Eur J Cardiothorac Surg 2006;30:538-542
© 2006 Elsevier Science NL
Fascin immunoreactivity for preoperatively predicting lymph node metastases in peripheral adenocarcinoma of the lung 3 cm or less in diameter
Phil Jo Choia,
Doo Kyung Yangb,
Choon Hee Sonb,
Kyung Eun Leec,
Jae Ik Leed,
Mee Sook Rohc,e,*
a Department of Thoracic and Cardiovascular Surgery, Dong-A University College of Medicine, Busan, South Korea
b Department of Internal Medicine, Dong-A University College of Medicine, Busan, South Korea
c Medical Research Center for Cancer Molecular Therapy, Dong-A University College of Medicine, Busan, South Korea
d Department of Thoracic and Cardiovascular Surgery, Gachon Medical School, Incheon, South Korea
e Department of Pathology, Dong-A University College of Medicine 1,3-ga, Dongdaeshin-dong, Seo-gu, Busan 602-715, South Korea
Received 21 March 2006;
accepted 26 June 2006.
* Corresponding author. Address: Department of Pathology, Dong-A University College of Medicine 1,3-ga, Dongdaeshin-dong, Seo-gu, Busan 602-715, South Korea. Tel.: +82 51 240 2833; fax: +82 51 243 7396. (Email: msroh{at}dau.ac.kr).
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Abstract
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Objective: Fascin is an actin-bundling protein that induces cell membrane protrusions and increases the motility of normal and transformed epithelial cells. We evaluated the expression of fascin by performing immunohistochemistry to determine its role in the progression of small-size peripheral lung adenocarcinomas and to elucidate its utility as a preoperative novel therapeutic option. Methods: Immunohistochemistry for fascin was performed in 49 peripheral adenocarcinomas of 
3 cm in diameter on the conventional whole tissue (CWT) sections and 2 mm-sized tissue microarray (TMA) cores that were the substitute for preoperative biopsy samples. Staining of more than 5% of tumor cells was recorded as positive immunoreactivity. The distribution of fascin labeling was measured according to the percentage of fascin-positive cells: 5–25% (low grade) and >25% (high grade). Results: Overall, fascin immunoreactivity was detected in 30 (61.2%) out of the total 49 cases. The tumors with high-grade fascin immunoreacivity tended to more frequently show lymph node metastasis (P
= 0.0007), lymphovascular invasion (P
= 0.0084) and a higher stage (P
= 0.05). There was no significant association with age, gender, tumor size and the histological subtype. The 2 mm-sized TMA cores, which were considered as a substitute for percutaneous needle biopsy sample in this study, showed concordant results with the CWT section (
= 0.836). Conclusions: We suggest that fascin immunoreactivity may identify the subsets of peripheral adenocarcinomas of the lung 3 cm or less in diameter that have a different potential to metastasize to the regional lymph nodes. The evaluation of fascin immunoreactivity on the preoperative biopsy sample could be a novel therapeutic strategy for selecting the most appropriate therapy for small-size pulmonary adenocarcinomas.
Key Words: Lung adenocarcinoma • Fascin • Immunohistochemistry • Lymph node metastasis
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1. Introduction
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Many small adenocarcinomas can now be detected as a result of the recent advances in diagnostic radiology. Despite their small size, however, some of them are already advanced tumors at the time of diagnosis, or they may develop distant metastasis after complete surgical resection [1]. These results indicate that a small tumor size does not always predict a good outcome. Since the histological and biological heterogeneity of adenocarcinoma often makes it difficult to predict the outcome for operated patients, clarifying the morphological and molecular prognostic factors of this tumor is indispensable to select an appropriate treatment strategy. If we could select the patients with a small-sized adenocarcinoma who have an excellent prognosis, either preoperatively or intraoperatively, then the surgeon could decide on limited surgery intraoperatively based on the favorable factors. If unfavorable prognostic factors were recognized after the operation, additional postoperative modalities could be employed to prolong survival. Therefore, it is important for us to be able to distinguish between these two groups of patient.
