Japanese Journal of Clinical Oncology

INTRODUCTION

The classification of neuroendocrine tumors of the lung used to be complex and confusing. Recently, Travis et al. (1) reported the following spectrum of pulmonary neuroendocrine (NE) lesions: (i) minute NE lesions, (ii) common neoplasms with an NE light microscopic appearance, (a) typical carcinoid, (b) atypical carcinoid, (c) large cell NE carcinoma and (d) small cell lung carcinoma, (iii) non-small cell lung carcinoma with NE features and (iv) uncommon primary NE neoplasms. The classification of small cell lung carcinoma (SCLC) has changed over time. The World Health Organization (WHO) defined the subtypes of SCLC in 1981 as oat cell type, intermediate cell type and combined type (2). The International Association for the Study of Lung Cancer (IASLC) redefined the subtypes in 1988 as small cell carcinoma, mixed small cell/large cell carcinoma and combined small cell carcinoma (3). Almost all combined SCLCs contain a component of squamous cell carcinoma or adenocarcinoma; however combinations can occur with spindle cell carcinoma (4) and giant cell carcinoma (5)^. We present a case of double primary lung cancers with NE features, one tumor consisting of small cell/large cell lung cancer combined with spindle cell sarcomatous lesions and the other an adenocarcinoma with NE features in a different lobe.

CASE REPORT

a	c
	d
b	e

Figure 1. (a) Macroscopic findings for LLL tumors. A yellow-white well defined mass measuring 14 × 10.5 × 8 cm in the LLL was compressing the surrounding normal lung parenchyma with formation of a capsule-like structure. (b)-(e) Microscopic findings for LLL tumor. Small cell component consists of small cells with large N/C ratio, scanty cytoplasm and finely granular nuclear chromatin with high mitotic activity (b). Cells in small cell/large cell component have eosinophilic larger cytoplasm and vesicular nucleus with distinct nucleolus (c). Organoid components form well demarcated round to ovoid clusters resembling atypical carcinoid in the background of small cell/large cells or spindle cells (d). Sarcomatous components have spindle-shaped cells of various sizes (e). Hematoxylin and eosin stain; original magnifications, ×333 (b, c), ×167 (d), ×130 (e).

A 63-year-old man was admitted to the National Cancer Center Hospital East in October 1994 because of cough, sputum and hemoptysis. He had smoked 20 cigarettes daily over 40 years. The patient's mother died of leukemia at the age of 38. Laboratory data were within normal limits except for a low serum albumin (2.9 g/dl) and elevated CEA (7.2 ng/ml). Chest radiography revealed a round nodule in the left lower lobe (LLL). Bronchoscopic examination showed a white polypoid mass obstructing the LLL bronchus. The biopsy specimen contained malignant cells; however, the cell type could not be determined because of the small size of the specimen. Computed tomography of the chest showed no enlargement of the mediastinal or hilar lymph nodes. Magnetic resonance imaging (MRI) of the brain showed a 6 × 6 mm solitary mass in the right cerebellar hemisphere. The LLL was resected in December 1994. Part of the left upper lobe (LUL) of the lung was also resected because another nodule was found in the LUL during the operation. Radiation therapy to the brain was performed when the brain mass was found to have grown to 14 × 14 mm on MRI in January 1995, suggesting a brain metastasis. After 50 Gy of radiation to the brain, the nodule could no longer be detected on MRI of the brain. No chemotherapy was given. Left leg pain occurred in February 1997 due to bone metastasis. Computed tomography of the chest showed multiple pulmonary metastases. Dyspnea developed and the patient died in February 1997, 2 years and 4 months after the operation. An autopsy was not performed.

We performed an immunohistochemical analysis to the formalin-fixed, paraffin-embedded sections by the avidin-biotin complex (ABC) method (6). Biotinylated secondary antibody and ABC reagents were purchased from Dako Japan (Kyoto, Japan). Primary antibodies used were against keratin (AE1/AE3, Dako, Dakopatts, Glostrup, Denmark; CAM5.2, Becton Dickinson, San Jose, CA, USA), surfactant apoprotien A (Sp-A) (PE-10, Dako), surfactant apoprotein D (Sp-D) (6B2, Yamasa, Chiba, Japan), CD56 (Lu243, Nippon Kayaku, Tokyo, Japan), chromogranin A (Dako), synaptophysin (Dako), CD57 (Leu7, Becton Dickinson), gastrin-releasing peptide (GRP) (Dako), serotonin (5HT-H209, Dako), calcitonin (Dako), CEA (011, Mochida, Tokyo, Japan), vimentin (V9, Dako), myoglobin (Dako), desmin (D33, Dako), alpha-smooth muscle actin (1A4, Dako), S-100 protein (Dako), Factor VIII (Dako), p53 (Nichirei, Tokyo, Japan) and Rb (MK-15-1, MBL, Nagoya, Japan).

