Material and Method: Immunohistochemically, TTF-1, CK 5/6 and p63 were used in 72 cases of squamous cell carcinoma, 19 cases of adenocarcinoma, and 29 cases of non-small cell lung cancer whose final diagnosis was decided with the subsequent resection material. The specificity, sensitivity, and positive and negative predictive value were calculated for each marker.

Results: TTF-1 positivity was seen in none of the 72 squamous cell carcinomas but in all of 19 adenocarcinoma cases. CK5/6 negativity was seen in all cases of adenocarcinoma and in two cases of squamous cell carcinoma. p63 was positive in all squamous cell carcinomas and in 4 adenocarcinomas. Cytokeratin 5/6, p63 positivity and TTF-1 negativity were observed in 17 non-small cell lung cancers whose final diagnosis was squamous cell carcinoma. None of the 12 non-small cell lung cancers whose final diagnosis was adenocarcinoma exhibited positive staining for CK5/6. However, p63 staining was not seen in the biopsy but was focal in the surgical specimen in one case. All the 12 non-small cell lung cancers whose certain diagnosis was adenocarcinoma were positive for TTF-1. TTF-1, CK 5/6 and p63 seem to be useful for differentiating adenocarcinoma from squamous cell carcinoma with 100% specificity, 100% sensitivity and 100% specificity, 97% sensitivity and 87% specificity, and 100% sensitivity, respectively.

Conclusion: We concluded that TTF-1 is a reliable marker for subtyping lung cancer. Different staining patterns can be seen with CK5/6 and p63; however, if they are used together with TTF-1 and interpreted correctly, they can be of help for the final diagnosis even in cases in which the morphology is unclear.

Our study aimed to study the efficiency of Thyroid Transcription Factor-1 (TTF-1), cytokeratin 5/6 (CK5/6) and p63 in distinguishing between AC and SCC and to study the contribution of these markers to the diagnosis in NSCLC.

The cases were included in the study¹ if the biopsy had typical AC morphology^2, if the biopsy had typical SCC morphology³ when the pathologists agreed on the diagnosis of NSCLC and if the tumor was subsequently resected at our institution⁴ if blocks from biopsy and lobectomy specimens were available for immunohistochemical study. Fifty-five of the 175 cases did not meet these criteria and were excluded. The remaining 120 cases were included in this study. Using morphological evaluation, the cases were divided into four groups: AC, SCC, NSCLC-SCC whose final diagnosis was SCC in the subsequent resection material, and NSCLC-AC whose final diagnosis was AC in the subsequent resection material.

Immunohistochemistry
Four-micron-thick sections were obtained for IHC investigation. Immunohistochemistry was performed by a standard protocol on a Ventana Discovery XT automated stainer (Ventana Medical Systems, Tucson, AZ, USA). Only primary antibodies, cytokeratin 5/6 (Clone D5/16B4, Dako, 1:200 dilution), p63 (Clone 4A4, Dako, 1:700 dilution) and TTF-1 (Clone SPT24, NovoCastra, 1:50 dilution) were added manually and incubated in 37°C for 32 minutes. Pneumocytes were considered as internal controls for TTF- 1 positivity, and bronchial basal cells for p63 and CK5/6 positivity.

Immunohistochemically, cytoplasmic staining was considered positive for cytokeratin 5/6 and nuclear staining was considered positive for TTF-1 and p63. All the areas of biopsy materials and at least 10 high power fields in tumor surgical materials were evaluated and the mean percentage of the positive tumor cells was determined. The staining rate was divided into four categories as follows: 0, <1%; 1+, 1–33%; 2+, 34–66%; 3+, >66%. Intensity of staining was graded as negative (0, no reactivity); weak (1+, less than normal cells); moderate (2+, same as normal cells); and strong (3+, stronger than normal cells)^9,10. When the staining was heterogeneous, the prominent pattern became the basis. The demographic features (age, gender) were obtained from the hospital archive system. For NSCLC cases, we also assessed whether immunohistochemical staining on biopsies correlated with staining on the subsequent surgical specimens as lung cancers are known to show morphologic heterogeneity in different areas of the same tumor.

Data Analysis
The statistical analyses were performed with SPSS software, version 20.0. The specificity, sensitivity, positive predictive value and negative predictive value were calculated for each marker. In addition, the relationship between the staining on biopsy and resection materials was analyzed using Pearson Chi-Square test, Chi-Square test and Fisher’s Exact Test. p values of ≤0.05 were considered statistically significant.

