Journal of Surgical Oncology 45146161 (1990)

Small Cell Lung Carcinoma: Clinicopathological, Immunohistochemical, and Ultrastructural Study SHYA-WEN SHY, MD, MS, WE!-HWA LEE, MD, PhD, MING-CHIH CHOU, MD, PhD, YIEH-SHYONG LAI, MD, PhD, AND YEN-CHANG TU, MD From the Departments of Pathology 6.-W.S., Y.-S.l.) and Chest Surgery (M.-C.C.), Chung-Shan Medical and Dental College, Taichung, Taiwan and the Department of Pathology, Jri-Service General Hospital, National Defense Medical Center, Taipei, Republic of China (W.-H.L., Y.-C.T.1

Sixty-seven cases of small cell lung carcinoma (SCLA) in Tri-Service General Hospital (TSGH) during the past 16 years were studied. For patients with extensive stage of disease, the mean survival time and 2-year survival rate were 7.2 months and 3.1% versus 13.4 months and 16.7% for patients with limited stage. A better prognosis was obtained by treatment with a combination of intensive chemotherapy and radiotherapy. Immunohistochemical studies were performed by the peroxidase-antiperoxidase method. The positive rates in descending order were bombesin (80%), synaptophysin (74.3%), neurofilament (68.6%), neuron-specific enolase (60%), low molecular weight cytokeratin (54.3%), high molecular weight cytokeratin (25.7%), chromogranin-A (22.9%), adrenocorticotrophic hormone (0). Seven cases were examined and found to be ultrastructure; only 3 cases were found to contain neurosecretory granules. We emphasize that electron microscopy is not necessary as a routine diagnostic procedure, while light microscopy should be employed whenever possible; the immunohistochemical study should be considered within this context. KEYWORDS:cytokeratin, neurofilament, neuron-specific enolase, synaptophysin, chromogranin, bombesin

INTRODUCTION Lung cancer is the most frequently diagnosed neoplasm in the world. If the current increase in incidence continues, 200,000 people will be dead from lung cancer in the United States in the year 2000 [ 11. Small cell lung carcinoma (SCLA) is a highly malignant tumor and represents 2&25% of all human lung cancers [2]. Approximately 30,000 new cases and 27,000 deaths from SCLA were expected in the United States in 1987 [3]. This highly malignant tumor has distinct clinical, morphological, biological, biochemical, and cytogenetic properties [4-61. Patients with SCLA always have a poor prognosis regardless of any treatment. For patients with extensive stage of disease, the median survival time is about 6 months [7]. It has been reported that SCLA showed positive staining of neurofilament [6,8-111. In contrast, all other 0 1990 Wiley-Liss, Inc.

types of lung carcinoma were found to be cytokeratinpositive and neurofilament-negative. From these results they concluded that SCLA was derived from neural cells and that other types of lung carcinoma were derived from cells with epithelial differentiation. SCLA usually exhibits many neuroendocrine properties. It synthesizes peptide hormone and has the capacity for amine synthesis [ 12-15]. Furthermore, dense neurosecretory granules were identified in small cell lung carcinoma by electron microscopy [2,6,16,17]. All these accumulating data suggest that SCLA belongs to the neuroendocrine tumors. Accepted for publication July 24, 1990. Address reprint requests to Dr. Shya-Wen Shy, Department of Pathology, Chung-Shan Medical and Dental College, 113, Section 2 , Ta-Ching Street, Taichung, 40203, Taiwan.

Small Cell Lung Cancer

MATERIALS AND METHODS Materials Sixty-seven cases of SCLA were identified by the Pathology Department of the Tri-Service General Hospital. The time interval of study was the 16-year period between 1972 and 1988. Methods Clinical studies. The charts of these patients were reviewed, including age, sex, symptoms and signs, smoking history, chest roentgenogram, treatment, and prognosis. Light microscopic studies. All the specimens were fixed in 10% formalin, embedded in paraffin, and sectioned at intervals of 5pm. The sections were stained routinely with haematoxylin and eosin, and examined by a Nicon type 104 light microscope for histological appearance. Immunohistochemical studies. Paraffin-embedded tissue sections from all cases were used for indirect immunoperoxidase staining. Seven immunohistochemical markers were studied: cytokeratin (Enzo), neurofilament (Immunotech), neuron-specific enolase (Dako), chromogranin-A (Enzo), synaptophysin (Boehringer), ACTH (Dako), and bombesin (Biomeda). The concentration of primary antiserums, incubation time, and temperature of the reaction for each antibody are shown in Table I. Electron microscopic studies. Ultrastructural studies were performed in 7 cases of SCLA. Tissues for ultrastructural studies were fixed in 2.5% glutaraldehyde for 24 hours, post-fixed in 1% osmium tetroxide for 2 hours, then embedded in Epon-Araldite. One micrometer thick, methylene-blue sections were examined for orientation, and representative tumor areas were selected for ultra-thin sectioning. Thin sections were stained with uranyl acetate and lead citrate. All were examined by a Zeiss EM- 109 electron microscope. RESULTS Clinical Studies These 67 cases of SCLA represented 7.5% of all lung cancers. Males were predominant with a ratio of 5.7:1.