A yellow-white, well defined mass measuring 14 × 10.5 × 8 cm in S10 of the LLL was compressing the surrounding normal lung parenchyma with formation of a capsule-like structure; the center of the mass revealed extensive hemorrhage and necrosis. (Fig. 1a). The LLL showed poor aeration and moderate anthracosis and fibrosis, especially beneath the pleura, but no emphysema.

A round, white nodule measuring 2.1 × 1.7 × 1.5 cm and having well defined margins was found in S1+2 of the LUL. The tumor was solid and there was no evidence of hemorrhage or necrosis. A sharp pleural indentation was detected (Fig. 2a).

a	b
	c

Figure 2. (a) Macroscopic findings for LLL and LUL tumors. A white round nodule measuring 2.1 × 1.7 × 1.5 cm was present in the LUL (S1+2) with a clear margin. (b), (c) Microscopic findings for LUL tumor. The tumor consists of glandular structure lined by tall columnar cells with hyperchromatic nuclei and coarse-granular chromatin (b). There were organoid nests showing occasional rosette formation (c). Hematoxylin and eosin stain; original magnifications, ×83 (b), ×410 (c).

The LLL tumor was largely necrotic, but there were viable tumor cells in the periphery. It consisted of four components: small cell, large cell, organoid and sarcomatous. The small cell component was composed of oval to spindle-shaped cells slightly larger than lymphocytes and having a large N/C ratio, scanty cytoplasm and oval nuclei with finely granular chromatin but no nucleoli (Fig. 1b). The large cell component consisted of polygonal to round cells that were larger than the cells in the small cell component and they contained abundant eosinophilic cytoplasm and a vesicular nucleus with one or two prominent nucleoli (Fig. 1c). Small cells and large cells were frequently intermingled. Both types of cells had high mitotic activity [22/10 high power field (HPF)] and clusters of pure small cell carcinoma cells were also observed focally. In the organoid component, the neoplastic cells formed well demarcated round to ovoid clusters resembling atypical carcinoid in the background with small cell/large cells or spindle cells (Fig. 1d). The cells forming the organoid structure were polygonal to spindle shaped and had a finely granular nucleus with one or two prominent nucleoli. Mitotic activity was high (24/10 HPF). The sarcomatous cells were spindle shaped and their nuclei were variable in size and contained coarse chromatin with one or two prominent nucleoli (Fig. 1e). The small cell/large cell carcinoma, spindle cell sarcomatous carcinoma and organoid components were highly intermingled with areas of transition between them; the proportions of these components were about 65, 30 and 5%, respectively. No lymphatic permeation or vascular invasion was observed. The remaining non-neoplastic pulmonary parenchyma showed no evidence of carcinoid tumorlets or neuroendocrine cell hyperplasia.

The LUL tumor had a clear margin and showed expansive growth. It consisted of irregular-shaped glands lined by tall columnar cells. The tumor cells contained relatively abundant eosinophilic cytoplasm and hyperchromatic nuclei with coarse-granular chromatin and distinct eosinophilic nucleoli (Fig. 2b). Mitotic activity was high (25/10 HPF). The stroma was scant. The tumor had a focal solid growth area with organoid nests showing occasional rosette formation (Fig. 2c). Nuclear palisading was also seen in the periphery of the nests. No lymphatic permeation or vascular invasion was observed. Hilar lymph nodes contained no metastatic tumors. Mediastinal lymph nodes were not explored.

The results of the immunohistochemical analysis are shown in Table 1. The small cell/large cell component of the LLL was diffusely positive for CD56 (Fig. 3a) and chromogranin A and partially positive for keratin. The organoid component was positive for CD56, chromogranin A, synaptophysin (Fig. 3b), GRP and CEA. The sarcomatous component was positive for vimentin (Fig. 3c) and some spindle-shaped also cells showed an immunoreaction for keratin (Fig. 3d). The LUL tumor was positive for CD56, chromogranin A (Fig. 4), GRP, calcitonin, CEA and keratin. The positive rates for p53 were 79% of the cells in the small cell/large cell component, 13% in the organoid component, 80% in sarcomatous component and 8% in the LUL tumor. Rb was positive only in the LUL tumor (30%) and negative in all components of the LLL tumor.

a	c
b	d

Figure 3. Immunohistochemical findings for LLL tumor. Small cell/large cell component was diffusely positive for CD56 (a). Organoid component was positive for synaptophysin (b). Sarcomatous component was positive for vimentin (c) but some spindle-shaped cells showed immunoreaction for keratin (d). Original magnification, ×410.

