Shuji Hamazaki

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.

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.

The effect of formalin fixation on DNA and the extraction of high-molecular-weight DNA from fixed and embedded tissues

The effect of formalin fixation on DNA and extraction of DNA from fixed tissues was investigated to retrieve archival tissue samples stored in pathology departments for molecular biological studies. Aldehyde fixatives resulted in degradation of DNA at room temperature but not at 4 degrees C. The degradation also occurred in formalin when the pH or the salt concentration was low, or the formic acid level was high. Restriction endonuclease digestion of fixed DNA was incomplete after formalin fixation and this was also temperature-dependent. Thus, relatively intact DNA was obtained from the tissues fixed in buffered formalin at 4 degrees C or fixed with microwave irradiation. The use of modified tissue-lysing buffer containing 4M urea allowed extraction of high-molecular-weight DNA suitable for Southern blot analysis from fixed and embedded tissues. In conclusion, fixation with buffered formalin at 4 degrees C permitted extraction of DNA of sufficient quality for Southern blot analysis.

Oxygen Reduction and Lipid Peroxidation by Iron Chelates with Special Reference to Ferric Nitrilotriacetate

A certain iron chelate, ferric nitrilotriacetate (Fe3+-NTA) is nephrotoxic and also carcinogenic to the kidney in mice and rats, a distinguishing feature not shared by other iron chelates tested so far. Iron-promoted lipid peroxidation is thought to be responsible for the initial events. We examined its ability to initiate lipid peroxidation in vitro in comparison with that of other ferric chelates. Chelation of Fe2+ by nitrilotriacetate (NTA) enhanced the autoxidation of Fe2+. In the presence of Fe2+-NTA, lipid peroxidation occurred as measured by the formation of conjugated diene in detergent-dispersed linoleate micelles, and by the formation of thiobarbituric acid-reactive substances in the liposomes of rat liver microsomal lipids. Addition of ascorbic acid to Fe3+-NTA solution promoted dose-dependent consumption of dissolved oxygen, which indicates temporary reduction of iron. On reduction, Fe3+-NTA initiated lipid peroxidation both in the linoleate micelles and in the liposomes. Fe3+-NTA also initiated NADPH-dependent lipid peroxidation in rat liver microsomes. Although other chelators used (deferoxamine, EDTA, diethylenetriaminepentaacetic acid, ADP) enhanced autoxidation, reduction by ascorbic acid, or in vitro lipid peroxidation of linoleate micelles or liposomal lipids, NTA was the sole chelator that enhanced all the reactions.

Acute renal failure and glucosuria induced by ferric nitrilotriacetate in rats

Nitrilotriacetate (NTA), an effective metal-chelating agent, has been used as a substitute for polyphosphates in household laundry detergents. Nephrotoxicity and renal tumorigenicity have been reported in experimental animals that received high doses of NTA po for 4 weeks to 2 years. Since NTA exists in water as a variety of NTA-metal complexes, it was important to investigate the biological effects of NTA in a complexed form. In this study, acute and subchronic toxicity of a ferric iron chelate of NTA (Fe-NTA) was investigated in rats. When Fe-NTA was given ip, acute tubular necrosis and renal failure occurred following a single injection of 15 mg iron/kg. Repeated injections of sublethal doses produced degeneration and necrosis of the proximal tubular epithelium and was associated with polyuria, glucosuria, aminoaciduria, and azotemia. After 9 days of treatment, regeneration of the tubular epithelium with atypical cells was observed. Except for a parenchymal iron deposit, no marked changes were observed in other organs. None of these effects were observed in animals given noncomplexed NTA. In conclusion, the toxicity observed following high doses of NTA given po may be the result of an absorbed metal-NTA chelate.

Liver, kidney, and central nervous system toxicity of aluminum given intraperitoneally to rats: A multiple-dose subchronic study using aluminum nitrilotriacetate

In the first test, aluminum nitrilotriacetate (Al-NTA), aluminum chloride, aluminum potassium sulfate, or saline was injected ip, employing male Wistar rats. Each group consisted of ten rats. Al was given in a dose of 5 mg Al/kg body wt/day, for 14 days. Only those rats given Al-NTA showed morphological damage of the liver and kidney. Damages included diffuse midzonal coagulation necrosis of hepatocytes and acute proximal tubular necrosis of the kidney at Day 4. Seven of ten rats given Al-NTA died within 5 days. The second test was performed in metabolic cages. Al-NTA, in a dose of 1.5 to 2.0 mg Al/kg body wt/day, and NTA, of an equivalent dose, were injected ip for 54 days. Midzonal coagulation necrosis and some regenerative changes were observed in the hepatic parenchyma at Day 8. At the end of the study, complete regeneration occurred in the liver. Biochemical tests at Days 6, 13, and 28 showed high amounts of GOT, GPT, LDH, gamma-GTP, and ALP. Necrosis of proximal tubular cells of the kidney and some regeneration was noted at Day 8. Metabolic acidosis was demonstrated at Days 6, 13, and 28. Moreover, from Day 38 on, atrophy of the nerve cells of the cerebrum and demyelination of the brain stem were observed. Control rats given NTA did not exhibit any organ damage. It is concluded that a relatively small dose of Al can produce toxicosis when given with certain metal chelators.

