Mutation of von Hippel-Lindau tumor suppressor gene in a sporadic endolymphatic sac tumor.

Endolymphatic sac tumor (ELST) is a low-grade adenocarcinoma of the temporal bone that is presumed to originate from the endolymphatic system. Although ELSTs are extremely rare in the general population, a significant number of studies have documented the occurrence of ELST among patients with von Hippel-Lindau (VHL) disease. Because of the rarity of the tumor, however, few cases of ELST have been analyzed for mutations of the VHL tumor suppressor gene. In this study, we reported a Japanese male patient with sporadic ELST, along with a molecular genetic analysis of the VHL gene. The light microscopic and immunohistochemical features and clinical presentations were typical of ELST. Sequencing studies of the tumor DNA disclosed a G to T substitution of nucleotide 564, which resulted in an amino acid substitution (Trp to Cys). This is the first report of the VHL gene mutation in a sporadic Japanese case of ELST.

Primary Ewing's sarcoma/peripheral primitive neuroectodermal tumor at the vertex of the skull with elevated serum carcinoembryonic antigen: case report.

A primary Ewing's sarcoma arising in the skull is relatively rare. Although a small number of case reports noted elevated carcinoembryonic antigen (CEA) in patients with primary central nervous system (CNS) neoplasms, there is no report of Ewing's sarcoma/peripheral primitive neuroectodermal tumor (PNET) with elevated serum levels of CEA. A 7-year-old boy who had episodes of headache and vomiting had noticed a solid mass in the vertex of the head. Imaging studies revealed a large intra- and extracranial tumor at the vertex of the skull. Hematological examination demonstrated high serum levels of CEA: 91.09 ng/ml. The patient initially underwent an embolization of the bilateral middle meningeal arteries with Gelfoam particles. One week later, the patient was operated on and a subtotal resection of the tumor was performed. On histopathological and molecular genetic examination, the tumor was diagnosed as a Ewing's sarcoma/peripheral PNET. Immunohistochemical study showed strongly positive staining for CEA in the tumor cells. The serum level of CEA was normalized at 0.83 ng/ml after the tumor was removed and the boy underwent radiotherapy and 3 courses of chemotherapy. This is the first reported case of a primary Ewing's sarcoma/peripheral PNET at the vertex of the skull with elevated serum CEA.

Gastrointestinal stromal tumor of the stomach: report of a case.

We report herein the case of a 70-year-old woman found to have a gastrointestinal stromal tumor (GIST) of the stomach. Preoperative X-ray and endoscopic examination revealed a hemispheric submucosal tumor with central depression in the anterior wall of the gastric fornix. The tumor, which was 3 cm in diameter, was resected by a laparoscopy-assisted procedure. Histologic examination revealed that it was composed of spindle-shaped cells with elongated nuclei, and few mitoses. Most of the tumor cells showed immunoreactivity for vimentin and CD34, but not for alpha-smooth muscle actin, desmin, or S-100 protein. The PCNA index was 40.5%. Thus, the GIST did not show differentiation toward smooth muscle or neural cells. A gastrectomy was not performed because the small size of the tumor, and the paucity of the mitoses indicated that it was benign. Nevertheless, careful and long-term follow-up is needed to monitor for signs of possible local recurrence or distant metastases.

Metastasis of renal cell carcinoma to central nervous system hemangioblastoma in two patients with von Hippel-Lindau disease.

Here we report tumor-to-tumor metastases identified in two patients with von Hippel-Lindau (VHL) disease. The first patient had bilateral renal carcinomas and multiple cerebellar hemangioblastomas, and the second patient had a renal carcinoma and multiple hemangioblastomas in the retina, cerebellum and spinal cord. A cerebellar lesion from the first patient and a spinal lesion from the second patient contained two distinct components. The inner part of these tumors consisted of a nested mass of polygonal clear cells that expressed cytokeratin and epithelial membrane antigen, while the outer part of the tumors showed proliferation of capillaries and intervening foamy stromal cells that were negative for cytokeratin and epithelial membrane antigen. The tumors were thus considered to be hemangioblastomas complicated by metastatic lesions of renal cell carcinoma of clear cell type. These cases indicate that tumor-to-tumor metastasis should be considered when hemangioblastoma contains a clear cell carcinoma component in the setting of VHL disease, and that immunohistochemical staining for cytokeratin and epithelial membrane antigen is useful for the diagnosis.

Solid-pseudopapillary tumor of the pancreas: immunohistochemical localization of neuroendocrine markers and CD10.