Ages ranged from 21 to 75 years with an average of 61 years. Table I1 shows the symptoms and signs recorded at the time of diagnosis. Cough was the most frequent initial symptom. Table I11 is a tabulation of the initial roentgenographic findings, and Table IV shows the anatomic location of the lesion as determined by roentgenography and bronchoscopy. Both tables emphasize the central location of this malignancy. Three-fifths of the tumors were located in the right lung, with the most frequent location being the right upper lobe (35.8%). Routine chest roentgenogram most frequently presented as hilar enlargement (44.8%) and mediastinal widening (40.3%). Blood vessel and lymphatic invasion were usual. Regional and distant lymph nodes were involved in 67.2% of the cases and the bone in 32.8% (Table V). For patients with limited-stage SCLA, the mean survival time and 2-year survivals were 13.4 months and 16.7% versus 7.2 months and 3.1% for patients with extensive-stage SCLA. Only one patient survived for more than 3 years (Table VI). A better prognosis was obtained by utilizing combination chemotherapy and radiotherapy. Sixteen cases were in this therapeutic program with a mean survival time of 13.8 months (Table VII).

Light Microscopic Studies According to the 1981 World Health Organization (WHO) classification scheme of lung tumors (Table VIII), SCLA was divided into the following subtypes: oat cell carcinoma, intermediate cell type, and combined oat cell carcinoma. In this study, 45 cases belonged to the oat cell carcinoma (Fig. l), 21 cases were intermediate cell type (Fig. 2), and only 1 case was combined oat cell carcinoma. The tumor cell arrangement showed various patterns, i.e., sheet, ribbon, rosette, trabecular, and ductile. The crushing sign was noted in 35 cases and deposits of penvascular basophilic material (Fig. 3) was seen in 21 cases (Table IX). Immunohistochemical Studies Table X shows the results of staining for the seven immunohistochemical markers. In descending order of

TABLE I. Antibodies Utilized in the Immunohistochemical Study of SCLA Antibody NSE Cytokeratin Neurofilament Chromogranin-A Synaptophysin Bombesin ACTH

Supplier

Type

Dako Enzo Immunotec Enzo Boehringer Biomeda Dako

Polya Monob Mono Poly Mono Poly Poly

aPoly = polyclonal. bMono = monoclonal.

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Dilution 1:200 1:lOO 1:lO 1:2,000 1:lO 1:50

Prediluted

Temperature ("C)

Incubation (h)

28 28 28 28 28 37 28

1 1 1 1 1

0.5 1

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TABLE 11. Incidence of Symptoms and Signs at the Time of Diagnosis of 67 Cases SCLA

TABLE VI. Survival Data of SCLA Over a 16-Year Period at TSGH

Symptom

Survival time (months)

Cough Dyspnea Chest pain Body weight loss Hemoptysis

svcsa Others

Cases

Percentage

48 25 19 18 12 5 11

71.6 37.3 28.3 26.9 17.9 7.5 16.4

aSVCS = superior vena cava syndrome.

Cases 30 27 22 19 18 5

Percentage 44.8 40.3 32.8 28.4 26.9 7.5

TABLE IV. The Comparison of Anatomic Location of SCLA Location

TSCRMHa

BHTSS~

TSGHC

5 3d

26.9 7.7 31.7 17.3 16.4

35.8 25.4 4.5 20.9 13.4

Right upper lobe Right middle lobe Right lower lobe Left upper lobe Left lower lobe

1 4 36 6

aTSCRMH = Thoracic Service Cancer Registry of Memorial Hospital. BHTSS = Barnes Hospital Thoracic Surgical Service. CTSGH= Tri-Service General Hospital. dNos. = percentage.