Impairment of bone formation with aluminum and ferric nitrilotriacetate complexes

SummaryThe deleterious effects of aluminum(Al) and iron(Fe) on bone formation were studied in the presence of nitrilotriacetate (NTA) as a chelator. Both Al-NTA (1.0–1.5 mg Al/kg/day, n=12)- and ferric nitrilotriacetate (Fe-NTA) (2.0 mg/kg/day, n=4)—treated Wistar rats showed renal insufficiency blood urea nitrogen [BUN] levels of 25±8.8−20±0.7 compared to 12±0.7–11±0.4 mg/dl), osteomalacia with a relative osteoid volume of 31.5±5.6−13.2±2.4 compared to 4.6±1.8−0.83±0.12%, and bone growth retardation (3.1±0−3.0±0.2 compared to 3.4±0−3.3±0.1 cm) in 24 control rats. Dietary vitamin E(VE) supplementation prevented the Fe-NTA-induced impairment, but not the Al-NTA toxicity. Aluminum was deposited at the interface between osteoid and mineralized bone, while Fe was deposited in the osteoblasts and osteoclasts. There seems to be a positive correlation between hypophosphatemia and osteomalacia but carboxy-terminal parathyroid hormone (C-PTH) and calcium (Ca) levels in the serum were not related to the degree of osteomalacia. Administration of Al-NTA results in more bone Al deposition than that of aluminum chloride (AlCl3) (450±40 compared to 211±18 mg/kg fat-free dry weight). The Fe-NTA bone change is related to VE-preventable cellular injury, being consistent with the notion that Fe-NTA toxicity is caused by lipid peroxidation. Al-NTA can be used as an animal model of renal osteodystrophy. Osteodystrophy by Al in chronic renal failure may be mediated by the intrinsic chelator or chelating substance(s) retained in the body fluid due to renal insufficiency.

Effect of dietary vitamin E on ferric nitrilotriacetate-induced nephrotoxicity in rats

The effect of dietary vitamin E on renal tissue damage and lipid peroxidation was investigated following treatment with ferric nitrilotriacetate (Fe-NTA) in rats. Almost 100% renal proximal tubular necrosis was observed in the vitamin E-deficient rats following Fe-NTA treatment (5 mgFe/kg body wt, ip) as early as 12 hr. In the vitamin E-supplemented rats, no injury was observed in the proximal convoluted (cortical) tubules, although some injury was seen in the medullary outer stripe (mostly pars recta of the proximal tubules) 24 hr after Fe-NTA treatment. The tissue lipid peroxidation was dose-dependently increased 1 hr after a single ip injection of Fe-NTA (1 to 10 mg Fe/kg body wt). Vitamin E-deficient animals had an increased tissue content of thiobarbituric acid-reactive substance following Fe-NTA treatment, whereas vitamin E-supplemented animals showed suppressed lipid peroxidation. This study indicates that vitamin E provides some protection against the nephrotoxicity and associated lipid peroxidation induced by Fe-NTA.

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.

Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis, and Stroke-like Episodes

We present an autopsy report on a 14‐year‐old girl with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke‐like episodes (MELAS), placing emphasis on the mitochondrial enzymatic histochemistry of the 3 types skeletal muscle and cardiomyocytes. Generalized muscular atrophy, cardiac hypertrophy, cerebral cortical laminar necrosis, basal ganglia calcification and liver steatosis were observed. In the skeletal muscles, modified Gomoris trichrome staining demonstrated scattered ragged red fibers, and histochemical staining for mitochondrial enzymes showed intense positivity in the subsarcolemmai zones of some muscle fibers. Some of the hypertrophic cardiomyocytes also showed a ragged red appearance with the modified Gomoris trichrome stain. Histochemical staining for mitochondrial enzymes showed patchy loss of enzymatic activity in the myocardium. Electron microscopically, extreme accumulation of enlarged mitochondria and severe loss of myofibrils was observed in both skeletal muscle fibers and cardiomyocytes. The arteriolar smooth muscle cells also showed a mild increase in mitochondria. Acta Pathol Jpn 39: 599 606, 1989.