361 @ 1990 The Japanese Society of Pathology

Atypical Endocrine Granules in Atypical Endocrine Tumor (AET) of the Lung An Immunoelectron Microscopic Study

Masashi Fukayama, Etsuko Furukawa, Yumiko Shiozawa, Yukiko Hayashi, and Nobuaki Funata

Highly dense granules are a hallmark for recognizing atypical endocrine tumor (AET) of the lung. We report a case of AET with many atypical neurosecretory-type granules : moderately dense granules (mean size 373.7 nm) and “target” granules with a central dense core (425.1 nm), both apparently larger than the highly dense granules (223.3 nm). lmmunoelectron microscopical studies demonstrated that all three types of granule were positive for gastrin-releasing peptide (GRP), human chorionic gonadotropin a-subunit (hCGa), calcitonin or serotonin. Although the size profiles of positive granules were similar for calcitonin and hCGa, they were different from those of GRP or serotonin granules. The presence of atypical granules and the different size profiles of hormonal products i n AET indicate that caution is required in ultrastructural evaluation of granules in lung carcinomas. Acta Pathol Jpn 40: 361-366, 1990. Key words: Atypical endocrine tumor, Lung, Immunoelectron microscopy

INTRODUCTION I t has become apparent that endocrine tumor of the lung is morphologically diverse. McDowell ef a/. (1) were first to clearly define atypical endocrine tumor (AET) of the lung: AET had previously been classified histologically as poorly differentiated carcinoma of the non-small cell category, but was proven by electron microscopy to contain numerous densecored granules, an ultrastructural hallmark of endocrine cell differReceived August 21, 1989. Accepted for publication December 20, 1989. Department of Pathology, Tokyo Metropolitan Komagome Hospital, Tokyo. Mailing address: Masashi Fukayama, M.D. (jgaEA), Department of Pathology, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113, Japan.

entiation (2). Since then, several workers have reported the frequency and biological behavior of AET (3-5). AET cannot be diagnosed without the use of electron microscopy or special staining procedures, and was reported to comprise a t least 3% of all lung tumors in a consecutive series of surgical resections (3). AET resembles non-small cell carcinoma in clinical presentation, median survival, and response to ordinary lung cancer chemotherapy (4, 5). However, two reports (5, 6) have documented that a few patients showed a dramatic response to streptorotocin/5-fluorouracil, like that seen in cases of metastatic carcinoid. Therefore, it is important t o clarify the features of AET as an endocrine tumor as well as to diagnose the tumor correctly. We screened our archive of fixed frozen sections of lung carcinomas with monoclonal antibodies against synaptophysin and against small-cell lung cancer (7). One of 61 lung cancers, which were originally classified as non-small cell carcinoma by ordinary histological examination with mucin histochemistry, showed diffuse staining with all monoclonal antibodies, revealing that it was AET. Subsequent examination by electron microscopy demonstrated atypical granules of the neuroendocrine type. In the present study, we investigated these granules using immunoelectron microscopy for hormonal products to clarify their characteristics.

CASEREPORT A 5-cm-diameter tumor was resected from the lower lobe of the left lung of a 56-year-old woman by left pneumonectomy. She had smoked 15 cigarettes daily for 10 years. The patient had no history of carcinoid syndrome. The tumor was diagnosed histologically as poorly differentiated adenocarcinoma. The neoplastic cells with large polygonal cytoplasm were arranged in trabeculae or nests. There were, however, occasional

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Endocrine Granules in AET of the Lung (Fukayama et a/.)

stored a t -80°C. For ult rast ruct ural observation, the ultra t hin sect ions were double-stained with uranyl acetate and lead citrate, and also with periodic acid-methenamine silver (PAM). Control sections for PAM staining were from five cases of carcinoid tumor. For immunohistochemical studies, monoclonal antibodies against small cell carcinoma (8-1 1) and synapto physin (12) were used to define the endocrine differentiation of the carcinoma. Hormonal products examined were gastrin-releasing peptide (GRP), sero tonin, human chorionic gonadotropin a-subunit (hCGa), calcitonin and adrenocorticotropic hormone (ACTH). Double immunostaining was performed to assess the frequency of synchronously positive cells in the tumor, with use of monoclonal anti-hCGa-antibody and polyclonal antibodies against other products. To investigate the nature of the granules in the AET, immunoelectron microscopy was carried out using the antibodies for the hormonal products mentioned above. The staining procedures for immunohistochemistry (13), double immunostaining and immunoelectron microscopy (14) have been reported elsewhere. Morphometrical analyses were performed with an image-analyzer, and the area and maximum diameter of the granules were evaluated. Because the distribution pattern of these parameters was apparently log-normal, statistical analysis was performed on logarithms of the values, using Welch’s t test and F-test.

Figure 1. Histological features of the present case of AET. Nests of large polygonal cells with abortive lumina. Note intracytoplasmic and intercellular mucus (arrows). Alcian blue, pH 2.5 and PAS double staining.

lumina filled with alcianophilic and PAS-positive mucus. lntracytoplasmic mucus was also identified (Fig. 1). There were metastases to the hilar lymph nodes. The patient was discharged after radiotherapy but died six months after the operation.

MATERIALS AND METHODS In our routine sampling of pulmonary tumors starting in 1986, small cubes of tumor tissue were fixed with phosphate-buffered 2.5% glutaraldehyde for electron microscopy, and thin slices of tumor tissue were fixed with periodate- lysine- pa raforma ldehyde (PLP) for immunohistochemistry. The former were routinely postfixed in 2.0% osmium tetroxide, dehydrated in a graded ethanol series and embedded in Epon 812. The latter were embedded in OCT compound, frozen quickly in dry ice-hexane after overnight fixation of PLP, and then

RESULTS Electron microscopy The cytoplasm of the carcinoma cells was relatively rich with many granules of various sizes. Ribosomes and mitochondria were abundant, and the Golgi apparatus and rough endoplasmic reticulum were well devel-

Table 1. Morphometrical Parameters of the Granules in Atypical Endocrine Tumor of the Lung Number of Granules Electron Microscopy Highly dense Moderately dense Target lrnrnunoelectron Microscopya GRP HCG Ca Icitonin Serotonin

A pm2)

122 57 100

3.031 8.702 12.388

:

138 114 137 64

4.390 6.109 6.383 9.813

:

:

:

: :

MxD (nm)

:

-1.5184 t0.2385 -1.0604 t0.2839 -0.9070 t0.2166

223.3 373.7 425.1

: :

- 1.3575bt0.1878 -1.2140 t0.1813 -1,1950 f0.1749 -1.0082bt0.2363b

261.6 304.0 318.7 385.7

: -0.5824bt 0.0949 : -0.5171 +0.0909 : -0.4966 t0.0885 : -0.41 37b+O.l181b

:

-0,6511 k0.1222 -0.4275 t0.1365 -0.3715 t0.1057

A, Area ; MxD, Maximum diameter. a The data were obtained from round granules, excluding positive target granules. b Significant differences (p

Atypical endocrine granules in atypical endocrine tumor (AET) of the lung. An immunoelectron microscopic study.

Highly dense granules are a hallmark for recognizing atypical endocrine tumor (AET) of the lung. We report a case of AET with many atypical neurosecre...
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