TABLE V. The Comparison of Metastatic Site of SCLA Site Bone Liver Brain Lymph node Kidney

Limited

Extensive

9 1 .7a 54.2 16.7 0 0 13.4

40.6 21.9 3.6 3.6 0 7.2

a N ~=~percentage. . bM.S.T. = Mean survival time

TABLE 111. Chest Roentgenographic Findings of 67 Cases SCLA Hilar enlargement Mediastinal widening Tumor mass Pleural effusion Obstructive sign Others

6 12 24 36 60 M.S.T. (monthsIb

Clinical staging

TSCRMHa

BHTSS~

TSGH‘

49 58 37 88 28

24.1 27.8 16.7

32.8 11.9 14.9 61.2

“SCRMH = Thoracic Service Cancer Registry of Memorial Hospital. bBHTSS = Barnes Hospital Thoracic Surgical Service. ‘TSGH = Tri-Service General Hospital. dNos. = percentage.

frequency, bombesin was observed in the tumor cells of 80% of SCLA (Fig. 4), synaptophysin in 74.3% (Fig, 5 ) , neurofilament in 68.6%, neuron-specific enolase in 60% (Fig. 6), low molecular weight cytokeratin in 54.3%, high molecular weight cytokeratin in 25.7%, chromogranin-A in 22.9%, and ACTH in none.

Electron Microscopic Studies Ultrastructural observation was performed in seven cases of SCLA. The tumor cells also consisted of dark nuclei, which were irregularly filled with chromatin clumps of various sizes, the shape of the nuclei was found to be remarkably irregular, and the nucleoli were relatively small. The most striking feature of the electron microscopic investigation was the finding of many round dense neurosecretory granules (Fig. 7). The size and number of these granules were relatively variable in an individual case. In this study, the electron-dense neurosecretory granules were identified in 3 cases of SCLA. The crushing sign noted by light microscope could also be found by electron microscope. DISCUSSION Clinical Studies SCLA is a rapidly growing early metastasizing tumor. It constitutes a variable percentage of total bronchogenic carcinomas from Moersch and Mcdonald’s [18] 9% to Auerbach et al. and Yesner et al.’s [19,20] 20-25%. In this study, we collected 67 cases of SCLA reported during the past 16 years, which constituted 7.5% of all bronchogenic carcinomas. SCLA occurs in middle-aged and elderly people. Previous reports showed that the mean age at the time of diagnosis was 56 years [5].In our study, the ages ranged from 21 to 75 years with 63.5% between the ages of 50 and 70 years. The mean age was 61 years. There was a strong predilection for males, early studies indicated a ratio of 11:l [4] or 19:1 [6]. Our study confirmed the fact that SCLA is almost exclusively a disease of the male and of smoking. There were 57 males and 10 females in our

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TABLE VII. Survival Results After Different Therapies for SCLA Over a 16-Year Period at TSGH* Survival time (months)

6 12 24 36 M.S.T. (months)

Therapentic methods RT

CT

S

CT+S

RTfCT

RT+CT+S

66.7a 40 13.3 0 10.7

42.9 0 0 0 4.6

66.7 0 0 0 5.8

80 40 0 0 10.4

75 62.5 12.5 6.3 13.8

80 40 0 0 11.2

* RT = radiotherapy; CT = chemotherapy; S

= surgery; M.S.T. = mean survival time.

No. = percentage.

TABLE VIII. 1981 World Health Organization (WHO) Histological Classification of Lung Tumors

1. Squamous cell carcinoma (epidermoid carcinoma) Variant a. Spindle cell (squamous) carcinoma 2. Small cell carcinoma a. Oat cell carcinoma b. Intermediate cell type c. Combined oat cell carcinoma 3. Adenocarcinoma a. Acinar adenocarcinoma b. Papillary adenocarcinoma c. Bronchiolo-alveolar carcinoma d. Solid carcinoma with mucus formation 4. Large cell carcinoma Variants a. Giant cell carcinoma b. Clear cell carcinoma 5. Adenosquamous carcinoma 6. Carcinoid tumor 7. Bronchial gland carcinoma a. Adenoid cystic carcinoma b. Mucoepidermoid carcinoma c. Others 8. Others

group, a ratio of 5.7:l. Eighty-six percent of the patients had a history of cigarette smoking. All smokers had used tobacco for more than 10 years and the majority had smoked for 30 to 50 years. SCLA usually produces symptoms that bring the patient to clinical attention and is less likely than are other lung carcinomas to be detected while asymptomatic. Cough was the most frequent symptom (71.6%); later symptoms in the order of frequency were dyspnea (37.3%)’ chest pain (28.3%), body weight loss (26.9%), hemoptysis (17.9%), and superior vena cava syndrome (7.5%). Infrequently, the initial presentation may be a syndrome resulting from ectopic hormone production, such as Cushing’s syndrome, but no Cushing’s syndrome was noted in our series. The chest roentgenogram often is characteristic of SCLA. There is usually a rounded area of increased density in the hilar or paratracheal region. Other authors