The reported sensitivity of CK5/6 for pulmonary SCC ranges from 73% to 100% and the sensitivity of CK5/6 is reported to go down as there is a decrease in the tumor differentiation^7,13,21. CK5/6 positivity was observed in 43 of 48 SCC cases in the study by Kim et al.^16, but in all 32 SCC cases in the study by Nicholson et al.²². One of the reasons for the wide range of CK5/6 sensitivity can be the fact that some studies regard focal or weak staining as positive but others do not. In our study, no CK5/6 staining was detected in only two cases while 75% of SCC cases were diffuse and strong positive. All the NSCLC-SCC cases were CK5/6 positive but six of them were focal. In addition, the staining was diffuse in two surgical specimens but it was focal in their former biopsies. According to our findings, CK5/6 was a highly specific but not very sensitive marker in distinguishing between SCC and AC when focal and weak staining were considered to be positive. Additionally, CK5/6 positivity can be considered as a finding supporting SCC even if it is focal in the cases where histological differentiation is not clear.

The utility of p63 for the identification of lung SCCs is well-known, and its sensitivity ranges from 73% to 100%^5,7,13. However, p63 specificity is not high because p63 can be stained focal and/or weak in some ACs^12,13,16. In our study, p63 positivity was observed in 4 AC cases which had TTF-1(+)/ CK5/6 (-). Staining was focal in three cases but diffuse in one. Nevertheless, intensity of staining was weak in the case that had diffuse staining like the other two cases that had focal staining. This finding is in agreement with the literature. All of the NSCLC-SCC in our study were positive for p63 and their staining rate and intensity were the same both in NSCLC areas and surgical specimens. None of the NSCLC-AC cases had staining except one, in which staining was not seen in the biopsy while focal (1–33%) staining was seen in the surgical specimen. In a study contucted by Warth et al.^2, p63 positivity was seen in 16.3% of the histomorphologically unclear ACs and staining intensity was lower than in SCC. Our study suggests that an AC diagnosis can be made despite p63 positivity in some cases, as in the literature. If the p63 positivity is focal and weak or moderate, TTF-1 positivity and CK5/6 negativity are enough for AC diagnosis even if AC is not clear histomorphologically. In our study, SCC had CK5/6 negativity and AC had p63 positivity in a few cases. However, TTF-1 was positive in all the ACs and negative in all the SCCs. Although the specificity, sensitivity, PPV and NPV of TTF-1 were 100%, a statistically significant difference was found in only the specificity value due to the limited number of cases. Our results need to be supported by studies including large series.

Subtyping of NSCLCs is very important due to current treatment modalities. Immunohistochemistry can be used in subtyping of samples with limited tissue and cases in which the morphology is unclear. TTF-1, CK 5/6 and p63 are the markers that can be used for this purpose. Different staining patterns can be seen with CK5/6 and p63; however they can be of help for the final diagnosis even in the cases in which morphology is unclear, if they are used together with TTF-1 and interpreted correctly.

Acknowledgments
We gratefully acknowledge Hatice Uluer in the Department of Biostatistics and Medical Communication of Ege University for performing the statistical analyses.

1) Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin. 2010;60:277-300.

2) Warth A, Muley T, Herpel E, Meister M, Herth FJ, Schirmacher P, Weichert W, Hoffmann H, Schnabel PA. Large-scale comparative analyses of immunomarkers for diagnostic subtyping of nonsmall- cell lung cancer biopsies. Histopathology. 2012;6:1017-25.

3) Whithaus K, Fukuoka J, Prihoda TJ, Jagirdar J. Evaluation of napsin A, cytokeratin 5/6, p63, and thyroid transcription factor 1 in adenocarcinoma versus squamous cell carcinoma of the lung. Arch Pathol Lab Med. 2012;136:155-62.

4) Gressett SM, Shah SR. Intricacies of bevacizumab-induced toxicities and their management. Ann Pharmacother. 2009;43:490-501.

5) Pelosi G, Rossi G, Bianchi F, Maisonneuve P, Galetta D, Sonzogni A, Veronesi G, Spaggiari L, Papotti M, Barbareschi M, Graziano P, Decensi A, Cavazza A, Viale G. Immunhistochemistry by means of widely agreed-upon markers (cytokeratins 5/6 and 7, p63, thyroid transcription factor-1, and vimentin) on small biopsies of non-small cell lung cancer effectively parallels the corresponding profiling and eventual diagnoses on surgical specimens. J Thorac Oncol. 2011;6:1039-49.

6) Rossi G, Pelosi G, Graziano P, Barbareschi M, Papotti M. A reevaluation of the clinical significance of histological subtyping of non--small-cell lung carcinoma: Diagnostic algorithms in the era of personalized treatments. Int J Surg Pathol. 2009;17:206-18.

7) Kargi A, Gurel D, Tuna B. The diagnostic value of TTF-1, CK5/6, and p63 immunostaining in classification of lung carcinomas. Appl Immunohistochem Mol Morphol 2007;15:415–20.

8) Noh S, Shim H. Optimal combination of immunohistochemical markers for subclassification of non-small cell lung carcinomas: A tissue microarray study of poorly differentiated areas. Lung Cancer 2012;76: 51–5.

