Hirano H, Maeda H, Takeuchi Y, Susaki Y, Kobayashi R, et al. (2014) Immunohisthochemical Analysis of P-Stage I Large Cell Neuroendocrine Carcinoma of the Lung: Analysis of Adhesion Molecules and Proliferative Activity. J Cancer Biol Res 2(1): 1034.

LCNEC: Large Cell Neuroendocrine Carcinoma; PDA: Poorly Differentiated Adenocarcinoma; Ki-67 LI: Ki-67 Labeling Index

Large Cell Neuroendocrine Carcinoma (LCNEC), first described by Travis et al. in 1991, is a highly malignant tumor of the lung that has a very poor prognosis similar to that of small cell carcinoma of the lung [1]. LCNECs show histological features suggesting neuroendocrine differentiation, such as organoid nesting, trabecular growth, and rossette-like and perilobular palisading patterns. Neuroendocrine differentiation is confirmed by immunohistochemical staining of neuroendocrine markers such as synaptophysin, chromogranin A and CD56, as well as ultrastructual observation [2].

E-cadherin is a transmembrane protein that forms cellcell adhesion complexes with β-catenin. There are two forms of β-catenin, combined and free forms [3]. The combined form binds with the intracellular domain of E-cadherin and plays an essential role in cell-cell adhesion, while free β-catenin exists in the cytoplasm and can enter the nucleus while activiting the WNT signaling pathway. Reduced expression and dysfunction of E-cadherin, and nuclear β-catenin expression have been reported to be associated with poor prognosis in various cancers, including non-small cell lung cancer [3-6].

A LCNEC is highly malignant, though the related mechanism has not been fully clarified [7]. In order to elucidate the mechanism of high aggressiveness, we investigated the expressions of E-cadherin and β-catenin on the cell membrane and nuclear expression of β-catenin, as well as proliferative activity represented by Ki-67 labeling index using LCNEC specimens obtained form pathological (p)-stage I cases. As a control, p-stage I poorly differentiated adenocarcinomas (PDAs) of the lung where the solid-sheet component occupied more than 80% because adenocarcinomas of the lung consisting predominantly of solid sheets show the worst prognosis among the various reported histological types [8]. Furthermore, LCNECs consist of sheets or nests of tumor cells similar to the solid-sheet component of an adenocarcinoma.

Patients and tissue specimens

Tumors from 12 patients (11 males, 1 female) diagnosed with pathological (p)-stage I large cell endocrine carcinoma (LCNEC) of the lung and from 19 (17 males, 2 females) with p-stage I PDA of the lung were used for this study. Each patient underwent surgical resection of the tumor at Toneyama National General Hospital between 2002 and 2010 and none received neo adjuvant chemotherapy or radiation therapy prior to surgery. In all 19 p-stage I PDAs examined in this study, the PDA area occupied more than 80% of the entire tumor area of each tumor (Table 1). All patients underwent dissection of the bifurcation and ipsilateral mediastinal lymph nodes, and pathological examinations revealed no metastasis in any. Furthermore, computed tomography and magnetic resonance imaging confirmed no metastasis in these patients. All were categorized as p-stage I, according to the TNM classification of the International Union Against Cancer (7th edition) (tumor size <5.0 cm and no lymph node or distal metastasis, or tumor size <3cm and pleural invasion and no lymph node or distal metastasis,) [9]. Clinical information was obtained for each patient by reviewing their medical charts and is presented in Table 1.This study was approved by the Ethics Committee of Toneyama National General Hospital.

Histology

The largest diameter was used for tumor diameter. The tumors were fixed in 0.01M phosphate-buffered 10% formalin (pH 7.4) and several paraffin-embedded tumor blocks were made from each, then 5-µm in sections, was cut. Some sections were used for hematoxylin-eosin staining and others for immunohistochemistry. LCNEC was diagnosed on the basis of the histological criteria proposed by Travis in 1991 (Figure 1) [1], with immunohistochemistry findings showing neuroendocrine markers such as synaptophysin, chromogranin A and CD56.

Immunohistochemistry

Immunohistochemical staining was performed using an avidin-streptavidin immunoperoxidase method. A antigen retrieval by incubation of deparaffinized sections in cell condition 1 solution at a mild degree and subsequent immunohistochemical staining were carried out using an automated Benchmark system (Ventana Medical System, Tucson, AZ, USA) according to the manufacturer’s instructions. Antibodies used for immunohistochemistry are shown in Table 2.