have commented that the mediastinal widening and hilar enlargement is the frequent characteristic picture. The percentages shown in Table I11 were at variance with those reported in a roentgenologic review series of I14 cases at the Mayo Clinic [21], i.e., 13% (compared to our 40.3%) with mediastinal widening, and 63% (vs. 44.5%) with a hilar enlargement. The very high metastatic potential for SCLA was supported by Hansen and Muggia’s study [22]. In our study, 84% of the patients were known to have been metastasized, including the regional or distant lymph nodes (67.2%), bone (32.8%), liver (11.4%), and brain (14.9%). The prognosis of SCLA corresponds to the clinical staging. The TNM staging system that is standard for non-small cell lung carcinoma [23] is considered inadequate for the classification of SCLA because of the high incidence of metastases. The two-stage classification of the Veterans Administration Lung Cancer Study Group (VALCSG) into limited stage and extensive stage is used. Limited stage is generally defined as a tumor confined to one hemithorax and its regional lymph nodes (hilar and mediastinal lymph nodes) with or without local extensions or involvement of the ipsilateral supraclavicular nodes. Extensive stage denotes tumor beyond these limits [24]. Choi et al. [7] reported that SCLA in the limited stage had a 2-year survival rate of 16.3% and a 5-year survival rate of 6.1%. In contrast, for patients with extensive-stage SCLA, the 2-year survival rate was 3.4% but none had survival for more 5 years. Wilson’s study [25] represented the mean survival time of limited-stage SCLA as 16 months versus 10 months for the patients with extensive-stage SCLA. In our study, 64% of the patients belonged to the extensive stage with a mean survival time of 7.2 months. The other patients which were in the limited stage had a mean survival time of 13.4 months. Regarding the survival rate, for patients in the limited stage, the 2-year survival rate was 16.7%, but only 3.1% had the same survival time in extensive-stage SCLA. The treatment of SCLA is still a difficult problem. The therapeutic approaches for limited-stage SCLA have

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Small Cell Lung Cancer TABLE M. Histologic Pictures of 67 Cases SCLA ~

Histologic uicture

Cases

Percentage

45 21 1 17 35 44 41 8 11 21

67.2 31.3 1.5 25.4 52.2 65.7 61.2 11.9 16.4 31.3

Oat cell Intermediate Combined Necrosis Crushing sign Sheet Trabeculae Ribbon Rosette Perivascular basophilic deposition

TABLE X. The Results of Immunohistochemical Staining of SCLA (35 Cases) Markers ~~~

Positive cases

Positive rate (%)

24 21 9 19 0 28 8 23

68.6 60 25.7 54.3 0 80 22.9 65.7

~

Neurofilament NSE Cytokeratin (HMW)” Cytokeratin (LMWIb ACTH Bombesin Chromogranin Synaptophysin

aHMW = high molecular weight. bLMW = low molecular weight.

evolved from thoracic radiotherapy alone to a combination of chemotherapy and thoracic radiotherapy. In an attempt to increase the long-term survival, the role of radiotherapy in limited-stage SCLA has been extensively studied [26-311. The addition of chest radiotherapy to combination chemotherapy increases the median survival time and decreases the recurrence rate [31-33]. Our study confirmed the fact that better prognosis is obtained by the treatment of combination chemotherapy and radiotherapy. The mean survival time was 13.8 months in our study.

Light Microscopic Studies The three major classification systems for SCLA published before are summarized in Table XI. The first major classification system was proposed by the World Health Organization (WHO) in 1967 [34]. In this system

