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The role of p63, thyroid transcription factor-1, and napsin A immunostaining in the subclassification of nonsmall cell lung carcinomas: a tool in treatment selection

Shafeek, Mona George; Mostafa, Naglaa Ali

doi: 10.1097/01.XEJ.0000406598.96484.89
ORIGINAL ARTICLES
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Background New developments in the treatment of primary lung cancer have necessitated further pathologic subclassification of nonsmall cell lung carcinomas (NSCLC). Certain therapeutic agents should not be used in squamous cell carcinoma or neoplasms with a dominant squamous component. The aim of this study was to identify the value of immunohistochemical markers p63, thyroid transcription factor-1, and napsin A (novel aspartic proteinase of the pepsin family) in the classification of NSCLC into squamous and nonsquamous subtypes. Thirty cases proved to be NSCLC were selected for this study.

Results Out of 14 squamous cell carcinomas, 12 (85.7%) reacted with p63, whereas only one (7.1%) reacted with thyroid transcription factor (TTF)-1, showing a weak focal staining pattern, and none with napsin A. Out of the 12 adenocarcinomas, eight (66.7%) cases stained for TTF-1, 10 (83.3%) for napsin A, and only one (8.3%) for p63. Out of the four large cell carcinomas, only two (50%) cases reacted with TTF-1, and none with napsin A and p63. Comparing the two markers of lung adenocarcinoma, napsin A showed a higher sensitivity (83.3%) and specificity (100%) than TTF-1 (66.7 and 83.3% respectively).

Conclusion The current results indicate that the use of p63, TTF-1, and napsin A markers may be useful to subclassify NSCLC and to improve therapeutic selection of patients with lung cancer.

Department of Pathology, Faculty of Medicine, Zagazig University, Zagazig, Egypt

Correspondence to Naglaa Ali Mostafa, MD, Department of Pathology, Faculty of Medicine, Zagazig University, Zagazig, Egypt Tel: +2 01096026022; fax: +2 0552307830; e-mail: naglaramda@yahoo.com

Received July 5, 2011

Accepted July 21, 2011

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Introduction

Lung cancer was the most commonly diagnosed cancer and the leading cause of cancer death in men in 2008 globally. Among women, it was the fourth most commonly diagnosed cancer and the second leading cause of cancer death (Jemal et al., 2011).

Nonsmall cell lung carcinoma (NSCLC), essentially consisting of squamous cell carcinoma (SCC), adenocarcinoma (AC), and large cell carcinoma (LCC), account for approximately 80% of all lung cancers (Travis, 2002). Traditionally, the distinction between small cell carcinoma and NSCLC had a considerable therapeutic implication; however, the subclassification of NSCLC into glandular and squamous subtypes had no therapeutic impact. Recently, targeted therapy has been used successfully in subtypes of NSCLC (Jorda et al., 2009; Rossi et al., 2009b).

The role of bevacizumab (Avastin), a vascular endothelial growth factor receptor inhibitor, in combination with chemotherapeutic agents has been proved to be beneficial for patients with unresectable, locally advanced, recurrent or metastatic NSCLC (Herbst et al., 2007; Sandler, 2007). Patients with a pure or a partial squamous histology have an increased risk of life-threatening pulmonary hemorrhage. Hence, only patients with non-SCC or mixed subtypes of NSCLC, if the predominant cell type is nonsquamous, are eligible for bevacizumab (Bunn and Thatcher, 2008; Molina et al., 2008). Therefore, the detection of squamous cell components in pathology samples is desirable for treatment selection. P63, a member of the p53 family, is normally expressed in squamous and urothelial cells, basal layers of prostatic glands, breast, and bronchial epithelium. The chromosomal region 3q22-qter, in which the p63 gene resides, has been shown to be the most frequently amplified genome locus in SCC of the lung. The restriction of p63 expression to SCC and its structural similarity to p53 suggests its role in regulating the proliferation and differentiation of specific epithelial cell types (Camilo et al., 2006; Hou et al., 2010). Thyroid transcription factor-1 (TTF-1), which is essential for the development of the fetal diencephalons, is selectively expressed in the lung epithelial cells and in the thyroid follicular cells (Penman et al., 2006). It has been the predominant marker used to identify lung origin with a reported sensitivity up to 80% for lung AC. However, TTF-1 also stains other tissues and tumors such as thyroid, breast, uterine, ovarian cancers, and, to a lesser degree, primary lung SCC (Zhang et al., 2009; Yang and Nonaka, 2010). Napsin A is an aspartic proteinase that is expressed in the normal type II pneumocytes of the lung and the proximal convoluted tubules of the kidney. It is involved in the maturation of the surfactant protein B and is regarded as a potential marker for lung AC and renal cell carcinoma (Dejmek et al., 2007).