Immunohistochemical staining

Positive immunostaining was defined as 5% or more tumor cells with staining. The Ki-67 labeling index (Ki-67 LI) was determined as the percentage of the tumor that was Ki-67- positive by examining 500-1000 tumor cells in the area with the highest labeling index using the Win Roof software program (Mitani Co. Tokyo, Japan). E-cadherin is known to exist on the cell membrane, while β-catenin is located on both the cell membrane and in the nucleus. Immunostaining of E-cadherin and β-catenin on the cell membrane, and in the nucleus was examined in randomly selected fields, then graded according to the percentage (p) of tumor cells stained positively as follows: negative, p 70%. There are two patterns of membrane staining for E-cadherin and β-catenin, membranelinear and membrane-disrupted. The membrane-linear pattern is seen as a continuous line of staining along the cell membrane and the membrane-disrupted pattern is seen as discontinuous disrupted staining along the cell membrane. For the present study, the predominant pattern was defined as the pattern shown by more than 50% of the membrane-positive tumor cells showed.

Statistical analysis

Disease-free rates were plotted according to the KaplanMeier method and analyzed using a Log-rank test. Data for more than 3 samples are presented as the mean ± S.D. and analyzed using Student’s t-test. Differences in frequencies between the groups were analyzed using the χ2 test. All statistical analyses were performed using the Excel Statistics 2012 software package for Windows (SSRI, Tokyo, Japan), with P values <0.05 considered to be statistically significant.

Table 1: Clinicopathological data of patients with large cell neuroendocrine carcinoma and poorly differentiated adenocarcinoma

BI: Brikemann Index; M: Male; F: Female

Table 2: Antibodies used for the immunohistochemistry.

Abbreviations: Dako Japan, Tokyo, Japan; Novocastra, Newcast upon Tyne, UK.

Figure 2 shows the disease-free rates of patients with p-stage I LCNEC and those with p-stage I PDA after surgery. The solidsheet component occupied more than 80% of the tumor area in all PDAs analzed (Table 1). The disease-free rate of patients with LCNEC rapidly decreased over time and became significantly lower than that of patients with PDA (P<0.05).

There was no significant difference in regard to age, sex ratio,smoking history and Brinkman index for patients with a smoking history between LCNEC and PDA (Table 3). Recurrence occurred in all patients with an LCNEC during the follow-up period, in contrast to in only 1 of 19 patients with a PDA (Table 3).

Immunohistochemical staining for the neuroendocrine markers, such as synaptophysin, chromogranin A and CD56 showed that all LCNECs expressed at least 1 of those markers, supporting the previous diagnosis of LCNEC (data not shown). Results of immunohistochemistry using E-cadherin, β-catenin, and Ki-67 are presented in Table 4 and 5. E-cadherin and β-catenin were stained on the cell membranes of the tumor cells (Figure 3A-D), while β-catenin staining was also observed in the nuclei (Figure 3E). In addition, the membrane-linear pattern was shown as a continuous line of staining along the cell membrane (Figure 3A, 3B), and the membrane-disrupted pattern as discontinuous staining along the cell membrane (Figure 3C, 3D). There was no significant difference in frequency of grade 3+ staining for E-cadherin or β-catenin between the LCNECs and solidsheet components of the PDAs (Table 5). However, all LCNECs predominantly showed a disrupted pattern of membrane staining for both E-cadherin and β-catenin (Figure 3C and D) while most solid-sheet components of the PDAs predominantly showed a membrane- liner pattern, and the frequencies of predominance of the membrane-disrupted pattern for E-cadherin and β-catenin were significantly greater in LCNECs than in the solid-sheet components of the PDAs (Table 5). Furthermore, the frequency of nuclear staining of β-catenin was significantly greater in the LCNECs than in the solid-sheet components. Finally, the Ki-67 labeling index for the LCNECs was approximately 4-fold greater than that for the solid-sheet components, which was a significant difference (Table 5, Figure 3F).

Table 3: Comparison of clinicopathological data of patients with large cell neuroendocrine carcinoma and poorly differentiated adenocarcinoma.