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the SCLA was divided into four subtypes: the lymphocyte-like, fusiform, polygonal, and others. Subsequently, the Pathology Committee of the Working Party for the Therapy of Lung (WP-L) revised this classification to simplify the categories and defined more precisely diagnostic criteria [35]. In this classification, the lymphocyte-like subtype of SCLA was separated from the other (fusiform, polygonal, and other) subtypes, with the latter categories being grouped together as an intermediate subtype. A new major classification system was proposed by the WHO in 1981 [36], dividing SCLA into three subtypes: oat cell, intermediate cell, and combined. The oat cell subtype is composed of uniform small cells, generally 2-3 times larger than lymphocytes, with dense round or oval nuclei, diffuse chromatin, inconspicuous nucleoli, and very sparse cytoplasm (Fig. 7). The intermediate cell subtype consists of small cells, with nuclear characteristics similar to the oat cell subtype but with more abundant cytoplasm. The cells may be polygonal or fusiform and are less regular in appearance than those of the oat cell subtype (Fig. 2). Tumors showing mixtures of oat cell and intermediate cell types should be categorized as oat cell subtype. Tumors showing mixtures of the intermediate cell type and large cell elements should be categorized as intermediate cell subtype. In combined subtype there is a definite component of oat cell carcinoma with squamous and/or adenocarcinoma. At the light microscopic level, tumors in which both components are well differentiated are extremely rare and represent from 0 [37-40] to 3.5% [41] of all lung cancers. SCLA combined with squamous cell carcinoma has been reported by Moertel [42] and Abeloff et al. [43]. According to this criteria, in our study, 45 cases belonged to the oat cell subtype, 21 cases were intermediate cell subtype, and only 1 case which showed combined oat cell carcinoma and squamous cell carcinoma (Fig. 8) belonged to the combined subtype. In many instances the tumor grows as a sheet of cell with no apparent architecture, but in other cases, there may be rosette (Fig. 9), trabeculae (Fig. lo), ribbon, and ductile formation. Occasionally, palisading of the cells around the blood vessels may also be found (Fig. 3). Very characteristic is a basophilic outlining of the vessels, which is said to be the result of a deposition of nucleic acid [44]. The pathogenesis of the crushing sign

Fig. 1. Oat cell type of SCLA. The tumor cells (left) are small, round, and hyperchromatic (HE, X200). Fig. 2. Intermediate cell type of SCLA. The tumor cells are larger than the oat cell type. The cells are spindle (arrowheads) or polygonal with spare cytoplasm (HE, X200). Fig. 3. In the necrotic area of SCLA, there is basophilic material deposited on the wall of blood vessels (arrowheads) (HE, x 100).

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Neuron-Specific Enolase (NSE) (Fig. 11) is still controversial. It may be an artifact during the procedure of bronchoscopic biopsy, but, the crushing Enolase molecules in mammalian tissue are dimers sign can also be found in the non-biopsy specimen. In our composed of three immunologically distinct subunits, a, study, the crushing sign was noted in 33 cases, and the B, and r [55,56], The a-enolase is the most common form deposits of perivascular basophilic material were seen in and occurs in most tissues. The B-enolase is found to be 21 cases. Although the above two characteristics are not mainly localized in the heart and stratified muscle. In essential diagnostic criteria for SCLA, we suggest these 1978, Schmechel and associates [57] demonstrated the characteristic pictures may act as diagnostic references. presence of r-enolase (neurospecific enolase), an isoenzyme of 2-phospho-D-glycerate hydrase, in several tisImmunohistochemical Studies sues, including the APUD (Amine Precursor Uptake and In this study, staining was performed by the peroxi- Decarboxylation) system. Therefore NSE is assumed to dase-antiperoxidase (PAP) technique as described previ- be restricted in distribution to neurons of the central and ously [45]. Seven immunohistochemical markers were peripheral nervous system [58,59]. In addition, significant levels of the NSE have been shown in neuroendostudied and discussed as follows. Cytokeratin. Keratin proteins are one of the five crine cells and their tumors [57,60]. The NSE had been major types of cytoskeletal proteins that form the 6-11 reported to be useful as a tumor marker for SCLA nm cytoplasmic intermediate filament [46-5 13. Although [61-64]. In order to better define the use of NSE as a originally thought to occur only in keratinizing epithe- marker for neuroendocrine tumors such as SCLA, we lium, they have been demonstrated by immunohis- studied 35 cases of SCLA by using the PAP technique tochemical studies also in non-keratinizing epithelial with an antiserum directed at NSE. Twenty-one cases of cells, such as the glandular epithelial cells. The cyto- SCLA were stained positively. We conclude that PAP keratins are therefore specific for epithelial differentia- staining for NSE content may be a useful adjunct to tion. Nineteen keratin polypeptides have been identified morphologic analysis in diagnostically identifying the in human tissues by two-dimensional gel electrophoresis SCLA and that our results in diagnostically identifying {52,53]. All types of keratin polypeptides can be simply the SCLA and that our results support the concept of a called keratins. Their molecular weights ranged from 40 functionally unified APUD system, as reflected in the kD (kilo-daltons) to 68 kD (Table XII). Cytokeratin tumors originating from it. proteins can generally be divided into the two main Neurofilament (NF). NFs are restricted in normal subgroups of low molecular weight (LMW) and high tissues and cells of neural crest and neuroendocrine molecular weight (HMW). As a rule, LMW cytokeratins origin. Isolation of NF from neural cells has revealed that are distributed in simple epithelium whereas HMW in mammalian tissues these filaments consisted of three cytokeratins are present in complex and stratified epithe- subunit proteins (molecular weight: 68-70 kD, 150 kD, lium. In this study, we present the result obtained with and 200 kD) called neurofilament triplet proteins [65,66]. two such antibodies: 35BH11 and 34BE12. The 35BH11 They have been reported to be present in tumors of neural decorates a LMW cytokeratin while 34BE12 decorates a origin including the central nervous system and the HMW cytokeratin. The positive rate was 54.3% with peripheral sympathetic system [65-701. Osborn and LMW cytokeratin and 25.7% with HMW cytokeratin. Weber [71] suggested that virtually 100% of paraganMuijen 161 reported that all types of lung carcinoma, gliomas, ganglioneuromas, ganglioneuroblastomas, and including SCLA, were positive with anti-cytokeratin. neuroblastomas were stained positively by using the 68 Ramaekers et al. [54] reported that SCLA was positively kD protein, but the 200 kD protein demonstrated a more stained with cytokeratin antibodies, including a mono- restricted pattern of distribution. Even some ganglion clonal antibody specific for cytokeratin 18. Nevertheless, cells may totally lack NF [71-741. Muijen’s study [6] Lehto’s study [8] showed that the SCLA was negatively showed that all cases of SCLA were negatively stained stained with cytokeratin antibodies. The difference may with anti-neurofilament. Inchul Lee et al. [75] reported be due to the different primary antibodies which was used that the positive rate was 16.7% with NF in SCLA. by the authors. Selective masking of cytokeratin deter- Lehto’s report [8] showed that all cases of SCLA were minants may result in the failure of some cytokeratin positively stained with anti-neurofilament. In this study, antibodies to decorate cytokeratin filaments. anti-neurofilamentmonoclonal antibody was used, which