The aim of this study was to identify the value of p63, TTF-1, and napsin A in the classification of NSCLC into squamous and nonsquamous subtypes.

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Materials and methods

The archive of the Pathology Department, Zagazig University Hospital, was searched for primary lung tumor tissue blocks from January 2007 to December 2010. The 30 cases selected included 21 men and nine women, ranging from 42 to 70 years of age (median 50). All cases were diagnosed by clinical data, chest radiograph, and computed tomography. All specimens were surgically resected or obtained by bronchoscopic biopsies. Small cell carcinomas were excluded from the study. The tumors were classified according to the World Health Organization histological typing of tumors (Brambilla, 2004). The specimens included 14 SCCs, 12 ACs, and four LCCs.

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Immunostaining

Immunostaining was performed on 4-μm formalin-fixed, paraffin-embedded tissue sections using the standard avidin–biotin peroxidase complex (ABC) procedure as described by Lim et al. (2003). Antigen retrieval was performed for each section (microwave 750 W, 10 mmol/l citrate buffer, pH 6.0 for 15 min).

Nonspecific binding was blocked with 10% normal rabbit serum. The commercially available monoclonal antibodies used were as follows: anti-p63 antibody (clone 4A4, dilution 1 : 50, Dako), anti-TTF-1 (clone 8G7G3/1, dilution 1 : 200, Dako, Glostrup, Denmark), and anti-napsin A (clone IP64, dilution 1 : 800, Novocastra, Newcastle, UK). The immunostaining was developed using 3,3′-diamino-benzidine as the chromogen and Mayer's hematoxylin as the counter-stain.

As negative controls, the primary antibody was replaced by nonimmune rabbit serum. Normal alveolar cells served as positive internal controls for TTF-1 and napsin A, whereas the adjacent noninvolved normal bronchial mucosa was used for p63 (Lim et al., 2003; Ueno et al., 2003).

For the evaluation of immunostaining results of both TTF-1 and p63, only distinct nuclear staining of tumor cells was considered positive. For napsin A, only granular cytoplasmic staining was interpreted as positive. The results were estimated semiquantitatively and expressed on a plus−minus scale on the basis of the percentage of stained tumor cells as follows: 0–10%, −; 11–50%, 1+ (focal); 51–100%, 2+(diffuse) (Ueno et al., 2003).

Sensitivity, specificity, and positive predictive value were calculated as follows: Sensitivity=true-positive results/(true-positive results+false-negative results). Specificity=true-negative results/(true-negative results+false-positive results). Positive predictive value=true-positive results/(true-positive results+false-positive results). The designations of true and false are based on the study hypothesis that p63 is expressed in SCCs and true positivity is expected for TTF-1 and napsin A in ACs (Kargi et al., 2007).

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Results

Immunoexpression of p63 was found in 12 of 14 (85.7%) SCCs, graded as 1+ in two (14.3%) and 2+ in 10 (71.4%) cases. The two negative cases were well-differentiated SCCs. In the moderately differentiated cases, p63 reactivity was focal, mainly in the peripheral cells (Fig. 1), whereas in poorly differentiated SCCs, a strong and diffuse expression was observed in the majority of the tumor cells (Fig. 2). Only one out of 12 ACs (8.3%) was weak and focally positive for p63 (Fig. 3), whereas all LCCs were nonreactive (Table 1).

