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Immunohistochemical study of Napsin A and thyroid transcription factor-1 in non-small-cell lung carcinoma

Tawfik, Heba M.a; Farghaly, Essam A.a; Gad, Yaser A.b

Egyptian Journal of Pathology: July 2012 - Volume 32 - Issue 1 - p 47–51
doi: 10.1097/01.XEJ.0000415808.32127.71
ORIGINAL ARTICLES
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Background Accurate typing of non-small-cell lung carcinomas is needed in the treatment for pulmonary adenocarcinoma. The histological subtype of non-small-cell lung carcinoma is important for selecting appropriate chemotherapy for patients with advanced disease, which has increased the need for immunohistochemical studies. Because of the very small biopsy samples of lung tissue, the distinction between poorly differentiated lung adenocarcinoma and squamous cell carcinoma (SqCC) is very difficult. The value of histochemical and immunohistochemical markers to help separate poorly differentiated adenocarcinoma from SqCC in resection specimens has been well established; however, the optimal use of markers in small tissue samples has only recently been examined, and the correlation of marker expression in small tissue samples with histologic subtype determined on resection specimens has not been well documented.

Aim of the work The aim of this study was to differentiate between bronchoscopic biopsies of moderately to poorly differentiated adenocarcinoma and SqCC of the lung by using different immunohistochemical markers.

Materials and methods Immunohistochemical analysis was performed for Napsin A and thyroid transcription factor-1 (TTF-1) using bronchoscopic fresh lung tissues of 34 adenocarcinomas and 18 SqCCs.

Results Pulmonary adenocarcinomas were Napsin A positive in 27 (79%) of 34 cases compared with 22 (64.7%) of 34 cases that were TTF-1 positive. There were eight Napsin A-positive/TTF-1-negative and two TTF-1-positive/Napsin A-negative tumors. Regarding SqCCs, Napsin A was positive in two (11%) of 18 cases compared with one (5%) of 18 cases that were positive for TTF-1.

Conclusion The combined use of Napsin A and TTF-1 results in improved sensitivity and specificity for identifying pulmonary adenocarcinoma in primary lung tumors and helps to distinguish it from poorly differentiated SqCC.

aDepartment of Pathology, Faculty of Medicine, Minia University, Minya

bChest Department, Assiut University, Asyut, Egypt

Correspondence to Heba M. Tawfik, PhD, Department of Pathology, Faculty of Medicine, Minia University, Minya 61111, Egypt Tel: +20 106 767 744; e-mail: aliayman@yahoo.com

Received December 10, 2011

Accepted December 25, 2011

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Introduction

Lung cancer is the leading cause of cancer-related death worldwide, and approximately 70–80% of cases fall under the classification of non-small-cell lung carcinoma (NSCLC; Lynch et al., 2004). Within this category, adenocarcinoma is the predominant subtype. Approximately 40–60% of patients present with locally advanced or metastatic disease at the time of diagnosis (Ueno et al., 2003).

The availability of targeted therapies has created a need for precise subtyping of NSCLCs. Unlike previous years, when such subtyping had no therapeutic relevance, differentiating between adenocarcinoma and squamous cell carcinoma (SqCC) is now important because new therapies have been developed that have different therapeutic or adverse effects depending on the histological type.

Although the majority of NSCLCs can be subtyped by examination of hematoxylin–eosin (H&E)-stained slides alone, difficulty is encountered in poorly differentiated tumors, especially in small biopsy specimens. Although earlier series have examined the role of immunohistochemical markers in the diagnosis of adenocarcinoma and SqCC, most researchers have studied all tumors regardless of differentiation, including those in which immunohistochemical stains are not needed for diagnosis (Bishop et al., 2010; Yang and Nonaka, 2010). For example, the epidermal growth factor receptor inhibitors gefitinib and erlotinib are more likely to be effective in adenocarcinomas than in SqCCs (Sanjay and Anna-Luise, 2011).

The antivascular endothelial growth factor agent bevacizumab is associated with a higher incidence of pulmonary hemorrhage in SqCCs than in nonsquamous carcinomas and is therefore contraindicated in SqCCs (Azzoli et al., 2009). The addition of the antifolate agent, pemetrexed, to conventional chemotherapy provides increased efficacy in nonsquamous carcinomas but not in SqCCs (Scagliotti et al., 2008).

Napsin A is an antibody that has recently emerged as a marker for pulmonary adenocarcinomas. Although Napsin A occasionally stains nonpulmonary adenocarcinomas, it is highly useful for separating primary lung adenocarcinomas from SqCCs (Suzuki et al., 2005).