Fig. 4. a: lmmunohistochemistry in SCLA, showing positive immunofluorescent staining for bombesin. b: The same case: haematoxylin and eosin stain. c: The same case: negative control of bombesin immunohistochemical staining. (a,b,c, X200).

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Fig. 5 . Immunohistochemistry in SCLA, showing positive staining for synaptophysin (X400) Fig. 6. lmmunohistochemistry in SCLA, showing positive staining for NSE (X400).

recognizes the 70 kD polypeptide of NF. The positive rate was 68.6%. The positive rate of SCLA varied, and was thought to be dependent on the fixatives and the specificity of the antibodies employed. Synaptophysin. Snyaptophysin is an integral membrane glycoprotein originally isolated from presynaptic vesicles of bovine neuron [76,77]. In mammalian bronchial ganglion cells and in nerves, synaptophysin is coexpressed with neurofilament, whereas in solitary neuroendocrine cells [78,79] of the bronchial mucosal lining, synaptophysin coexists with cytokeratin [75]. Synaptophysin has also been immunohistochemically demonstrated in most, but not all carcinoid and in a limited number of neuroendocrine tumors at various sites [SO-831, such as pheochromocytomas, neuroblastomas, pancreatic islet-cell tumors, and paragangliomas. Inchul

Lee et al. [75] reported that the positive rate of the neuroendocrine tumors was 83.8% (represented as 515 of well-differentiated neuroendocrine carcinoma, 8/10 of intermediate cell neuroendocrine carcinomas, and 212 of small cell neuroendocrine carcinomas). In our study, the anti-synaptophysin immunostaining was carried out by the PAP method in 35 cases of SCLA. The positive rate was 65.7% (23/25). Thus, we suggest that synaptophysin antibody notably clone SY-38, may be a useful diagnostic marker for neuroendocrine cells and tumors, including the SCLA. Chromogranin. Chromogranin was originally described in the catecholamine-containing granules of bovine adrenal medulla [84,85]. It comprised a group of acidic polypeptide of various sizes which forms a major part of the soluble proteins in the secretory granule of the

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Fig. 7. Electron microscopy in SCLA . The dense-core neurosecretory granules (*) are identified in the sparse cytoplasm. (X4,OOO) (N : nucleus). TABLE XI. Three Maior Classification Svstems for SCLA WHO (1967)a Fusiform Polygonal Lymphocyte-like Others

WP-L (1973)b

WHO (1981)

Ly mphocyte-like Intermediate cell

Oat cell Intermediate cell Combined

aWHO (World Health Organization). WP-L (Working Party for the Therapy of Lung Cancer).

