Ann Thorac Surg 2002;73:1732-1735
© 2002 The Society of Thoracic Surgeons
Original article: general thoracic
Prognostic significance of neuroendocrine differentiation in adenocarcinoma of the lung
Kenzo Hiroshima, MD*a,
Akira Iyoda, MDb,
Kiyoshi Shibuya, MDa,
Tetsuya Toyozaki, MDa,
Yukiko Haga, MDa,
Takehiko Fujisawa, MDb,
Hidemi Ohwada, MDa
a Department of Basic Pathology, Graduate School of Medicine, Chiba University, Chiba, Japan
b Department of Thoracic Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan
Accepted for publication February 6, 2002.
* Address reprint requests to Dr Hiroshima, Department of Basic Pathology, Graduate School of Medicine, Chiba University, 1-8-1 Inohona, Chuo-ku, Chiba 260-8670, Japan
e-mail: kenzo{at}med.m.chiba-u.ac.jp
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Abstract
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Background. The relative incidence of adenocarcinoma of the lung is increasing and some patients with lung carcinoma, detected at an early stage, still develop recurrent disease despite complete resection of the tumor. Recently, neuroendocrine differentiation in large cell carcinoma of the lung has been reported to be of prognostic significance. Therefore, we have evaluated the prognostic significance of neuroendocrine differentiation in adenocarcinoma of the lung.
Methods. A total of 90 resected specimens of adenocarcinoma of the lung measuring 3 cm or less (T1 N0 M0 or T2 N0 M0) were reviewed histologically and immunohistochemical staining was performed to determine the degree of neuroendocrine differentiation.
Results. Seven adenocarcinomas exhibited neuroendocrine differentiation in 10% or more of tumor cells. The disease-free survival rate for these patients was significantly lower than that of patients with tumors exhibiting neuroendocrine differentiation in less than 10% of tumor cells or with absent neuroendocrine differentiation (p < 0.0005). Other conventional pathologic factors such as vascular invasion (p < 0.0005), lymphatic invasion (p < 0.05), and pleural involvement (p < 0.05) were also of prognostic significance. In multivariate analysis, the presence of 10% or more neuroendocrine marker-positive tumor cells, vascular invasion, and lymphatic invasion were found to be significantly adverse prognostic factors (p = 0.0162, p = 0.0111, and p = 0.0173, respectively).
Conclusions. Neuroendocrine differentiation of tumor cells is a prognostic factor in lung adenocarcinoma. It is suggested that the identification of neuroendocrine differentiation as well as vascular invasion by tumor in small peripheral adenocarcinoma of the lung may predict the prognosis of these patients.
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Introduction
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The relative incidence of adenocarcinoma of the lung is currently increasing [1–3]. Complete surgical resection of the tumor offers the best chance of cure in this disease. Accurate staging of the lung carcinoma is the most important prognostic measure with the 5-year survival for surgically treated patients with T1 N0 M0 non-small cell carcinoma (NSCLC) being more than 75% [4–6]. However, some patients who undergo complete resection of lung carcinoma after an early diagnosis of the tumor may still develop recurrent disease after operation.
Large cell carcinomas of the lung are classified into four types based on light microscopic evidence of neuroendocrine morphology and an immunohistochemical or electron microscopic assessment of neuroendocrine differentiation as follows: (1) large cell neuroendocrine carcinoma exhibits both neuroendocrine morphology and evidence of neuroendocrine differentiation; (2) large cell carcinoma with neuroendocrine differentiation exhibits neuroendocrine markers but lacks neuroendocrine morphology; (3) large cell carcinoma with neuroendocrine morphology exhibits neuroendocrine morphologic features but lacks neuroendocrine markers; and (4) classic large cell carcinoma exhibits neither neuroendocrine morphology or differentiation [7]. We have previously reported that large cell carcinoma with neuroendocrine differentiation is clinically more aggressive than classic large cell carcinoma like large cell neuroendocrine carcinoma or large cell carcinoma with neuroendocrine morphology [8]. Neuroendocrine markers in NSCLC are expressed not only in large cell carcinoma but also in adenocarcinomas [9, 10]. The aim of this study was to evaluate the incidence and prognostic significance of neuroendocrine differentiation in pulmonary adenocarcinoma.
