Ann Thorac Surg 2004;77:1891-1895
© 2004 The Society of Thoracic Surgeons
Original article: general thoracic
Pulmonary large cell neuroendocrine carcinoma demonstrates high proliferative activity
Akira Iyoda, MDa,
Kenzo Hiroshima, MDb,
Yasumitsu Moriya, MDa,
Teruaki Mizobuchi, MDa,
Mizuto Otsuji, MDa,
Yasuo Sekine, MDa,
Kiyoshi Shibuya, MDa,
Toshihiko Iizasa, MDa,
Yukio Saitoh, MDa,
Takehiko Fujisawa, MDa*
a Department of Thoracic Surgery, Chiba, Japan
b Department of Basic Pathology, Graduate School of Medicine, Chiba University, Inohana, Chuo-ku, Chiba, Japan
Accepted for publication October 28, 2003.
* Address reprint requests to Dr Fujisawa, Department of Thoracic Surgery, Graduate School of Medicine, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan
e-mail: fujisawa{at}med.m.chiba-u.ac.jp
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Abstract
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BACKGROUND: In 1999, the World Health Organization classified large cell neuroendocrine carcinoma as a variant of large cell carcinoma and this has been categorized as lying between atypical carcinoid and small cell lung carcinoma in terms of clinical aggressiveness.
METHODS: We analyzed the proliferative activity of stage 1 large cell neuroendocrine carcinoma derived from patients with primary lung cancer who underwent surgical resection and compared the results with stage 1 classic large cell carcinoma cases. The mitotic rate was counted in ten high-power fields of light microscope. Immunohistochemical staining using anti-Ki-67 antibody was performed. The Ki-67 labeling index, expressed as a percentage of positive cells, was determined by light microscopy with random counting of at least 1,000 tumor nuclei. The expression of P53 and Bcl-2 was examined and compared.
RESULTS: The mitotic rate of large cell neuroendocrine carcinoma cases was significantly higher than that of classic large cell carcinoma cases. The Ki-67 labeling index of stage 1 large cell neuroendocrine carcinoma cases was significantly higher than that of stage 1 classic large cell carcinoma cases. Immunohistochemical expression of P53 in large cell neuroendocrine carcinoma and classic large cell carcinoma was comparable. However, large cell neuroendocrine carcinoma exhibited a significantly higher expression of Bcl-2 than classic large cell carcinoma. The disease specific disease-free survival for patients with stage 1 large cell neuroendocrine carcinoma was significantly lower than that for patients with stage 1 classic large cell carcinoma.
CONCLUSIONS: Large cell neuroendocrine carcinoma appears to be more clinically aggressive than classic large cell carcinoma with these findings indicating that large cell neuroendocrine carcinoma has a higher level of proliferative activity than classic large cell carcinoma.
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Introduction
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In 1999 the World Health Organization (WHO) [1] classified large cell neuroendocrine carcinoma (LCNEC) as a variant of large cell carcinoma. This tumor has been categorized as lying between atypical carcinoid and small cell lung carcinoma in terms of clinical aggressiveness [2, 3]. In our previous study, we found that LCNEC appears to be a more clinically aggressive tumor than classic large cell carcinoma (CLCC) [4]. In this study, we have therefore examined differences in biological behavior between LCNEC and CLCC defined according to the WHO classification.