Table 1. Immunohistochemical profiles of left lower lobe and left upper lobe tumors

Antigens	LLL tumor			LUL tumor
	S/L	Organoid	Sarcomatous
Keratin (AE1/AE3)	+ (partly)	-	±	+
Keratin (CAM5.2)	±	+	+	+++
Sp-A	+ (partly)	-	-	-
Sp-D	-	-	-	-
CD56	+	+	-	+
Chromogranin A	-	+	-	+
Synaptophysin	-	+	-	+++
CD57	-	-	-	-
GRP	-	+	-	+ (partly)
Serotonin	-	-	-	-
Calcitonin	-	-	-	+
CEA	-	+	-	+
Vimentin	-	-	+	-
Myoglobin	-	-	-	-
Desmin	-	-	-	-
Alpha-smooth muscle actin	-	-	-	-
S-100 protein	-	-	-	-
Factor VIII	-	-	-	-
p53*	79%	13%	80%	8%
Rb*	-	-	-	30%

LLL, left lower lobe; LUL, left upper lobe; S/L, small cell/large cell carcinoma; Sp-A, surfactant apoprotein A; Sp-D, surfactant apoprotein D; GRP, gastrin-releasing peptide. *The percentage of immunoreactive cells counted in 500 cells.

DISCUSSION

Our differential diagnosis of the LLL tumor was (i) small cell carcinoma with spindle cell component, (ii) large cell NE carcinoma and (iii) atypical carcinoid. The high mitotic activity (22/10 HPF) ruled out atypical carcinoid, although some parts of the LLL tumor showed features resembling atypical carcinoid, such as an organoid growth pattern, tumor cells containing moderately eosinophilic, finely granular cytoplasm and nuclei possessing finely granular chromatin. Small cell carcinoma was preferred over large cell NE cancer because the LLL tumor consisted mainly of two kinds of cells, small-sized tumor cells with a high N/C ratio and large-sized polygonal to round tumor cells with one or two prominent nucleoli. In addition, the small cells and large cells were highly intermingled and clusters of pure small cell carcinoma cells were also observed focally. We therefore concluded that the LLL tumor was a mixed small cell/large cell carcinoma, one of the subtypes of small cell lung carcinoma in the IASLC classification (3).

Figure 4. Immunohistochemical findings for LUL tumor. It was positive for chromogranin A. Original magnification, ×410.

Spindle cell carcinoma has been found most often in association with giant cell carcinoma and adenocarcinoma, less frequently with large cell carcinoma or squamous cell carcinoma and infrequently with small cell carcinoma (7). Tsubota et al. (4) reported a case of combined small cell (pure type) and spindle cell carcinoma of the lung. The spindle cell carcinoma was predominant and immunoreactive for smooth-muscle actin, but not for NE markers, in their case, whereas the small cell/large cell component in our case occupied more of the tumor than the spindle cells. Moreover, the spindle cell sarcomatous area in our case did not show clear NE features or differentiation to mesenchyme but exhibited epithelial differentiation, indicating poorly differentiated carcinoma or sarcomatoid change of carcinoma. The spindle cell sarcomatous component was immunohistochemically positive for p53 and the frequency of positive cells was almost same as in the small cell/large cell component, suggesting that although their phenotypes were different, a similar genetic abnormality may have occurred in both.

We considered three possible diagnoses for the LUL mass: (i) moderately differentiated adenocarcinoma, tubular type, with NE features, (ii) combined adenocarcinoma and large cell NE carcinoma and (iii) metastatic carcinoma from the LLL tumor. The LUL tumor exhibited glandular structures lined by tall columnar cells and organoid nests of large pleomorphic cells with rosette formation and nuclear palisading. The latter pattern may be seen in large cell NE carcinoma (1); however, the clear glandular differentiation favored adenocarcinoma rather than large cell NE carcinoma.

Immunohistochemical testing yielded different characteristics in the LUL and LLL tumor. Calcitonin and Rb were positive only in the LUL tumor. Furthermore, the LLL tumor exhibited no evidence of glandular differentiation, indicating that the LUL tumor was not a metastasis of the LLL tumor. Immunohistochemical study showed that both the organoid and the glandular component of the LUL tumor had NE features, indicating that it was an adenocarcinoma with NE features rather than a combination of adenocarcinoma and NE carcinoma.