Fascin is a highly conserved 55 kDa actin-bundling protein that plays an important role in the organization of several types of actin-based structures such as filopodia, spikes, lamelipodial ribs, dendrites and microvilli [2]. Its overexpression results in decreased cell-to-cell adhesion and increased epithelial cell motility [2]. The fascin expression is usually absent or very low in normal epithelial cells, but it is often upregulated in several types of human neoplasms such as ovarian, breast, pancreatic, colon, lung, skin, stomach, esophagus and urinary bladder tumors [3–11]. A high level of fascin in these neoplastic cells is associated with progressive high-grade tumors and tumors with great metastatic potential [3–11].
Herein we performed immunohistochemical detection of fascin protein in peripheral adenocarcinoma tissue samples of the lung that were 3 cm or less in diameter by using conventional whole tissue (CWT) sections to define the relationship of the fascin expression with the progression of small-sized lung adenocarcinoma. Moreover, we performed immunohistochemical staining for fascin with using the corresponding high density tissue microarray (TMA) core, which is considered as the most suitable and reliable substitute for a preoperative biopsy sample, to define whether the immunohistochemical detection of fascin could provide useful information as a novel preoperative therapeutic option.
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2. Materials and methods
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2.1 Patient and tissue specimens
Forty-nine cases of peripheral adenocarcinoma of the lung that had a maximal tumor dimension of 3 cm or less were retrospectively selected from the surgical pathology records of the Department of Pathology at Dong-A University Medical Center, and these records were complied between 1994 and 2004. No preoperative chemotherapy or radiotherapy had been performed in any of these cases. Standard lobectomy and lymph node dissections were performed in every case. The cases having any other malignancies that occurred before or after the primary lung cancer were excluded from our study. At the time of performing thoracotomy, the mediastinal lymph nodes were dissected as completely as possible including ipsilateral, paratracheal, lower mediastinal, subcarinal and N1 areas to arrive at an accurate pathological staging. The clinical records, pathological reports and follow-up information were also obtained when available. The institutional review board approved our study, and a written informed consent was obtained from all the patients for surgery and to use their resected samples for research. The hematoxylin and eosin (HE)-stained slides were reviewed in each case to confirm the original diagnosis that was based on World Health Organization criteria [12]. Further, all the cases were subdivided into three types: bronchioloalveolar carcinomas (BAC), adenocarcinomas with BAC components and invasive carcinomas (mixed), and invasive adenocarcinomas without BAC components (invasive). The postoperative pathological staging was determined according to the guidelines of the American Joint Committee on Cancer (AJCC) [13].
2.2 Construction of TMA
To define whether the immunohistochemical detection of fascin could provide useful information as a novel preoperative therapeutic option, a TMA was constructed, which is considered as the most suitable and reliable substitute for the preoperative biopsy sample. After making one slide from each specimen for examining the fascin expression staining on the CWT section, the TMA was designed as follows. After reviewing all of the HE-stained slides from the cases, one representative slide was selected from each case, and one area of the tumor was circled on the slide. Creation on the blocks was done with a precision instrument (Beecher Instruments, Silver Spring, MD, USA) that uses two separate core needles for punching the donor and recipient blocks, and a micrometer precise coordinate system for assembling the microsamples on a block. The selected area in the donor block was cored with a 2 mm diameter needle, and the tissue cores from each tumor were then mounted in linear arrays in the recipient paraffin TMA block. The final tissue microarray consisted of one block that contained 49 spots, with the sample spaced 2 mm apart.