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Material and methods
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One hundred two peripheral primary lung adenocarcinomas with a maximal tumor diameter of 3 cm or less and tumor staging of either T1 N0 M0 or T2 N0 M0 were resected at our institution between January 1991 and December 1997. Histologic typing was determined according to the World Health Organization classification [7], whereas TNM classification and stage of disease were determined according to the TNM classification of the International Union Against Cancer [11]. No preoperative treatment was administered and all tumors were completely resected. Formalin-fixed, paraffin-embedded tumor blocks were available for 90 of these 102 tumors and these were analyzed retrospectively. The specimens had been sliced at 5- to 10-mm intervals. They were evaluated microscopically by conventional staining including hematoxylin and eosin and elastic stains. Pleural involvement, vascular invasion, and lymphatic invasion by tumor were assessed. Pleural involvement was classified as p0, p1, p2, and p3; p0 included tumor with no invasion beyond the visceral elastic pleura; p1 included tumor with invasion beyond the visceral elastic pleura, although limited to the pulmonary pleura; p2 included tumor with invasion of the surface of the pulmonary pleura; and p3 included tumor invasion of the chest wall, diaphragm, mediastinal structures, or adjacent lobes [12]. Because only patients with T1 or T2 lesions were evaluated there were no p3 tumors included in this study. In addition, there were no cases of p2 tumors in this study. Vascular and lymphatic invasion was determined by the presence of identifiable tumor cells in the lumen of blood or lymphatic vessels.
Neuroendocrine differentiation was determined by immunohistochemical staining with a polyclonal antichromogranin A antibody (Nichirei Corporation, Tokyo, Japan), a monoclonal antisynaptophysin antibody (DAKO, Glostrup, Denmark), and a monoclonal antineural cell adhesion molecule (Zymed, San Francisco, CA) as previously described [8]. Combined large cell neuroendocrine carcinoma and adenocarcinoma was excluded from this study. Neuroendocrine differentiation was quantified for percent of immunopositive neoplastic cells in the sections with the greatest dimension. The percent positive cells was graded 0 through 2 (NE-0 = negative, NE-1 = 1% to 9% positive, NE-2 = 10% to 100% positive).
Clinical information collected from the medical records included patient sex, age, smoking status, tumor size, and patient outcome. The length of disease-free survival was defined as the interval between the date of operation and the time of first local or distant recurrence and was evaluated using the method of Kaplan-Meier [13]. The curves obtained were compared with the log-rank test. The prognostic impact of pleural involvement, vascular invasion, lymphatic invasion, and neuroendocrine differentiation was determined using Cox’s proportional hazards multivariable regression model [14]. A p value of less than 0.05 was considered to be statistically significant.
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Results
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Sixty-nine tumors (76.7%) were negative for all neuroendocrine markers examined (NE-0). Fourteen tumors (15.6%) had 1% to 9% positive cells with at least one neuroendocrine marker (NE-1). Seven tumors (7.8%) had 10% or more positive cells (NE-2) (Fig 1). Approximately 10% of the tumor cells stained in 1 patient, approximately 20% of the tumor cells stained in 2 patients, and the tumor stained diffusely with neuroendocrine markers in 4 patients. There was no tumor that stained only with monoclonal antineural cell adhesion molecule in NE-1 or in NE-2. The summary of characteristics of NE-2 tumors are listed in Table 1.
The NE-2 tumors included four moderately differentiated papillary adenocarcinomas, two moderately differentiated acinar adenocarcinomas, and one poorly differentiated acinar adenocarcinoma. In the acinar adenocarcinomas, the tumor cells were arranged in an organoid pattern with thin stroma that differed from large cell neuroendocrine carcinoma because of the existence of apparent glandular formation.
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Fig 1. Immunohistochemical staining of pulmonary adenocarcinoma of the lung with polyclonal antichromogranin A antibody. The tumor cells stain positive with antichromogranin A antibody (original magnification x50).
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The median follow-up period and disease-free 5-year survival rate for the entire group was 64 months and 89.6%, respectively. The disease-free 5-year survival rate for patients with NE-0 tumors (n = 69) was 92.5% and was comparable at 92.9% for patients with NE-1 tumors (n = 14). However, the disease-free 5-year survival rate for patients with NE-2 tumors (n = 7) was significantly lower at 57.1% (p < 0.0005; Fig 2).
Furthermore, 3 of the 4 patients whose tumor stained diffusely with neuroendocrine markers died within 5 years (Table 1). Other conventional pathologic factors such as vascular invasion (p < 0.0005), lymphatic invasion (p < 0.05), and pleural involvement (p < 0.05) also exhibited adverse prognostic significance (Table 2).