The relationship between LLL and LUL tumors is a matter of controversy. The LUL tumor was immunohistochemically positive for p53 the same as the organoid area in the LLL tumor, but positive cells were less frequent than in the small cell/large cell or spindle cell sarcomatous lesions. The NE marker study showed that the LUL tumor and the organoid area in the LLL might differentiate with NE feature better than small cell/large cell or spindle lesions. LUL tumor resembled the organoid component in the LLL in the immunohistochemical pattern of the NE markers (CD56, chromogranin A, synaptophysin, GRP). The same genetic change may have occurred in the areas of these two tumors exhibiting NE features, even though their morphology was different. Genetic analysis might resolve this issue.

The only case of multiple lung cancer with NE features reported previously was a case of bronchial carcinoid, small cell carcinoma and adenocarcinoma of the right lung described by Jung-Legg et al. (8) The series of synchronous double primary lung cancers reported by Carey et al. (9) included cases of combined non-small cell lung cancer and small cell lung cancer or carcinoid, but there were no cases of double cancer with NE differentiation demonstrated by immunohistochemistry. Since these cases were not immunohistochemically tested for NE markers, some of them may have been double cancer, both of which had NE differentiation. In any event, the occurrence of synchronous double cancers with NE features in both appears to be a rare event.

The survival time in our case was 2 years and 4 months without chemotherapy, longer than in ordinary small cell lung cancer (10). The median survival for limited stage small cell lung cancer treated by surgery alone has been reported to be about 6 months (11). Tsubota et al. did not comment on the outcome of their case of a combined small cell and spindle cell carcinoma of the lung. Small cell lung cancer has a poor prognosis although it is sensitive to chemotherapy and radiation. In contrast to small cell lung cancer, spindle cell carcinoma is also generally considered to have a poor prognosis and to be resistant to irradiation and chemotherapy (12,13). The metastatic brain tumor in our case was as sensitive to radiation therapy as small cell lung cancer; however, it recurred 2 years after surgery, a much longer latent period than in ordinary small cell lung cancer. No lymph node metastasis was observed in our case despite the large tumor, a finding also different from usual small cell lung cancer. The reason for the comparatively good outcome in our case remains unclear. Two-year disease-free survivors represented 13% of patients presenting with limited small cell lung cancer but only 2% of those with extensive disease (10). More than 80% of 2-year survivors of small lung cell lung cancer have been found to have received chest irradiation and almost all extensive disease patients had metastases confined to a single organ system (11). In our case, however, the brain was the only metastatic site at the time of presentation. Hardly any long-term survivors of resected small cell lung cancer have had lymph node metastases or distant metastases (14,15). The present case was exceptional, because the patient survived for a long time in spite of the brain metastasis and having been treated only by resection of the lung tumors and radiotherapy to the brain. Slow growth may have been one of the characteristics of the tumors in our case, despite their high mitotic activity.

In conclusion, we have reported a unique case of synchronous double primary lung cancers with a combination of small cell/large cell carcinoma and a spindle sarcomatous component in the LLL and adenocarcinoma with NE features in the LUL. At presentation the tumor had metastasized to the brain, but not to lymph nodes. Radiation to the brain after resection of the lung tumors was very effective. The survival time in our patient, who did not receive chemotherapy, was 2 years and 4 months. Multiple lung cancers with NE features are extremely rare and similar cases have not been reported in the literature.

Acknowledgments

This work was supported in part by a Grant from the Ministry of Health and Welfare for the 2nd term Comprehensive Strategy for Cancer Control and a Grant-in Aid for Cancer Research from the Ministry of Health and Welfare, Japan.

References

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Received September 11, 1998; accepted December 21, 1998
For reprints and all correspondence: Seiji Niho, Division of Thoracic Oncology, National Cancer Center Hospital East, 5-1, Kashiwanoha 6-chome, Kashiwa, Chiba 277-8577, Japan. E-mail: siniho{at}east.ncc.go.jp
Abbreviations: LLL, left lower lobe; LUL, left upper lobe; NE, neuroendocrine; SCLC, small cell lung carcinoma; WHO, World Health Organization; IASLC, International Association for the Study of Lung Cancer; CEA, carcinoembryonic antigen; MRI, magnetic resonance imaging; ABC, avidin-biotin complex; Sp-A, surfactant apoprotien A; Sp-D, surfactant apoprotein D; GRP, gastrin-releasing peptide

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