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Role of immunohistochemistry markers in neoplastic lung lesions

 Department of Paraclinical Sciences, Faculty of Medical Sciences, The University of The West Indies, Trinidad and Tobago

Date of Submission08-Apr-2019
Date of Decision11-Apr-2020
Date of Acceptance22-Apr-2020
Date of Web Publication22-Jun-2021

Correspondence Address:
Srikanth Umakanthan,
Department of Paraclinical Sciences, Faculty of Medical Sciences, The University of The West Indies, Mount Hope Campus
Trinidad and Tobago
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_187_19

 > Abstract 

Objectives: The objective of the evaluate was to study and determine the usefulness of immunohistochemistry (IHC) staining in neoplastic lung lesions.
Materials and Methods: We evaluated seven IHC stains in fifty lung cancers that included adenocarcinoma (AC), squamous cell carcinoma (SCC), small cell carcinoma, and carcinoid tumors.
Results: P63 was expressed in all the cases of SCCs and thyroid transcription factor-1 (TTF-1) was expressed in all cases of ACs. CK 5/6 was expressed in 77.77% of SCCs and CK 7 was expressed in 92.59% of ACs. Synaptophysin and chromogranin-A were expressed in 100% of neuroendocrine (NE) carcinomas.
Conclusion: P63 and TTF-1 are sensitive markers for SCCs and ACs. Synaptophysin and Chromogranin-A are sensitive markers for NE carcinomas.

Keywords: Adenocarcinoma, immunohistochemistry, lung

How to cite this URL:
Umakanthan S, Chalapathi Rao A V, Mohammed W. Role of immunohistochemistry markers in neoplastic lung lesions. J Can Res Ther [Epub ahead of print] [cited 2021 Nov 29]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=319037

 > Introduction Top

Lung cancer is the most common cause of mortality in cancer-related cases among men and women worldwide.[1] Historically, pathologists and oncologists have classified lung cancers into small-cell lung cancer (SCLC) and non-SCLC (NSCLC). NSCLC is mainly composed of adenocarcinoma (AC), squamous cell carcinoma (SCC), and large cell carcinoma. The differentiation between AC and SCC is important because of the different target-specific treatments.[2] Morphology alone might not be conclusive, especially in cases due to poor differentiation, small biopsies, heterogeneity of tumors, and the artifacts. Advanced evaluation using immunohistochemistry (IHC) and molecular genetics have been useful in solving such cases. Owing to the most practical solution, usually IHC provides a suitable conclusion.[3]

The aim of this study is to determine the usefulness, efficiency, and contribution of IHC stains in distinguishing NSCLC and SCLC. The report interpretation of IHC results and their impact on diagnostic accuracy is also evaluated.

This project is the first study based on lung tumors to be conducted in the Caribbean region. It determines the importance of IHC as the best cost-effective modality in confirming the histologic type of lung cancer in the present regional laboratory setting.

 > Materials and Methods Top

Ethical approval

Ethical approval was granted by The University of the West Indies, Trinidad and also by the North Central Regional Health Authority, Trinidad.

Study design

The analysis included processed tissue samples of fifty cases involving lung biopsies and tissue specimens obtained by wedge resection from Eric Williams Medical Sciences Complex from 2011 to 2016 were included in the study. The diagnosis of these histologic sections from the tissues was viewed for all cases and diagnosis given in adherence with the 2015 World Health Organization classification of lung tumors. All tumors had at least focal area of definite line of morphologic differentiation to be classified under the standard criteria.


IHC was performed by The DakoCytomation EnVision +™ System. Horseradish Peroxidase (HRP) is a two-step IHC staining technique that allows the user to choose a short or long protocol depending on the sensitivity required. This system is based on an HRP-labeled polymer, which is conjugated with secondary antibodies. Primary antibodies produced in rabbit react well with the labeled polymer. Quench any endogenous peroxidase activity by incubating the specimen for 5 min with the peroxidase blocking reagent. The specimen is then incubated with an appropriately characterized and diluted rabbit primary antibody, followed by the incubation with the labeled polymer using two sequential 30-min incubations. It should be noted that, for antibodies requiring enzyme digestion or target retrieval, it may be necessary to increase incubation times of the primary antibody and labeled polymer by 5–10 min. A 5–10 min incubation with 3-3' diaminobenzidine (DAB+) substrate-chromogen added after the labeled polymer creates an enzymatic conversion, resulting in the formation of a visible product at the antigen site. The result of the peroxidase activity on the chromogenic substrate is to render the soluble chromogen into a brown-colored insoluble product that precipitates at the site of the enzyme activity.