Fig. 1

Fig. 1

Fig. 2

Fig. 2

Fig. 3

Fig. 3

Table 1

Table 1

TTF-1 was expressed in eight of 12 (66.7%) ACs; among them, two cases showed a strong diffuse immunostaining (Fig. 4). The expression was also observed in other types of NSCLCs, including one of 14 (7.1%) SCCs (Fig. 5) and two of four (50%) LCCs as shown in Table 1.

Fig. 4

Fig. 4

Fig. 5

Fig. 5

Napsin A immunoreactivity was found in 10 out of 12 ACs (83.3%) (Fig. 6). Two out of four TTF-1-negative ACs were positive for napsin A, whereas none of the SCCs or LCCs was positive. The expression of napsin A and TTF-1 in relation to the differentiation grade of AC is shown in Fig. 7. Among the poorly differentiated ACs, a higher positivity was found for napsin A compared with TTF-1 (60 and 40%, respectively).

Fig. 6

Fig. 6

Fig. 7

Fig. 7

A striking difference was found regarding the proportion of positive cells for TTF-1 and napsin A in cases of AC. Reactivity in >50% of the tumor cells was found in only two of eight and seven of 10 positive cases of TTF-1 and napsin A, respectively (Table 1).

Sensitivity, specificity, and diagnostic positive predictive value of the three markers are summarized in Table 2.

Table 2

Table 2

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Discussion

Until recently, the differentiation of NSCLC variants had an academic but not a therapeutic implication, because all were treated according to similar strategies (Schrump et al., 2008). Bevacizumab, when combined with carboplatin and paclitaxel, improves the overall survival in patients with advanced or recurrent NSCLC. However, this drug is not recommended in patients with SCC or neoplasms with a dominant squamous component (Cohen and Weydert, 2011). In certain patients, the distinction of squamous and non-SCC including AC and LCC cannot be made by the assessment of morphology alone. In this situation, immunohistochemical markers may provide additional diagnostic information, especially in small biopsy and cytologic materials (Stoll et al., 2010; Travis et al., 2010).

Previous studies have been performed to evaluate the usefulness of p63 for the distinction of primary pulmonary SCC. The results of Wu et al. (2003), Au et al. (2004), and Kargi et al. (2007), who used formalin-fixed, paraffin-embedded tissue sections, revealed p63 positivity in between 81 and 100% of NSCLC with squamous differentiation. Similar results were obtained by Jorda et al. (2009) and Kalhor et al. (2006) using cytologic material. They observed a positive expression in 88 and 100% of SCCs, respectively. The results of the current study were consistent with the previous studies as 12 of 14 SCCs reacted with p63, reflecting the high sensitivity of this marker (85.7%). Although the two well-differentiated SCCs were negative, all poorly differentiated SCCs showed a strong and diffuse reactivity of most tumor cells. This result confirmed that p63 is a promising marker to indicate a minimal squamous differentiation as reported by Shimada et al. (2009). However, p63 positivity in pulmonary tumors is not limited to SCCs. For ACs, Wang et al. (2002) reported a p63 positivity in 65% of the cases but in a focal pattern in contrast to the diffuse staining in poorly differentiated SCCs. Other investigators have shown considerably lower percentages of p63-positive pulmonary ACs ranging in most studies from 12.1 to 18.3% (Lim et al., 2003; Massion et al., 2003; Sheikh et al., 2004). In other studies, p63 was not detected in any of the ACs (Kargi et al., 2007). This discrepancy in p63 expression could be related to differences in the techniques used and variations in the antibody dilutions used in different studies (Au et al., 2004).

Recently, to increase the specificity of p63, Conde et al. (2010) scored positive results when high-intensity staining was present in >50% of the tumor cells. Also, Ang et al. 2010 reported that p63 may be positive (>20% of tumor cells) or focal (<20% of tumor cells) in 6 and 23% of ACs, respectively, whereas this tumor type very rarely exhibits (1.6%) diffuse staining (>50% of tumor cells).