To clarify the neuroendocrine differentiation and CD10 expression in solid-pseudopapillary tumors (SPTs) of the pancreas, we performed immunohistochemical analysis in 19 such tumors, including one solid-pseudopapillary carcinoma (SPC), along with 20 pancreatic neuroendocrine tumors (PNTs), six acinar cell carcinomas (ACCs), and one pancreatoblastoma (PB). We used antisera directed against CD56, synaptophysin, protein gene product 9.5, the alpha-subunit of Go protein, chromogranin A, CD10, trypsin, chymotrypsin, various cytokeratins (CKs), CA19-9, vimentin, and alpha-1-antitrypsin (AAT). All SPTs exhibited immunoreactivity for CD56 and CD10, and 15 expressed other neuroendocrine markers focally with the exception of chromogranin A. Frequent clustering of synaptophysin-positive cells was noted. Two cases contained a peculiar nodule that cytomorphologically and immunohistochemically resembled PNT. CD10-positive cells were scarce in one SPC. PNTs were CD56-positive, but often with faint intensity, and staining for other neuroendocrine markers, including chromogranin A, was diffusely positive. CD10 was detected, mostly in a focal pattern, in five PNTs. Pan-CK, CK8, CK18, and CK19 were more frequently demonstrated in PNT than SPT. Vimentin and AAT were often identified in PNT as well and were not specific for SPT. ACCs were CD56-negative, with the exception of one case designated as a mixed acinar-endocrine carcinoma. PB was focally positive for CD56 at the periphery of the tumor nests. Four ACCs and one PB exhibited focal CD10 reactivity. This study demonstrated the unique immunohistochemical features of SPT. Our results also suggest that SPT exhibits, at least focally, neuroendocrine differentiation, and that these neuroendocrine markers and CD10 are diagnostically useful.

Spinal atypical teratoid/rhabdoid tumor in an infant.

Atypical teratoid/rhabdoid tumor of the central nervous system in infancy and childhood was established as an entity based on histological, immunohistochemical, and cytogenetic studies. We report the case of a 7-month-old girl who presented with progressive paraplegia and hypesthesia of her legs. Imaging studies revealed a spinal cord mass occupying the entire spinal canal below the T(7) level. Through a T(12)-L(3) laminectomy, the intramedullary tumor was partially debulked. Histologically, the tumor specimen had rhabdoid cells, and immunostaining showed vimentin and cytokeratin positivity. No abnormality of chromosome 22q was detected with the fluorescence in situ hybridization method.

Increased incidence of follicular lymphoma in the duodenum.

The incidence of indolent lymphomas in the lymph nodes and extranodal regions is quite different. Follicular lymphoma (FL) is most common in the nodes, and it seems to be least common in the gastrointestinal (GI) tract, where mucosa-associated lymphoid tissue lymphoma arises most frequently. The authors report that the incidence of FL is unexpectedly high in the duodenum compared with other portions of the GI tract. FL was detected in only eight of 222 cases of GI lymphoma (3.6%). However, five cases of FL arose in the duodenum, which accounted for 38.5% of 13 duodenal lymphomas. Only in two patients did FL arise in either the stomach or the colorectum, and in the remaining patients FL was widespread with lymphomatous polyposis. Duodenal FL was composed of neoplastic follicles with small cleaved cells in dominance, and the immunophenotype of the lymphoma cells was CD10+, BCL-2+, CD20+, CD75+, CD79+, CD3-, CD5-, cyclin D1-, CD23-, and CD45RO-. All the patients were women age 37 to 66 years (average age, 52.4 yrs). In all patients the lymphoma was present around the ampulla of Vater, and four of five patients showed multiple small-size polyps. Although lymphoma cell infiltration was confined to the submucosa in the four patients examined, the regional lymph nodes were involved partially in two patients without distant metastasis. All patients are alive at 2 to 50 months of follow up (average, 27 mos), which is comparable with the prognosis for indolent nodal lymphomas. These results suggest that the duodenum has a distinct background of histogenesis of the lymphomas and that biopsy specimens from the duodenum with multiple polyps should be examined carefully.

Ganglioglioma in a patient with Turcot syndrome. Case report.

A 33-year-old woman with Turcot syndrome harbored a brain tumor and colon cancer and had a familial history of this syndrome. On histological examination, the brain tumor was found to have large and diffusely scattered ganglion cells within a diffuse background of astrocytic cells in a fibrillary matrix. The tumor was diagnosed as a ganglioglioma. No germline mutation in the adenomatous polyposis coli gene was detected using a protein truncation assay. These findings indicate that this patient had brain tumor-polyposis syndrome Type 1 of Turcot syndrome. This is the first report of a ganglioglioma related to Turcot syndrome.

