granulomas, in a similar pattern of distribution to those of the bacterial component of MDP and the bacterial product of IT-27. Moreover, ELISA analysis of the SG5 antibody revealed a specific reactivity to culture supernatant of P. acnes. By the immunization of mice with a crude bacterial extract of P. acnes, we recovered one clone of hybridoma named PAB antibody, that possessed an identical reactivity to that of SG5 antibody, as far as examined by ELISA and immunohistochemistry. Furthermore, PCR detection of P. acnes DNA resulted in 92% (36/39) positive in the lymph nodes with sarcoidosis and 41% (12/29) in the control lymph nodes, with a significant difference (p < 0.001, by Fisher's Probability Test) between these two groups.(ABSTRACT TRUNCATED AT 250 WORDS)