These results are largely in accordance with those of the present study. Only one of 12 (8.3%) ACs was weak and focally (11–50%) positive for p63 whereas all LCCs were nonreactive, indicating a high specificity of this marker for SCC (93.8%). Therefore, several approaches have been proposed to improve the subtyping of NSCLCs, including the use of a combination of markers (Rossi et al., 2009a, Kim and Kwon, 2010).

Comparing the reactivity to TTF-1 and napsin A as markers for primary lung ACs, high-sensitivity values were found in the current study. Napsin A was positive in 10 of 12 (83.3%) whereas TTF-1 reactivity was found in only eight (66.7%) AC cases. Moreover, the present study revealed a striking difference in the proportion of positively stained cells for TTF-1 and napsin A in lung AC. Diffuse reactivity (in >50% of tumor cells) was found in two of eight and in seven of 10 cases for TTF-1 and napsin A, respectively.

Similar results were reported by Hirano et al. (2003) and Jagirdar (2008). They suggested that napsin A is superior to TTF-1 in that its expression is stronger, more diffuse, and more sensitive in the primary lung AC with sensitivity levels of 90 and 90.7%, respectively, whereas the TTF-1 sensitivity level was around 80%.

Regarding the expression of TTF-1 and napsin A in relation to the differentiation grade of AC, our findings of higher expression for napsin A compared with TTF-1 in well-differentiated ACs (100 and 66.6%, respectively) has been documented by previous studies (Ueno et al., 2003; Bishop et al., 2010). Also, decreased expression of both markers in poorly differentiated AC was reported. Bishop et al. (2010) and Zhang et al. (2010) demonstrated that napsin A had more positivity (69 and 33.3%, respectively) than TTF-1 (44 and 28.6%, respectively) among poorly differentiated ACs. Similarly, the present study found napsin A and TTF-1 positivities of 60 and 40%, respectively.

The differential expression of TTF-1 and napsin A in different studies may be caused by the quantity of tumor cells available for staining and a relatively small number of cases studied. This observation still needs to be examined further in a large-scale study (Jagirdar, 2008).

Rossi et al. (2009a) demonstrated TTF-1 reactivity in NSCLCs as LCCs and rare cases of SCCs. The increased TTF-1 positivity in lung SCC has been noted when a new TTF-1 monoclonal antibody is used (Matoso et al., 2010). However, napsin A expression examined by in-situ hybridization revealed that the napsin mRNA is expressed in primary lung AC and in some LCCs but not SCCs (Ueno et al., 2003). Recently, Zhang et al. (2010) reported that the sensitivity and specificity of napsin A for primary lung AC (84.9 and 93.8%, respectively) were higher than the sensitivity and specificity of TTF-1 (84.4 and 83.9%, respectively). Furthermore, napsin A-positive cells were also observed in the glandular component of adeno-SCC specimens, whereas cells in the squamous component were all negative. The present study, in keeping with the previous reports, confirmed a higher specificity of napsin-A for AC versus SCC (100%) than TTF-1 (83.3%).

The most recent study by Fatima et al. (2011) using fine-needle aspiration cell block materials has concluded that dual TTF-1/napsin A has a sensitivity of 74% and a specificity of 87% for diagnosing AC, and hence is useful in differentiating AC from SCC.

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Conclusion

  • The use of p63 immunostaining can effectively improve the identification of SCCs.
  • TTF-1 remains a desirable marker for ACs of the lung. Napsin A, with a higher sensitivity and specificity, advocates for its use in conjunction with TTF-1.

Therefore, we recommend that the use of p63, TTF-1, and napsin A immunostaining can effectively improve the classification of NSCLC into squamous and nonsquamous subtypes, especially in small biopsy specimens. This may eventually be considered for targeted therapy that must exclude SCCs.

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Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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