Patient age, gender, smoking status, or the size of the tumor did not significantly affect patient survival rate. The disease-free 5-year survival rate for patients with well-differentiated adenocarcinoma (n = 26) was 100%, but there was no significant difference in histologic differentiation.
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Fig 2. The disease-free survival curve for pulmonary adenocarcinoma measuring 3.0 cm or less with T1 N0 M0 or T2 N0 M0. The disease-free survival for patients with NE-2 tumors is significantly lower than that those for patients with NE-0 and NE-1 tumors (p < 0.0005). NE-0 was defined as tumor negative for all neuroendocrine markers, NE-1 as tumor having 1% to 9% positive cells with at least one neuroendocrine marker, and NE-2 as tumor having 10% or more positive cells with at least one neuroendocrine marker. (NE = neuroendocrine.)
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We performed multivariate analysis for patient survival with respect to pleural involvement, vascular and lymphatic invasion, and neuroendocrine differentiation. Because the disease-free survival rates of patients with NE-0 and NE-1 tumors were not significantly different, these groups were combined together and compared with patients with NE-2 tumors. Analyses indicated that the presence of 10% or more tumor cells exhibiting positivity for neuroendocrine markers was a significant adverse prognostic factor (p = 0.0162), along with evidence of vascular invasion (p = 0.0111) and lymphatic invasion (p = 0.0173) (Table 3).
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Comment
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Many specific histologic measures have been assessed as possible prognostic indicators in lung carcinomas. Blood vessel invasion by tumor cells predicts recurrence in completely resected T1 N0 M0 NSCLC [6], whereas the presence of a high mitotic index in NSCLC indicates an adverse prognosis [15, 16]. The expression of C-erbB-2 or the presence of the K-ras oncogene mutation predicts a shortened survival for patients with lung adenocarcinoma [17]. Resected localized bronchiolar lung carcinomas measuring 2 cm or less have a favorable prognosis [18]. In addition, the size of central fibrosis in peripheral adenocarcinoma of the lung is reported to be of prognostic significance [19].
There are some data available regarding the relationship between neuroendocrine differentiation of lung carcinoma and prognosis. Linnoila and colleagues [20] reported that neuroendocrine differentiation did not predict recurrence in patients with resected NSCLC. Sundaresan and associates [21] reported no correlation between neuroendocrine differentiation and survival. However, Berendsen and co-workers [22] reported that NSCLC biopsies containing more than 50% positive-staining tumor cells with the MOC-1 antibody, which reacts with neuroendocrine tissues, is considered to be a negative prognostic factor. We have previously reported that pulmonary large cell carcinoma with neuroendocrine features (large cell neuroendocrine carcinoma, large cell carcinoma with neuroendocrine morphology, and large cell carcinoma with neuroendocrine differentiation) has a poorer prognosis than classic large cell carcinoma [8].
Skov and colleagues [10] reported that more than 10% of tumor cells were chromogranin A positive in 19% of pulmonary adenocarcinoma but found that survival curves constructed for various degrees of tumor chromogranin A positivity were comparable. In contrast, we found that pulmonary adenocarcinoma exhibiting 10% or more positive cells for neuroendocrine markers had a poorer prognosis than tumors with less than 10% positive cells. Especially, 3 of the 4 patients with tumors that stained diffusely with neuroendocrine markers died within 5 years after the resection of the tumor. Multivariate analysis in this study indicated that the presence of neuroendocrine differentiation in adenocarcinoma was a significant and independent adverse prognostic factor for disease-free patient survival. The difference between our study and that of Skov and colleagues may well be secondary to differences in staging, as their patients were in stage III or IV and inoperable, whereas our patients were at an early stage and underwent complete surgical resection. Indeed, we specifically studied pulmonary adenocarcinoma measuring 3 cm or less at stage T1 N0 M0 or T2 N0 M0 to minimize the confounding effects of prognostic factors such as lymph node and distant metastasis or direct invasion of adjacent structures. Because the number of NE-2 tumor in this study is small, further analysis of the prognostic importance of neuroendocrine differentiation in adenocarcinoma of the lung is required.
In conclusion, neuroendocrine differentiation of tumor cells is a prognostic factor in early pulmonary adenocarcinoma. The identification of neuroendocrine differentiation in adenocarcinoma of the lung may be a good marker to predict the prognosis of these patients as well as vascular invasion by tumor.
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Acknowledgments
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We thank Michiko Hanazono, Ayaka Sato, Kazuko Abe, and Tamiyo Taniguchi for their technical assistance. This study was partially supported by the Smoking Research Foundation.
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Abstract
Introduction