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Material and methods
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We analyzed pathologic stage 1 cases diagnosed as LCNEC and CLCC derived from patients with primary lung carcinoma who underwent surgical resection with lymph node dissection at Chiba University Hospital between 1987 and 2000. We excluded so-called combined carcinomas with cytologic features of mixed small cell carcinoma. Large cell neuroendocrine carcinoma has both neuroendocrine morphology and evidence of neuroendocrine differentiation by immunohistochemistry and(or) electron microscopy. In contrast, CLCC lacks neuroendocrine morphology or differentiation. Immunohistochemical staining was performed on large cell carcinomas using a polyclonal antichromogranin A antibody (Nichirei Corporation, Tokyo, Japan), a monoclonal antisynaptophysin antibody (DAKO, Glostrup, Denmark), and an anti-neural cell adhesion molecule (NCAM) antibody (Zymed, California, USA). Neuroendocrine differentiation was detected by positive immunohistochemical staining for chromogranin A, synaptophysin, or NCAM. The mitotic rate was counted in ten high-power fields of light microscope. Immunohistochemical staining using an anti Ki-67 antibody (DAKO, Glostrup, Denmark) was performed for those cases. The Ki-67 labeling index, expressed as the percentage of positive cells, was determined by light microscopy with random counting of at least 1,000 tumor nuclei. The counter was blinded on the clinicopathologic data of these patients. Immunohistochemical staining using antibodies directed against P53 (DAKO, Glostrup, Denmark) and Bcl-2 (DAKO, Glostrup, Denmark) was also performed and expression levels compared. Patient gender, age, smoking index, surgical procedure, TNM stages, chemotherapy, and patient outcome were determined from the medical records.
Statistical analysis
The Fischer's exact test was used to compare binomial proportions. The 
2 test was used to assess differences in gender and immunohistochemical positivity for P53 and Bcl-2. The unpaired t test was used to detect significant differences between groups in patient age, smoking index, tumor size, and tumor mitotic rates. The Mann-Whitney U test was used for analyzing Ki-67 labeling indices. Survival time was calculated from the date of surgery until the time of first local or distant recurrence (disease specific disease-free survival), and was evaluated using the method of Kaplan and Meier. The curves obtained were compared with the log-rank test. A p value less than 0.05 was considered statistically significant.
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Results
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Twenty cases of stage 1 LCNEC and 13 cases of stage 1 CLCC were included in the study with patients being predominantly male. There was no significant difference between each tumor category and smoking indices or tumor size (Table 1).
The mitotic rate of LCNEC cases was significantly higher than that of CLCC cases (Table 2).
The Ki-67 labeling index of LCNEC cases was significantly higher than that of CLCC cases (Table 2, Figs 1
and 2
). Immunohistochemical staining for P53 was comparable between LCNEC and CLCC (Table 3, Figs 3 and 4).
However, immunohistochemical staining for Bcl-2 was significantly higher in LCNEC than CLCC (Table 3, Figs 5
and 6
). The 5-year disease specific disease-free survival rate of stage 1 LCNEC cases was 51.7%, which was significantly lower than that for those with stage 1 CLCC (p = 0.0321) (Fig 7) . Moreover, the 5-year disease specific disease-free survival rate of stage 1 LCNEC cases without adjuvant chemotherapy was 42.9%, which was significantly lower than that for those with stage 1 CLCC without adjuvant chemotherapy (p = 0.0225) (Fig 8) .
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Fig 1. Immunohistochemical staining for anti Ki-67 antibody in large cell neuroendocrine carcinoma. (x40).
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Fig 3. Immunohistochemical staining for anti P53 protein antibody in large cell neuroendocrine carcinoma. (x40).
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Fig 4. Immunohistochemical staining for anti P53 protein antibody in classic large cell carcinoma. (x40).
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Fig 5. Immunohistochemical staining for anti Bcl-2 antibody in large cell neuroendocrine carcinoma. (x40).
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Fig 7. The disease specific disease-free survival analysis indicates that LCNEC cases with stage 1 disease had a significantly poorer prognosis than CLCC cases with stage 1 disease. (LCNEC = large cell neuroendocrine carcinoma; CLCC = classic large cell carcinoma.)
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Fig 8. The disease specific disease-free survival analysis indicates that LCNEC cases without adjuvant chemotherapy had a significantly poorer prognosis than CLCC cases without adjuvant chemotherapy. (LCNEC = large cell neuroendocrine carcinoma; CLCC = classic large cell carcinoma.)