It is a functional aspartic proteinase that is expressed in normal lung parenchyma in type II pneumocytes and in the proximal and convoluted tubules of the kidney (Mori et al., 2002). It is present in the lysosomes of type II pneumocytes and alveolar macrophages (probably secondary to phagocytosis) and to a lesser degree in pancreatic acini and ducts (Mori et al., 2001). The proteinase is expressed abundantly in the cytoplasm of normal lung cells (type II pneumocytes and Clara cells) and kidney cells (proximal and convoluted tubules) and in lung adenocarcinomas and renal cell carcinomas (Ueno et al., 2004).

It has been demonstrated that the expression of Napsin A is regulated by thyroid transcription factor-1 (TTF-1), a member of the Nkx2 family of transcription factors that also regulates the expression of surfactant protein B (Johansson, 2004). Previous studies using resected tumor tissues demonstrated that Napsin A was equal to or better than TTF-1 and surfactant protein A immunostains for determining lung origin in well to moderately differentiated adenocarcinomas (Kargi et al., 2007). Further, previous studies demonstrated that Napsin A and TTF-1 expression levels were decreased in poorly differentiated lung adenocarcinomas compared with well-differentiated carcinomas (Hirsch et al., 2008).

TTF-1 has been the predominant immunohistochemical marker used to identify lung origin and has a reported sensitivity of 75–80% for lung adenocarcinomas (Jagirdar, 2008). However, TTF-1 also stains other tissues and tumors, such as thyroid tissue, metastatic breast carcinoma, neuroendocrine tumors such as small-cell lung carcinoma and carcinoid, and to a lesser degree primary lung SqCC (Bishop et al., 2010; Yang and Nonaka, 2010).

TTF-1 is a nuclear protein that, in the lung, regulates surfactant expression. It is a highly specific marker for carcinomas of lung and thyroid origin and is currently the most widely used marker for confirming pulmonary origin of adenocarcinomas of the lung. TTF-1 is positive in most adenocarcinomas and small-cell carcinomas of the lung, and it is also seen in a subset of small-cell carcinomas from nonpulmonary organs (Justin et al., 2010).

A combination of the two stains (TTF-1 and Napsin A) has been proposed to achieve higher sensitivity and specificity for lung adenocarcinoma, and the combination might have greater reliability (Bishop et al., 2010).

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Materials and methods

Patients

A total of 52 lung cancers (34 adenocarcinomas and 18 SqCCs) were selected from the Chest Department of Assiut and Minia University Hospitals for this study during the period 2008–2010. The cases were selected after reviewing the full clinical history details for each case and histopathologically evaluating them. Most of the cases that were moderately to poorly differentiated adenocarcinoma and SqCC were selected.

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Bronchoscopy

Fiberoptic bronchoscopy was performed using a bronchoscope model (Pentax EB-1830T3). All patients were made to fast for 4 h before bronchoscopy and were premedicated with atropine sulfate 0.6 mg, intramuscularly and diazepam, orally, 1 h before bronchoscopy. Occasionally it was necessary to give a supplementary dose of diazepam 5 mg intravenously during the procedure to extremely apprehensive patients. The procedure was carried out under aseptic operating conditions with the operator wearing sterile gloves and masks. The fiberscope was ready for use after complete sterilization of its insertion tube and channels by vertical immersion of the shaft of the scope in 2% activated glutaraldehyde disinfectant solution (Cidex) for 20 min. Glutaraldehyde was instilled into the inner channels of the scope with a syringe. Next, the scope and its inner channels were washed in sterile water and an alcohol wipe was used for sterilizing the surface of the scope that could not be immersed. Image quality and movement of the distal tip of the fiberscope were checked immediately before starting the procedure. The shaft of the fiberscope was lubricated with lignocaine gel 2% before insertion of the scope to give additional surface anesthesia. The patient lay comfortably in a semirecombent position on a couch facing the operator. Lignocaine 2% solution (2 ml) was sprayed into the nose and nasopharynx. After a few minutes the fiberscope was introduced transnasally below the inferior turbinates and then passed gently through the nasopharynx, down the epiglottis to the vocal cords. In case of difficulty in transnasal introduction of the bronchoscope due to marked septal deviation or narrowing of the nasal passages, the bronchoscope was passed transorally after placing a mouth gag between the upper and lower jaws. When the scope was positioned above the vocal cords, surface anesthesia was applied by two bolus injections of 2 ml of lignocaine 2% vigorously flushed through the inner channel of the fiberscope. This commonly produced brief bouts of cough, of which the patient was forewarned. Pause, to allow the topical anesthesia to take effect. After a minute the fiberscope was gently passed through the vocal cords and into the trachea. An additional 2 ml of lignocaine 2% was injected through the central channel of the fiberscope into the trachea, right and left bronchi, and upper and lower lobes. Systemic examination of the airways was then conducted in all cases. A thorough inspection of the larynx was followed by examination of the vocal cords, including their movements during phonation. Next, the trachea and carina were examined, including mobility of the carina during deep breathing and coughing. This was followed by examination of the bronchial tree, usually starting with the right side, and a detailed exploration of the right main bronchus and upper, middle, and lower lobe bronchi down to the subsegmental levels. The fiberscope was then withdrawn to the level of the main carina and examination of the left side was carried out. As with the right side, the left main bronchus and the upper lobe, ligula, and lower lobe bronchi were examined down to subsegmental levels. Any endobronchial lesions visualized were recorded according to their sites, number, and levels in the bronchial tree. Three specimens were obtained from any visualized lesion during bronchoscopy. These were used for histological and immunohistochemical examination.