adrenal medulla. Then it was found to be widely distributed in endocrine tissues [86-881. Earlier reports suggested that chromogranin represented a broad, potentially universal marker of neuroendocrine cells and tumors [89-951. However, recent findings showed that the member of the chromogranin family of secretory proteins comprise at least three distinct types, termed A, B, and C [96,97]. Chromogranin-A was the largest of the polypeptides (molecular weight: 68 kD) and makes up about 40-50% of the total soluble granule proteins of the bovine adrenal medulla [89], while another report as-

serted that chromogranin-B was the most abundant type in human adrenal medulla [91]. Lloyd et al. [98] reported that the glucagon and pancreatic polypeptide-producing tumors were positively stained by chromogranin, whereas insulinomas and somatostatinomas apparently did not express it. In the lung, chromogranin-A has been immunohistochemically demonstrated in normal epithelial neuroendocrine cells [99]. Such studies in pulmonary neuroendocrine neoplasms were also performed by Glatstein et al. [27]. The positive rate was 40% with chromogranin-A in SCLA. In our study, the chromogranin-A immunohistochemical staining was performed in 35 cases of SCLA; only 8 cases were positively stained. Chromogranin-A could not be detected in most cases of SCLA. The reason for this lack of reactivity may be related to the processing of the specimen; formaldehyde fixation and paraffin embedding may destroy partially the antigenic sites of the granules. It is also possible that in poorly differentiated cells with sparse cytoplasm the quantity of granules is under the detection level of the immunohistochemical technique. Our data do not indicate that chromogranin-A is a useful marker in the diagnosis of SCLA.

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Fig. 8. Combined type of SCLA, showing the small cell carcinoma (left) combined with squamous cell carcinoma (right) (HE, x200).

Fig. 9. SCLA. The tumor cells are arranged with rosette formation (arrowheads) (HE, X200).

Adrenocorticotrophic Hormone (ACTH) Several distinct clinical syndromes have been recognized in association with the production of certain ectopic hormones [ 100,1011. Broder 11021 suggested that lung carcinoma may be the most frequent malignancy associated with ectopic hormone production. SCLA is almost always, if not entirely, responsible among all types of lung cancer for causing endocrine disturbance. Several hormones are produced by SCLA such as calcitonin, serotonin, ACTH, antidiuretic hormone, gastrin-releasing peptide and neurophysin [ 1031. Several clinical syndromes such as Cushing’s syndrome [ 1041 and syndrome of inappropriate antidiuretic hormone secretion

(SIADH) are noted to be associated with such ectopic hormone production [105]. The SCLA may also be associated with the carcinoid syndrome resulting in the urinary excretion of excessive amounts of 5-hydroxyindole acidic acid (5-HIAA) or 5-hydroxytryptophan (5HTP) . Ectopic hormone production of ACTH is most commonly associated with SCLA; approximately 30% of untreated patients with SCLA exhibit elevation of serum SCTH, but only 5% of such patients have clinical manifestations [103]. Inchul Lee et al. [75]represented the positive rate of ACTH immunohistochemical staining as well-differentiated neuroendocrine carcinomas 3/5,

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Fig. 10. SCLA. The tumor cells grow in a pattern of cord or trabeculae (arrowheads) (HE, X 200).

Fig. 11. SCLA. The crushing sign is shown (arrowheads), but intact diagnostic tumor cells are also apparent (centre) (HE, x 100).

TABLE MI. Major Cytokeratin Polypeptides in Human Tissues

No. 2

10

11

12

13

14

15

16

17

18

19

MWa 68 65.5 63 59 58 56 54 52.5 64 56.5 D H ~ 7.8 7.8 7.5 7.3 7.4 7.8 6.0 6.1 5.4 5.3

56 5.3

55 4.9

54 5.1

50 5.3

50 4.9

48 5.1

46 5.1

45 5.7

40 5.2

1

3

4

aMW = molecular weight (X10-3). bpH = isoelectric PH value.

5

6

7

8

9

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intermediate cell neuroendocrine carcinomas 6/10, and the small cell neuroendocrine carcinomas 1/2, while Blobel’s study showed all negatively stained. In our study, the ACTH immunohistochemical staining was performed in 35 cases of SCLA, but none was positively stained. Pariente et al. showed that the serotonin or kinin content of a carcinoid tumor is related to the number of neurosecretory granules present in the cytoplasm. In our opinion it is uncommon for the patient with SCLA to develop a well-developed immunohistochemical reactivity, which may be related to the number of neurosecretory granules present in the sparse cytoplasm, just as it is not always corresponding a clinical symptom for the insufficient plasma level.