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Comment
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Large cell neuroendocrine carcinoma was classified as a variant of large cell carcinoma by the World Health Organization in 1999. Large cell neuroendocrine carcinoma is an aggressive tumor, with the work of Travis and colleagues [5] indicating that the survival for LCNEC was worse than that for atypical carcinoid and was not different from that for small cell lung carcinoma. Jiang and colleagues [2] reported 22 cases of LCNEC and indicated that the 5-year survival for patients with LCNEC was 44.8% and that there was a significant difference in overall survival for LCNEC patients versus nonsmall cell lung carcinoma patients. Dresler and colleagues [6] showed that the 5-year survival for stage 1 LCNEC patients was 18% and all-stage 5-year survival was 13% such that LCNEC had a remarkably poor prognosis even in very early stage disease. In a previous study [4] we found that LCNEC has a poorer prognosis than CLCC. Therefore, in this study we compared various biologic parameters of LCNEC with CLCC in stage 1 disease.
Our study demonstrated that LCNEC cases exhibited an increased Ki-67 labeling index and increased mitotic rate than CLCC cases even at stage 1 of the disease process. The Ki-67 nuclear antigen is associated with cell proliferation and is detectable in the nucleus of cycling (G1, S, G2, and M-phase) cells but is absent in resting (G0 phase) cells. It is believed that biologically active tumors express high levels of Ki-67 nuclear antigen [7, 8]. Thus, our findings suggested that LCNEC cases had a higher level of proliferative activity than CLCC cases. The expression of P53 is a significant prognostic factor in pulmonary carcinomas. However, our findings revealed that there was no significant difference in P53 expression between LCNEC and CLCC [9, 10].
On the other hand, our study demonstrated a significant difference in Bcl-2 expression between LCNEC and CLCC [11]. Bcl-2 is an important regulator of apoptosis and acts to suppress apoptosis. Bcl-2 expression is therefore regarded as a significant negative prognostic factor. Jiang and colleagues [12] reported that cases of pulmonary neuroendocrine carcinoma exhibited increased Bcl-2 expression. The increased Bcl-2 expression documented in this study may therefore adversely affect the prognosis of LCNEC cases.
Large cell neuroendocrine carcinoma was classified as nonsmall cell lung carcinoma and, regarding the use of adjuvant chemotherapy in general, no significant data to date exist beyond abstract form for benefit in the treatment of nonsmall cell lung carcinoma [13]. However, Przygodzki and colleagues [14] analyzed the expression of P53, K-ras-2, and C-raf-1 in pulmonary neuroendocrine tumors and revealed that LCNEC was more akin genetically and immunohistochemically to small cell lung carcinoma although it was categorized as nonsmall cell lung carcinoma. In previous survival analyses, our findings reveal that the neuroendocrine features of large cell carcinomas are independent prognostic factors in pulmonary neuroendocrine tumors [15]. In addition, large cell carcinoma with neuroendocrine features, which contained not only LCNEC but also large cell carcinoma with neuroendocrine differentiation, and large cell carcinoma with neuroendocrine morphology should be categorized as high grade neuroendocrine tumors [15]. Small cell lung carcinoma is sensitive to chemotherapy and Shepherd and colleagues [16] reported that adjuvant chemotherapy after surgery resulted in prolonged survival and cure for a significant number of patients with stage1 small cell lung carcinoma. Graziano and colleagues [17] studied the relationship between the neuroendocrine differentiation of nonsmall cell lung carcinoma and the response to chemotherapy and found that nonsmall cell lung carcinoma with neuroendocrine differentiation was associated with an increased likelihood of responding to chemotherapy. We have previously reported that adjuvant chemotherapy can prolong the overall survival of patients with stage 1 large cell carcinoma with neuroendocrine features [18]. The result of this study suggests that patients with LCNEC should receive adjuvant chemotherapy following surgical resection even in stage 1 disease.
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Acknowledgments
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This work was supported in part by a Grant-in-aid for Scientific research (A) (2)15209045, (C) (2) 15591467, (C) (2)14571256, (C) (2) 13671376 of the Japanese Ministry of Education, Culture, Sports, Science, and Technology.
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References
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Abstract
Introduction