2.3 Immumohistochemistry for fascin
The CWT and TMA section slides were subjected to immunohistochemical staining as follows. The immunohistochemical study for fascin was performed on formalin-fixed, paraffin-embedded, 4 µm-thick tissue sections, using the avidin–biotin–peroxidase complex method. The primary antibody was a mouse monoclonal antibody directed against fascin (DakoCytomation, Carpinteria, CA, USA) used at 1:50 dilution. Deparaffinization of all sections was performed through a series of xylene baths, and rehydration was performed with a series of graded alcohol solutions. To enhance the immunoreactivity, microwave antigen retrieval was performed at 750 W for 30 min in citrate buffer (pH 6.0). After blocking the endogenous peroxidase activity with 5% hydrogen peroxidase for 10 min, incubation with the primary antibody was performed for 1 h at room temperature. An EnvisionTMChemTM Detection Kit (DakoCytomation) was used for the secondary antibody at room temperature for 30 min. After washing the tissue samples in Tris buffered saline for 10 min, 3,3'-diaminobenzidine was used as a chromogen, and then Mayer's hematoxylin counterstain was applied.
2.4 Immunohistochemical assessment
The fascin immunoreactivity in each tumor was verified by the labeling of endothelial cells of the microvessels in every specimen. Fascin-positive samples were defined as those showing a cytoplasmic staining pattern of the lesional tissue. Staining more than 5% of tumor cells was recorded as positive immunoreactivity. The distribution of fascin labeling was measured according to the percentage of fascin-positive cells: 5–25% was considered low grade and >25% was considered high grade. The overall result was recorded for the CWT section. The same observer (M.S. Roh), who was kept blinded on what particular tissue section she was working on, evaluated the TMA cores. Each of the CWT and TMA sections was scored in the manner described above.
2.5 Statistical analysis
The association between the clinicopathological characteristics and the fascin grade were analyzed using contingency tables. Statistical significance was evaluated with 
2 tests. The statistical difference was considered significant for a P value was less than 0.05. Concordance between the CWT and TMA sections was evaluated using statistics. A positive (or negative) score of a TMA core and a positive (or negative) score of a CWT section were considered a match, respectively. value in excess of 0.7 was considered as strong associations between the methods. The data were analyzed with the Statistical Package, SPSS 11.0 for Windows (SPSS Inc., Chicago, IL, USA).
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3. Results
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3.1 Clinicopathological characteristics
The patients consisted of 26 men and 23 women, and they ranged in age from 40 to 76 years (median, 58 years). The tumor size ranged from 0.7 to 3 cm, with eleven cases involving tumors 2 cm, while 38 cases involved tumors >2 cm. Histologically, the tumors were all adenocarcinomas that showed a mixture of the various histological subtypes, including the acinar, papillary, solid and bronchioloalveolar. When all the cases were subdivided into the three types, there were 1 in the BAC, 19 in the mixed and 29 in the invasive groups. There were 33 negative cases and 16 positive cases of lymph node metastases. There were 29 negative cases and 20 positive cases of lymphovascular invasion. According to the AJCC staging system, 33 patients were stage IA, 6 were stage IIA and 10 were stage IIIA.
3.2 Immunohistochemical findings of fascin
Fascin immunoreactivity appeared as a fine, granular to diffuse cytoplasmic staining in both the normal and tumor cells.
In the normal lung, the non-neoplastic bronchial and alveolar epithelial cells were consistently non-reactive for fascin. Likewise, the surface ciliated and mucous cells of the bronchial epithelium and the seromucinous glands of the bronchial wall did not show fascin immunostaining. However, positive staining was observed in the endothelial cells of bronchial and alveolar walls, lymphocytes and stromal cells in the interstitium (Fig. 1A).
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Fig. 1. Immunohistochemical stainings for fascin on the conventional whole tissue sections. (A) In the normal lung, fascin was invariably expressed on microvessels endothelial cells as well as the stromal cells of the interstitium, whereas the bronchoalveolar epithelial cells were non-reactive for fascin. (B) A mixed type adenocarcinoma showed as distinctly fascin-positive in the invasive component (left) and as fascin-negative in the bronchioloalveolar component (right). (C) In an adenocarcinoma with lymphovascular invasion, the tumor emboli in the lymphovascular spaces showed positive reactivity for fascin.