9) World Health Organization classification of tumours. Pathology and genetics of tumours of the lung, pleura, thymus and heart. Travis WD, Brambilla E, Mqller-Hermelink HK, Harris CC, editors. Lyon7: IARC Press; 2004.

10) Fatima N, Cohen C, Lawson D, Siddiqui MT. TTF1 and napsin A double stain: A useful marker for diagnosing lung adenocarcinoma on fine-needle aspiration cell blocks. Cancer Cytopathol. 2011;119:127-33.

11) Rekhtman N, Ang DC, Sima CS, Travis WD, Moreira AL. Immunohistochemical algorithm for differentiation of lung adenocarcinoma and squamous cell carcinoma based on large series of whole-tissue sections with validation in small specimens. Mod Pathol. 2011;24:1348-59.

12) Conde E, Angulo B, Redondo P, Toldos O, García-García E, Suárez-Gauthier A, Rubio-Viqueira B, Marrón C, García- Luján R, Sánchez-Céspedes M, López-Encuentra A, Paz-Ares L, López-Ríos F. The use of p63 immunohistochemistry for the identification of squamous cell carcinoma of the lung. PLoS One 2010;5:1-6.

13) Mukhopadhyay S, Katzenstein AL. Subclassification of nonsmall cell lung carcinomas lacking morphologic differentiation on biopsy specimen: Utility of an immunohistochemical panel containing TTF-1, napsin A, p63, and CK5/6. Am J Surg Pathol 2011;35:15-25.

14) Travis WD, Brambilla E, Noguchi M, Nicholson AG, Geisinger KR, Yatabe Y, Beer DG, Powell CA, Riely GJ, Van Schil PE, Garg K, Austin JH, Asamura H, Rusch VW, Hirsch FR, Scagliotti G, Mitsudomi T, Huber RM, Ishikawa Y, Jett J, Sanchez-Cespedes M, Sculier JP, Takahashi T, Tsuboi M, Vansteenkiste J, Wistuba I, Yang PC, Aberle D, Brambilla C, Flieder D, Franklin W, Gazdar A, Gould M, Hasleton P, Henderson D, Johnson B, Johnson D, Kerr K, Kuriyama K, Lee JS, Miller VA, Petersen I, Roggli V, Rosell R, Saijo N, Thunnissen E, Tsao M, Yankelewitz D. International association for the study of lung cancer/american thoracic society/european respiratory society international multidisciplinary classification of lung adenocarcinoma. J Thorac Oncol. 2011;6:244–85.

15) Loo PS, Thomas SC, Nicolson MC, Fyfe MN, Kerr KM. Subtyping of undifferentiated non-small cell carcinomas in bronchial biopsy specimens. J Thorac Oncol 2010;5:442-7.

16) Kim MJ, Shin HC, Shin KC, Ro JY. Best immunohistochemical panel in distinguishing adenocarcinoma from squamous cell carcinoma of lung: Tissue microarray assay in resected lung cancer specimens. Ann Diagn Pathol. 2013;17:85-90.

17) Chang YL, Lee YC, Liao WY, Wu CT. The utility and limitation of thyroid transcription factor-1 protein in primary and metastatic pulmonary neoplasms. Lung Cancer. 2004;44:149–57.

18) Jerome Marson V, Mazieres J, Groussard O, Garcia O, Berjaud J, Dahan M, Carles P, Daste G. Expression of TTF-1 and cytokeratins in primary and secondary epithelial lung tumours: Correlation with histological type and grade. Histopathology. 2004;45:125–34.

19) Tan D, Li Q, Deeb G, Ramnath N, Slocum HK, Brooks J, Cheney R, Wiseman S, Anderson T, Loewen G. Thyroid transcription factor-1 expression prevalence and its clinical implications in nonsmall cell lung cancer: A high-throughout tissue microarray and immunohistochemistry study. Hum Pathol. 2003;34:597– 604.

20) Monica V, Ceppi P, Righi L, Tavaglione V, Volante M, Pelosi G, Scagliotti GV, Papotti M. Desmocollin-3: A new marker of squamous differentiation in indifferentiated large-cell carcinoma of the lung. Mod Pathol. 2009;22:709–17.

21) Downey P, Cummins R, Moran M, Gulmann C. If it’s not CK5/6 positive, TTF-1 negative it’s not a squamous cell carcinoma of lung. APMIS. 2008;116:526–9.

22) Nicholson AG, Gonzalez D, Shah P, Pynegar MJ, Deshmukh M, Rice A, Popat S. Refining the diagnosis and EGFR status of nonsmall cell lung carcinoma in biopsy and cytologic material, using a panel of mucin staining, TTF-1, cytokeratin 5/6, and P63, and EGFR mutation analysis. J Thorac Oncol. 2010;5:436-41.