Formalin-fixed, paraffin-embedded, H&E-stained sections were then examined for adequacy of cellular diagnostic tissue and selected for this study. The final diagnosis was also confirmed in surgical pathology reports of the corresponding biopsy of the resected tumors.

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Immunohistochemical staining

Formalin-fixed and paraffin-embedded tissue samples were sectioned at 4 µm thickness. The sections were deparaffinized in xylene and rehydrated through a graded series of ethanol concentrations. Endogenous peroxidase activity was blocked by 10 min incubation with 3% hydrogen peroxide and rinsed in water. Antigen retrieval was achieved with 1 mmol/l EDTA pH 8 (Sigma Chemical Co., St Louis, Missouri, USA) in a microwave oven at 850 W for 8 min. Following two washes in PBS, sections were incubated in 10% normal goat serum for 15 min. Samples were then incubated overnight using two antibodies, the first primary antibody TTF-1 (RTU, clone SPT24; Leica Microsystems) and the second primary antibody Napsin A (IP64 clone, 1:400; Leica Microsystems). After three additional washes, sections were incubated for 30 min with a polyvalent biotinylated goat anti-rabbit antibody at room temperature. Following three additional washes in PBS, samples were incubated with streptavidin-conjugated peroxidase for 30 min. The reaction product was visualized by incubation for approximately 10 min with 3,3-diaminobenzidine tetrahydrochloride, followed by washing in distilled water. The sections were counterstained in Harris hematoxylin for 1–2 min. Stained slides were dehydrated in ascending grades of alcohol, cleared in xylene, and mounted with DPX.

Positive controls of known lung adenocarcinoma and negative controls with primary antibody replaced with Tris buffer were run with the study slides. Expressions of TTF-1 as a nuclear stain and Napsin A as a cytoplasmic stain were identified easily in tumor cells and quantified as positive or negative. TTF-1-stained alveolar lining cells and Napsin A-stained cytoplasm of alveolar macrophages were internal positive controls.

Two observers (H.T. and H.M.) evaluated Napsin A and TTF-1 immunoreactivities of tumor cells independently and blindly, without knowledge of the patients’ identity or clinical outcome.

For each stain, the percentage of positive cells was recorded. The presence of more than 10% expression of the marker within tumor cells was considered positive. Cases with less than 10% overall staining and with no focal areas of positive staining were considered negative.

Cases that were mostly negative but contained small areas of tumor in which nearly all cells stained positive were classified as focally positive.

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Results

The results of immunohistochemical for Napsin A and TTF-1 are summarized in Table 1. Napsin A was immunoreactive in 27 (79%) of 34 pulmonary adenocarcinomas (Fig. 1a) compared with 22 (64.7%) of 34 that were positive for TTF-1 (Fig. 2a). There were eight Napsin A-positive/TTF-1-negative and two TTF-1-positive/Napsin A-negative tumors.

Table 1

Table 1

Fig. 1

Fig. 1

Fig. 2

Fig. 2

With regard to SqCCs, Napsin A was positive in two (11%) of 18 cases that were negative (Fig. 1b) compared with one (5%) of 18 cases that were TTF-1 positive; the remaining were negative (Fig. 2b).

In addition, specificity, sensitivity, positive predictive values, and negative predictive values for the immunohistochemical stain were also calculated (Table 2; Loong, 2003).

Table 2

Table 2

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Discussion

Primary lung cancer is one of the most malignant solid tumors, and lung cancer incidence and thus mortality is still increasing. In particular, primary lung adenocarcinoma in the peripheral lung is increasing (Takashi et al., 2003).