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In the adenocarcinoma, fascin immunoreactivity was detected in 30 out of a total of 49 cases (61.2%). There were 17 (38.8%) and 13 (26.5%) cases having low grade and high grade fascin expressions, respectively. The fascin immunoreactive tumors sometimes showed heterogeneity in the percentage of labeled cells and the intensity of immunostaining. The BAC showed negative reactivity for fascin. Some tumors, especially for the mixed type, showed distinct fascin positivity in the invasive components and fascin negativity in the bronchioloalveolar components (Fig. 1B). Other tumors of the invasive type showed diffuse or weak staining patterns in the center of the tumor, whereas increased immunoreactivity was seen at the edge of the tumor. The tumor emboli in the lymphovascular spaces frequently showed positive reactivity for fascin (Fig. 1C). The surrounding stroma sometimes showed strong fascin immunoreactivity even without a fascin expression in the tumor cells.
3.3 Correlation between fascin immunoreactivity on the CWT sections and the clinicopathological characteristics
The various clinicopathological characteristics of the patients and their tumors were compared according to the fascin immunoreactivity (Table 1
). The analysis revealed that the fascin expression was correlated with lymph node metastasis, lymphovascular invasion and the pathologic stage (P
= 0.05), according to the grade of fascin immunoreactivity. The tumors with high-grade fascin immunoreacivity tended to more frequently show lymph node metastasis (P
= 0.0007), lymphovascular invasion (P
= 0.0084) and a higher stage (P
= 0.05). There was no significant association with age, gender, tumor size and the histological subtype.
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Table 1. The relationship between clinicopathological characteristics and the expression of fascin on the conventional whole tissue sections of 49 lung adenocarcinoma patients
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3.4 Correlation between the CWT and TMA section immunoreactivity
The cores from all 49 cases were readable. The relationship between the immunoreactivities of CWT sections and TMA cores is shown in Table 2
and Fig. 2
. Thus, the false negative rate among all the TMA cores taken from the CWT sections with positive fascin immunoreactivity is 10.0% (3 in 30). The false positive rate among all the TMA cores taken from the CWT sections with negative fascin immunoreactivity is 5.3% (1 in 19). As was expected, none of the cases had high-grade TMA cores in the presence of a negative CWT sections and none of the cases had negative TMA cores in the presence of high-grade CWT sections. The Pearson correlation coefficient between the CWT section results and TMA core results was 0.836.
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Table 2. The relationship between the conventional whole tissue section and the tissue microarray core for fascin immunoreactivity
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Fig. 2. Immunohistochemical stainings for fascin on the conventional whole tissue sections (A–C) compared with those of the tissue microarray cores (D–F). Negative (A and D), low grade (B and E) and high grade (C and F).
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4. Discussion
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In the present investigation, we have shown the prevalence of fascin immunoreactivity in the peripheral adenocarcinomas of the lung that were 3 cm or less in diameter, and we found a significant correlation between fascin immunoreactivity and the pathological findings. These results suggest the possible involvement of this protein in the development and progression of small-sized lung adenocarcinoma and especially lymph node metastasis.
Tumor invasion and metastasis are the results of several sequential steps and they are caused, in part, by the motile properties of tumor cells that can overcome cell-to-cell and cell-to-matrix adhesion and so invade the surrounding tissue [14,15]. Obviously, it is impossible to evaluate this feature of malignant tumors in fixed sections of tissues. Recent studies have shown that fascin, an important regulatory protein of cellular motility, is associated with the progression and metastasis of several common tumors [3–11]. Pelosi et al. [7] have reported that they observed fascin expression in 78% of 96 adenocarcinomas of stage I non-small cell lung cancer and fascin was an independent prognostic factor; a diffusely strong distribution of fascin was associated with a higher grade and contralateral thoracic or distant metastases. Hashimoto et al. [10] have reported that they observed fascin down-regulation and decreased motility and invasiveness in an esophageal squamous cell carcinoma cell line by using the vector-based siRNA. Although there still remains the question of which pathway contributes to the overexpression of fascin protein in lung adenocarcinoma, down-regulation of tumor-specific fascin may become a novel therapeutic strategy.