Bombesin Moody et al. and Polak et al.’s reports [106,107] show that bombesin is a tetradecapeptide originally isolated from frog skin by Erspamer. In dogs, small and possibly more physiological amounts of bombesin caused potent release of gastrin and stimulated gastric-acid secretion, contraction of the gall bladder, relaxation of the choledochal junction, and stimulation of a protein-rich pancreatic secretion. Peptides corresponding closely in structure to the biologically active carboxyl terminal region of amphibian peptide bombesin have now been isolated from several mammalian species, including man. In mammals, it is present mainly in nerve cells and fibers. The recent studies show that bombesin-like neuropeptides are natural, plentiful components of the mammalian brain, stomach, intestine, and fetal lung. Several groups of human neuroendocrine tumors were examined for the presence of bombesin by Chejfec et al. [ 1081. The results showed that bombesin was present in 68% of bronchial carcinoids, 65% of pulmonary neuroendocrine carcinomas, 62% of neuroendocrine carcinomas of the skin, 5-1 0% of pheochromocytomas and extra-adrenal paragangliomas, and 35% of gastrointestinal carcinoid and neuroendocrine carcinomas. The same studies were performed in a wide variety of non-neuroendocrine tumors, which showed all to be negative for bombesin. Moody et al. [ 1061reported that the bombesin was present in all cell lines of SCLA, but not in other lung cancer. The high levels of intracellular bombesin characterized human SCLA. Blobel et al.’s study [13] showed that the small cell type neuroendocrine carcinomas of the bronchopulmonary tract were all positively stained by bombesin immunohistochemical staining. Another report [75] also showed the same result. In our study, the bombesin immunohistochemical staining was performed in 35 cases of SCLA. The positive rate was 80% (28/35). Bombesin appears to be the most reliable marker for the diagnosis of SCLA. The receptors for bombesin in cells of SCLA in culture have been localized by using a dimeric peptide ligand and

electron microscopical immunocytochemistry . These receptors appear to be involved in the autocrine regulation of tumor cell proliferation [109]. It has been suggested that SCLA was derived from bombesinergic precursor cells, and the presence of bombesin peptides in the blood of people at risk for lung cancer (such as heavy smokers) could allow the early detection of SCLA [106].

Electron Microscopic Studies The group of cells which are widely dispersed throughout the body and share some common characteristics, including dense-core secretory granules and the ability to take up amine precursors and modify them by decarboxylation, are known as APUD cells. The presence of APUD-type cell in the lung was proved by Feyrter in 1954 [1 101. They have variously been termed Feyrter cells, Kultschitzky cells, argyrophil-fluorescent-granulated cells, enterochromaffin-like cells, neurosecretory cells, and pulmonary argyrophil cells. These endocrine cells can be demonstrated in the lungs of fetuses and children, but they cannot be demonstrated in the lungs of adults [ 110,ll I]. The persistence of such cells in adult lungs had been uncertain until 1965 when Bensch and co-workers [ 1121 described the ultrastructural features of the cells in the bronchial glands. The cells contain the neurosecretory-type granules which are always found in the other cells of APUD system. In 1967, Bensch [ 161 first reported on the ultrastructure of neurosecretory-type granules in SCLA. This was later confirmed by Hattori et al. [113,114]. The cells show scanty cytoplasm with numerous mitochondria, polysomes, and cytoplasmic vesicles, but the lysosomes are very scanty. The neurosecretory granules in the cytoplasm can be found in about one in six cells. The granules are similar to those seen in the bronchial carcinoid, but fewer in number and smaller in size; in general, the diameter of neurosecretory granules varies, 50-240 pm. SCLA is thought to arise from the normal APUD cells of the lungs [ 115,1161. Hattori [5] reported that SCLA contained the neurosecretory granules in a ratio of 95%, while other reports showed ratios of 75% (Muijen) [6], 66% (Li) [17] and 26% (Sidhu) [115]. In this study, 7 cases of SCLA were subjected to ultrastructural studies. The neurosecretory granules were identified in only 3 cases (43%). The previous studies and our studies showed that the neurosecretory granules were not always identified in the SCLA. We suggest that electron microscopic study is not necessary as a routine diagnostic procedure unless the light microscopic and immunohistochemical studies are not useful in the diagnosis of SCLA.

CONCLUSIONS SCLA has a clinical course of high-grade malignancy, early metastatic potential, low 2-year survival rate, and low median survival time. It has a characteristic cell

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structure as well as tumor structure such as the small round hyperchromatic cell with rosette formation, the crushing sign, and the perivascular basophilic deposits. Immunohistochemically, the immunoreactivity for cytokeratin, neurofilament, neuron-specific enolase, chromogranin, and synaptophysin indicate that SCLA has both epithelial and neurosecretory features and may originate from the bronchial APUD-type cell. Bombesin and synaptophysin are two reliable new markers in the diagnosis of SCLA. It is our hope that both clinicians and pathologists be well acquainted with this highly malignant tumor and have extensive efforts aimed at defining a new diagnostic and therapeutic approach to this tumor.

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