The differentiation of adenocarcinoma from SqCC is critical in stratifying NSCLC patients to chemotherapeutic regimens that offer the most benefit and avoid potentially life-threatening side effects (Hirsch et al., 2008).

Most NSCLCs can be easily subtyped as adenocarcinoma or SqCC on biopsies without using additional special stains or immunohistochemical analysis. In fact, the current criteria for subtyping are based on the latest WHO classification of lung carcinomas (Colby et al., 2004), which is based primarily on H&E morphology. Difficulty in classification, however, is not infrequently encountered in small biopsy specimens, either because of poor sampling or because of the presence of only a small amount of tumor that may not show features of differentiation.

Our study shows that the use of a pair of immunohistochemical stains, including TTF-1 and Napsin A, in such cases allows correct subclassification in more than three-fourths of them. TTF-1 stained in only 5% of tumors and Napsin A stained in 11%; the small sample size of SqCC does not allow statistical analysis. These findings are important because, in contrast to previous years, clinicians currently demand more precise classification of NSCLCs for treatment with targeted therapies.

The WHO has published guidelines for the classification of lung cancer in resection specimens based on examination of the entire tumor (Colby et al., 2004); however, 70–80% of lung cancers present at advanced stages and are unresectable; therefore, these guidelines are not directly applicable. As there are no standardized guidelines for subclassifying NSCLC in small tissue biopsies, histochemical and immunohistochemical markers for adenocarcinoma and SqCC can suggest a subtype in the absence of definitive morphological evidence.

Part of the difficulty in accurately subtyping NSCLC using small samples arises from problems inherent to the biopsy procedure, particularly sampling error and tumor heterogeneity. Advances in sampling techniques, such as ultrasound-guided biopsies and fine needle aspirates, may help ameliorate these problems. Heterogeneity in histological grade within a particular tumor further confounds accurate diagnosis, as sampling of a poorly differentiated area cannot, by definition, differentiate adenocarcinoma or SqCC (Colby et al., 2004).

Accuracy in the cytomorphological diagnosis of adenocarcinoma is reportedly 80%, whereas diagnostic accuracy for SqCC is reported to be 87% (Edwards et al., 2000). The challenges encountered in cytomorphological diagnosis of NSCLC and distinguishing adenocarcinoma from SqCC are mainly due to the finding that adenocarcinoma can occasionally undergo coagulative necrosis, giving the cells a pseudokeratinized appearance along with dark pyknotic nuclei; conversely, SqCC can develop nonspecific degenerative vacuoles that can mimic secretory vacuoles of adenocarcinoma (Schreiber and McCrory, 2003).

Previous studies using histological specimens indicated that Napsin A has a sensitivity equal to or greater than that of TTF-1 in well to moderately differentiated lung adenocarcinomas (Dejmek et al., 2007). Therefore, its use has been advocated in conjunction with TTF-1 in the differential diagnosis of lung adenocarcinomas.

The sensitivity and specificity for each individual marker calculated in this study are similar to those reported in earlier studies, including those on small samples such as fine needle aspirates, core biopsies, and cytological specimens.

Compared with TTF-1 and surfactants A and B, the expression of Napsin A has not been extensively studied in neoplastic tissues. In lung tumors, previous investigators found that the sensitivity and specificity of Napsin A was at least equal to and often greater than that of TTF-1 for identifying adenocarcinomas (Jagirdar, 2008).

We found that Napsin A had higher sensitivity for pulmonary adenocarcinomas when compared with TTF-1, and, like TTF-1, Napsin A positivity decreased with increasing tumor grade. Nevertheless, the number of Napsin A-positive tumors was greater for each grade.

In summary, Napsin A appears to be a useful marker when combined with TTF-1 because it provides increased sensitivity and specificity for classifying primary lung tumors as adenocarcinoma.

This is particularly important in view of recent advances in the treatment of NSCLCs and, in particular, of adenocarcinomas of the lung and because the amount of diagnostic tissue is often limited (e.g. bronchoscopic or transthoracic biopsies).

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Conclusion

We showed that Napsin A was expressed in a large proportion of primary lung adenocarcinoma and showed its association with the differentiation grade. The results suggest that Napsin A is a promising marker for differential diagnosis of adenocarcinoma in the lung.

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Acknowledgements

Heba M. Tawfik, Essam A. Farghaly, and Yaser A. Gad developed the concept and designed the experiments. Heba M. Tawfik performed the pathology part of the paper, diagnosis of the cases by H&E staining, and performed the immunohistochemical analysis with translation of the immunohistochemical results. Essam A. Farghaly and Yaser A. Gad had collected the samples endoscopically and diagnosed them clinically and together conducted the statistical study.

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Conflicts of interest

None declared.

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