In the present investigation, we found significant correlations between lymphovascular invasion and lymph node metastasis and the fascin expression grade; this suggests a role for this molecule in the process of metastasis for pulmonary adenocarcinoma. In another report, fascin immunoreactivity was closely correlated with the occurrence of lymph node metastases and the number of involved lymph nodes in pulmonary neuroendocrine tumors [16]. In gastric carcinoma, the fascin expression was associated with lymph node metastasis and a peculiar prevalence of the fascin expression in the metastatic tumors of lymph nodes was observed [9]. However, the precise role of the fascin expression in the progression of primary lung adenocarcinoma to lymph node metastasis is still unclear. Pelosi et al. [7] have reported that the correlation between fascin upregulation and a tumor's metastatic potential may be, at least in part, justified by the peculiar prevalence of fascin expression in most neoplastic emboli inside the blood vessels, which is independent of the tumor type, and also in most endothelial cells of the pulmonary microvessels, but not of the major vessels. The metastasis mechanism could involve either direct vascular permeation by the actively migrating tumor cells that have strong fascin expression or the development of newly formed microvessels that are highlighted by strong fascin immunoreactivity. Further work is needed to test the possibility of using fascin as a more suitable marker of metastatic potential, and this may predict vascular permeation and lymphatic and/or distant metastases of small-size peripheral adenocarcinomas of the lung.
The recent advances in detecting small peripheral lung adenocarcinomas have displayed the need to assess the value of performing limited types of surgical intervention for this disease [17]. However, even at the same stage and with similar pathologic characteristics, the 3 cm or smaller lung adenocarcinomas that are surgically treated can have a variable prognosis depend on the biologic characteristics of the tumor [1]. Using tumor size alone, based on non-invasive staging, seems to be inaccurate for choosing the most appropriate treatment strategy for parenchymal lesions. It is clear that the presence of nodal metastasis is of paramount prognostic importance. With this in mind, researchers are looking for ways to accurately estimate the extent of malignancy for each small tumor. If the morphological and molecular prognostic markers that define a biologically distinct subgroup could be detected on a preoperative biopsy sample, then this would be of value from a therapeutic perspective. However, the biopsies of peripheral pulmonary adenocarcinomas that are obtained by percutaneous routes are often small. The question then arises whether the biopsy sample is representative of the entire lesion. As shown in this study, the 2 mm-sized TMA cores, which had a size that was similar to that of the lesion in the percutaneous needle biopsy sample, were excellent representatives of the CWT sections, so there seems no reason why there should be a difference between the resection and biopsy sample as long as the latter are sampled in a non-biased manner. Since fascin is not expressed in normal lung epithelium, the fascin expression can be used to support the diagnosis of adenocarcinoma, particularly in small tissue samples. Furthermore, because fascin usually has opposite intertumor and intratumor staining properties (25% vs >25% of immunoreactivity), the assessment of fascin in the biopsy specimen might assist in distinguishing between potentially aggressive disease and potentially non-aggressive disease when screening small lung adenocarcinomas.
In conclusion, this study indicates that fascin immunoreactivity may identify the subsets of peripheral adenocarcinomas of the lung 3 cm or less in diameter that have different potential to metastasize to regional lymph nodes. The evaluation of fascin immunoreactivity on preoperative biopsy samples could be a novel therapeutic strategy for selecting the most appropriate therapy for treating pulmonary adenocarcinomas.
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Acknowledgments
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This work was supported by the Korea Science and Engineering Foundation through the MRCCMT at the Dong-A University.
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Abstract
1. Introduction