Epithelioid trophoblastic tumor: morphological and immunohistochemical study of three lung lesions.

Epithelioid trophoblastic tumor (ETT) is a term proposed for an unusual variant of trophoblastic tumor that is closely related to choriocarcinoma but shows monomorphic growth of highly atypical trophoblastic cells instead of the typical dimorphic pattern of choriocarcinoma. We report here 3 cases of ETT, all of which were lung lesions probably originating from uterine trophoblastic disease. The antecedent pregnancies of the 3 cases were hydatidiform mole, invasive mole, and term pregnancy, respectively. The tumors were composed of highly atypical mononucleate cells, which mainly involved alveolar spaces, forming nests with central eosinophilic necrosis. Multinucleate giant cells were found within the nests, but they were fewer in number than in typical choriocarcinoma. The tumors were not associated with extensive hemorrhage or necrosis, except for 1 case, in which the ETT was combined with typical dimorphic choriocarcinoma. Immunohistochemically, multinucleate giant cells and occasional mononucleate tumor cells showed positivity for human chorionic gonadotropin. Staining for human placental lactogen was positive in rare multinucleate giant cells, and in 1 case, tumor cells showed diffuse positivity for placental alkaline phosphatase. Because ETT has a remarkably epithelioid appearance in cytological and architectural features, differentiation from the epithelial malignancies is problematic. Trophoblastic markers are frequently expressed in nontrophoblastic tumors, and reactivity for those markers alone is not sufficient for exclusion of other tumors. Rather, evidence of ETT comes from a combination of morphological features, immunohistochemical study, and clinical history.

Sex differences in oxidative damage in ddY mouse kidney treated with a renal carcinogen, iron nitrilotriacetate.

Iron-induced free radical injuries in male and female ddY mice, especially the sex difference and its mechanisms, were studied after an i.p. injection of a renal carcinogen, ferric nitrilotriacetate. Male mice were much more susceptible to iron-induced free radical injuries than female mice. Oxidative modification of proteins and DNA occurred more strongly in males than in females, as measured by protein carbonyl content and 8-hydroxydeoxyguanosine, respectively. Histochemical detection of 4-hydroxy-2-nonenal-modified proteins using an antibody and DNA fragmentation as detected by the TUNEL method also showed that males are more severely damaged than females, especially in the proximal convoluted tubules. These results could not be explained by the difference in iron status between male and female mice. In fact, the toxic so-called 'free' iron in serum and kidney were not different between male and female mice and storage iron, such as ferritin and hemosiderin, was also comparable in both kidneys. In previous studies we proposed the glutathione cycling hypothesis to explain the sex differences. The half-life of glutathione in the kidney was significantly shorter in males (29 min) than in females (57 min), as determined by the glutathione decrease after buthionine sulfoximine treatment, a specific inhibitor of glutathione synthesis. The specific activity of gamma-glutamyltranspeptidase (EC 2.3.2.2) in female mice was 73% of that in male mice. These results suggest that the faster glutathione turnover in males could account for the higher susceptibility to oxidative injury by supplying the reducing equivalent that reduces Fe(III) to Fe(II), thereby facilitating iron-catalyzed free radical reactions.

Induction of renal cell carcinoma in male Wistar rats treated with cupric nitrilotriacetate.

Systemic administration of copper, an essential trace metal in the human body, has never been reported to be carcinogenic in animals. We investigated the induction of tumors by the cupric complex of nitrilotriacetic acid (Cu-NTA) in male Wistar rats. Thirty-two animals received ip injections of Cu-NTA, 3 to 5 mg of copper/kg body weight 5 days a week for 12 weeks, and were kept under close observation. For comparison, 31 animals received ip injections of ferric nitrilotriacetate (Fe-NTA), 5 to 10 mg of iron/kg body weight, and 16 animals received nitrilotriacetic acid (NTA) alone at the molar dose equivalent to Cu-NTA for the same period of time. Sixteen animals were left untreated as controls. Fourteen animals in the Cu-NTA group died of hepatic failure during the treatment period, and renal cell carcinoma (RCC) was induced in eight animals (25%). Of these, four animals died of either pulmonary metastasis or intraperitoneal hemorrhage. A total of 12 RCC were obtained, of which six tumors were > or = 5 mm. The Cu-NTA group yielded fewer RCC and required a longer latent period for their incubation than the Fe-NTA group. Furthermore, the Cu-NTA group showed one hepatocellular carcinoma and one high-grade sarcoma of hepatic origin. No renal or hepatic tumor was observed in the NTA or control groups. The nontumorous part of the kidney treated with Cu-NTA presented hemosiderosis caused by copper-induced hemolytic anemia. This is the first report that systemic administration of copper compounds can induce malignant tumors in animals. Not only copper but also iron may play a role in the Cu-NTA-induced renal carcinogenesis model.