Abbreviations: *, One patient died of the other cause. NS: Not Significance

Table 4: Results of immunohistochemistry.

Abbreviations: The staining grade was determined according to the percentage (p) of positive cells: -, pp≥5%; 2+; 70%>p≥30%; 3+, p≥70%

Table 5: Comparison of the immunohistochemical results between large cell neuroendocrine carcinomas and solid-sheet components of poorly differentiated adenocarcinomas.

Abbreviations: NS; Not Significant. The staining grade was determined according to the percentage (p) of positive cells: -, pp≥5%; 2+; 70%>p≥30%; 3+, p≥70%.

The disease-free rate of patients with an LCNEC was much lower over time, as compared to patients with a PDA, the latter of which is predominantly composed of a solid-sheet component. The prognosis for patients with a PDA composed, predominantly of a solid-sheet component has been reported to be the worst among adenocarcinomas of various histological types [8]. In addition, it has been shown that the prognosis of patients with an LCNEC is as poor as of those with small cell carcinoma of the lung [10], while the prognosis of small cell carcinoma is worse than that of non-small cell carcinoma of the lung [2]. Therefore, the present result is in agreement with these previous studies.

There was no significant difference in regard to the membrane expression of E-cadherin and β-catenin between LCNECs and PDAs. However, all LCNECs predominantly showed the disrupted membrane-staining pattern whereas most solidsheet components of the PDAs predominantly showed the liner membrane-staining pattern. A disrupted membrane-staining pattern of E-cadherin and β-catenin indicates dysfunction of intercellular adhesion [11]. Therefore, the abnormal staining pattern of E-cadherin and β-catenin in LCNEC seems to be correlated to its aggressiveness.

There are two forms of β-catenin, combined and free [12,13]. The combined form binds with the intracellular domain of E-cadherin and plays an essential role in cell-cell adhesion. On the other hand, free β-catenin exists in the cytoplasm and can enter the nucleus, where it activates the WNT signaling pathway and switches on transcription of target genes such as c-myc and cyclin D1, resulting in proliferation and metastasis of tumor cells [12,14]. Nuclear β-catenin expression was found in 4 of 13 LCNECs, but none of 19 PDAs. Therefore, nuclear β-catenin expression in some LCNECs seems to be associated with their aggressiveness.

Ki-67 LI is considered to be a reliable proliferative marker for estimating malignancy grade [15] Mitosis is frequently found in LCNECs [2]. In agreement with those findings, we found that the Ki-67 labeling index of LCNECs was high, indicating the high proliferative potential of LCNEC tumor cells. In some LCNECs expression of nuclear β-catenin may partly contribute to their high proliferative activity.

In conclusion, the abnormal membrane expression of E-cadherin and β-catenin, as well as nuclear β-catenin expression and high proliferative potential are associated with LCNEC aggressiveness.

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A Large Cell Neuroendocrine Carcinoma (LCNEC) of the lung is highly malignant.  Reduced or abnormal expression of adhesion molecules, such as E-cadherin and  b-catenin, on the cell membrane is associated with the aggressiveness of its tumor  cells, while nuclear b-catenin activates the WNT signaling pathway. To examine the  mechanism of LCNEC aggressiveness, we used immunohistochemistry to examine the  expressions of E-cadherin and b-catenin in the membrane, as well as the nuclear  expression of b-catenin and Ki-67 labeling index in 12 pathological (p)-stage I LCNEC  specimens. As a control, we used solid-sheet components from 19 p-stages I solid  predominant Poorly Differentiated Adenocarcinomas (PDAs), as that tumor is the most  aggressive among non-small cell carcinomas of various histological types. The diseasefree rate of patients with LCNEC was much lower than that of patients with PDA. In the  LCNECs, there was no significant difference in the frequency of membrane-expression  of E-cadherin and b-catenin, though all specimens predominantly showed disrupted   patterns of membrane staining for both E-cadherin and b-catenin, while 16 of 19  PDAs predominantly showed a linear pattern. Nuclear b-catenin staining was found   in 4 of 13 LCNECs, but in none of the PDAs. The Ki-67 labeling index of the LCNEC  specimens was about 4-fold greater than that of the PDAs. The present results suggest  that abnormal membrane expression of E-cadherin and b-catenin, nuclear b-catenin  expression, and high proliferative potential are associated with LCNEC aggressiveness.