Absence of ras mutations and low incidence of p53 mutations in renal cell carcinomas induced by ferric nitrilotriacetate.

Renal cell carcinomas induced in male Wistar rats by iron chelate of nitrilotriacetate (Fe-NTA) were examined for mutations in ras oncogenes and p53 tumor suppressor gene. Fourteen primary tumors and two metastatic tumors from 11 animals were evaluated. Exons 1 and 2 of the H-, K-, and N-ras genes were amplified by polymerase chain reaction (PCR), and the presence of mutations was examined by direct sequencing. Exon 5 through exon 7 of p53 gene, including the 3' half of the conserved region II and the entire conserved region III through V, were surveyed for point mutations by PCR-single stranded conformation polymorphism (SSCP) analysis. Direct sequencing of the ras genes showed no mutations in codon 12, 13, or 61 among the tumors evaluated. SSCP analysis of p53 gene exon 6 indicated conformational changes in two primary tumors. One tumor had a CCG-to-CTG transition at codon 199, and the other had an ATC-to-att transition at codon 229 and two nonsense C-to-T transitions. These results suggest that neither ras genes nor p53 gene play a major role in the development of renal cell carcinomas induced by Fe-NTA.

Cloning of the rat homologue of the von Hippel-Lindau tumor suppressor gene and its non-somatic mutation in rat renal cell carcinomas.

Recently, von Hippel-Lindau (VHL) gene mutations were detected in non-inherited, sporadic human renal cell carcinomas (RCs) at a high frequency. In order to determine whether or not the VHL gene is also a critical gene in rat RCs, we cloned and sequenced the rat homologue of human VHL gene and searched for mutations of the VHL gene in rat RCs. Mutations in the VHL gene were not detected in spontaneous RCs of the Eker rat model or in ferric nitrilotriacetate-induced rat RCs using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method. These data indicate that mutation of the VHL tumor suppressor gene is not an event in rat renal carcinogenesis, at least in our present systems.

Iron binding, a new function for the reticulocyte endosome H(+)-ATPase.

The significance of the H(+)-ATPase in iron absorption by rabbit reticulocytes is explored using isolated endosomes, effects of inhibitors, and the purified proton pump. We have recently reported H(+)-ATPase-mediated iron transfer across a liposomal membrane (Li et al., 1994). In this report, the effect of H(+)-ATPase inhibitors on iron mobilization is investigated at pH 6.0 in the presence of 15 microM FCCP in order to dissociate 59Fe(III) from transferrin and eliminate the kinetic effects of acidification by the ATPase. Iron transport by isolated endosomes is decreased 50% by the cation pore inhibitor dicyclohexylcarbodiimide (DCCD) for ascorbate-mediated iron mobilization and increased by 40-50% when NADH and ferrocyanide are used as electron donors. In contrast, the ATPase hydrolysis inhibitors N-methylmaleimide (NEM) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD) increase iron mobilization when NADH and ferrocyanide are used as reductants but have negligible effects for ascorbate. The differential inhibition or enhancement by DCCD, NEM, and NBD with respect to the reductants used for mobilization indicates that the H(+)-ATPase may be involved in the multiple pathways or iron transport found in isolated rabbit reticulocyte endosomes. Effects of inhibitors of ATP hydrolysis suggest significant structural interactions between the proton pump and sites for iron binding and/or reduction. The isolated H(+)-ATPase binds iron as revealed by using nondenaturing electrophoretic and chromatographic methods. One class of iron binding sites is suggested to be the 17.5 kDa proton pore subunits of the H(+)-ATPase which also covalently react with DCCD.(ABSTRACT TRUNCATED AT 250 WORDS)

Degradation of DNA in dried tissues by atmospheric oxygen.

Recently, DNA of extinct creatures have been brought into analyses. The difficulty in this field of research is DNA degradation. Oxidative damage is considered to be one of the causes of the DNA degradation. We studied how DNA in dried tissue was affected by atmospheric oxygen using freeze-dried rat liver as a model. In tissues exposed to oxygen, DNA degradation occurred within several months and the amount of 8-hydroxy-2'-deoxyguanosine in DNA rapidly increased. The DNA degradation was inhibited by lipid extraction prior to the exposure to atmospheric oxygen. Purified lambda phage DNA was not affected by oxygen. Cellular DNA and RNA were degraded slowly in nitrogen air. These results suggest that both atmospheric and endogenous oxygens play a role in DNA degradation in dried tissues.

Influence of cigarette smoking and schistosomiasis on p53 gene mutation in urothelial cancer.

The mutation patterns of the p53 tumor suppressor gene have been shown to reflect the specific carcinogen(s) involved, or the epidemiological background in some cancers. To elucidate the impact of cigarette smoking or bilharzial infection on the p53 gene mutation pattern, 61 cases of urothelial cancer from Japan and 7 cases of bladder cancer with schistosomiasis from Egypt were examined for mutations of the p53 gene. In total, p53 gene mutations were detected in 20 Japanese cases (33%) and 6 Egyptian cases (86%). Although the incidence of p53 gene mutation was not significantly influenced by habitual smoking, a different mutation pattern was observed as follows: 4 of 10 mutations in smokers in Japan were A:T to G:C transitions, whereas such mutations were not detected in any of 10 mutations in nonsmokers, or in any of 6 mutations associated with schistosomiasis. Although no specific mutation pattern was detected for the squamous cell carcinomas with schistosomiasis, all 8 base substitutions observed in tumors with squamous cell carcinomas occurred at G:C sites, whereas base substitutions at A:T sites were observed in 33% (6 of 18) of mutations in transitional cell carcinomas. Our results suggest that cigarette smoking may have a significant impact on the mutations of the p53 gene in urothelial cancers. Furthermore, the distinct spectrum of the p53 gene mutation found in tumors with squamous cell carcinomas may reflect their unique etiological backgrounds.

The effect of formalin fixation on restriction endonuclease digestion of DNA and PCR amplification.

The effect of formalin fixation on DNA and on polymerase chain reaction (PCR) amplification was investigated. Lambda phage DNA fixed in buffered formalin showed incomplete digestion on restriction endonuclease treatment. The resistance to restriction digestion was dependent on the temperature of fixation, but not affected by salt concentration of the fixative. Lambda phage DNA fixed in unbuffered formalin showed poor PCR amplification due to degradation of DNA during fixation. Lambda phage DNA fixed in buffered formalin evaded degradation and suited for template of amplification. Feasibility of formalin-fixed tissues as sources for PCR amplification was also investigated with primers producing 128 bp fragment of c-Ki-ras exon 2. Although DNA from tissues fixed for 3 months showed amplification, there was no amplification from tissues kept in unbuffered formalin for longer than 6 months.

Organ distribution of mutant mitochondrial tRNA(leu(UUR)) gene in a MELAS patient.

Point mutations in the mitochondrial tRNA(leu(UUR)) gene have been recently reported in patients with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). To investigate the relationship between the degree of heteroplasmy and the organ damage, the ratio of mutant and wildtype genes was quantitated in 14 different organs obtained at an autopsy case of MELAS. The percentages of mitochondrial tRNA(leu(UUR)) gene carrying an A to G transition mutation at nucleotide 3243 were determined by the restriction enzyme digestion of the polymerase chain reaction products. The organs largely depending on oxidative phosphorylation for the sources of energy contained higher proportions of the mutant tRNA(leu(UUR)) gene than organs with a lower oxygen demand. However, the percentage of the mutant genes was similar in both symptomatic and asymptomatic organs with a higher oxygen demand.

Infrequent ras mutation in human stomach cancers.

Mutations of ras oncogenes in 37 human stomach cancers and 13 adenomas were investigated with regard to the histological phenotypes using polymerase chain reaction (PCR), allele-specific oligonucleotide hybridization and/or direct sequencing of the PCR products. The ras mutation was found only in one case (2.7%), the histology of which was poorly differentiated adenocarcinoma. We found no mutation in stomach adenomas. The mutation consisted of a guanine-to-adenine transition in the first base of codon 13 of c-Ki-ras which replaced wild-type glycine with serine, indicating that a putative glycine-to-aspartic acid change is not necessarily the critical event for c-Ki-ras gene activation in codon 13. These results further confirm the infrequency of ras mutation in stomach tumors and also suggest that ras mutations are not specific to the differentiated type of stomach cancer.