The detection of some antigens may require pretreatment of the tissue samples by either heating the slides in an antigen retrieval solution at high temperature or using an enzyme digestion procedure. These procedures restore the immunoreactivity of many epitopes that were masked or distorted by tissue fixation treatment.

IHC for p63, thyroid transcription factor-1 (TTF-1), CK 5/6, CK 7, CK 20, synaptophysin, and Chromogranin A was performed on all samples as shown in [Table 1]. Tissue samples with appropriate positive immunostaining were used as a control.
Table 1: Panel of immunohistochemistry markers used in lung neoplasms

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The final diagnosis of the histological type of NSCLCs after immunohistochemical staining was:

  1. SCC
  2. AC
  3. NSCLC-not otherwise specified (NOS)
  4. Neuroendocrine (NE)-small-cell carcinoma
  5. NE-carcinoid.

Presence and absence of immunoreactivity in the tumor cells were marked as 1 and 0, respectively. Diagnostic dilemmas were resolved by a second or third pathological opinion.

 > Results Top

The probable diagnosis is the histologic diagnosis of the samples based on the tumor morphology. These samples were further stained for IHC using monoclonal antibodies and the immunoreactivity was analyzed in each of the cases to attain a final diagnosis as detailed in [Table 2].
Table 2: Detailed review of all biopsy specimens, applied immunohistochemistry, revised diagnosis from probable to final diagnosis of histological types based on the 2015 World Health Organization classification of lung tumors

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Out of eight cases morphologically diagnosed as SCC, six were of SCC and one each of AC and NSCLC-NOS type following IHC evaluation. In additional, there were two cases of NSCLC-NOS and one case of AC-poorly differentiated which turned out to be SCC on IHC evaluation. Hence, a total of nine cases of SCC were diagnosed finally (after IHC evaluation).

Out of 26 cases morphologically diagnosed as AC, 23 were of AC, one of SCC, and 2 cases of NSCLC-NOS type following IHC evaluation. In addition, there was one case of SCC and three cases of NSCLC-NOS type which turned out to be AC on IHC evaluation. Hence, a total of 27 cases of AC were diagnosed finally (after IHC evaluation).

Out of 13 cases morphologically diagnosed as NSCLC-NOS, six cases were of NSCLC-NOS type, three of AC, two of SCC, and two of NE tumors following IHC evaluation. In addition, there were two cases of AC and one case each of SCC and NE which turned out to be NSCLC-NOS type. Hence, a total of ten cases of NSCLC-NOS were diagnosed finally (after IHC evaluation).

Out of three cases morphologically diagnosed as NE, two cases were of NE and one of NSCLC. In addition, there were two cases of NSCLC-NOS (morphologically large cell carcinoma) which turned out to be NE tumor following IHC evaluation. Hence, a total of four cases of NE tumors were diagnosed finally (after IHC evaluation).

P63 expression was evident in all (100%) the cases of SCC and in 11.11% of AC. Expression of antibody was not evident in NE and NSCLC-NOS type [Table 3] and [Figure 1].
Table 3: p63 specificity

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Figure 1: Squamous cell carcinoma positive for P63 (×10)

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TTF-1 was expressed in all (100%) cases of AC and one case of NE. TTF-1 was not expressed in SCC and NSCLS-NOS [Table 4] and [Figure 2].
Table 4: Thyroid transcription factor 1 specificity

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Figure 2: Adenocarcinoma immunohistochemically positive for thyroid transcription factor-1 (x10)

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CK 5/6 was evident in 77.77% of SCC and in 1 (3.70%) in AC. There was no expression in NSCLC-NOS and NE [Table 5] and [Figure 3].
Table 5: Cytokeratin 5/6 specificity

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Figure 3: Squamous cell carcinoma positive for CK5/6 (x10)

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CK 7 was positive in 25 cases (92.59%) in AC and 7 (77.77%) in SCC, 7 cases of NSCLC-NOS, and 2 cases of NE [Table 6] and [Figure 4].
Table 6: Cytokeratin 7 specificity

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Figure 4: Adenocarcinoma immunohistochemically positive for cytokeratin 7 (×10)

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CK 20 was positive in four cases (14.81) in AC and 1 (11.11) in SCC. There was no expression in NSCLC-NOS and NE [Table 7].
Table 7: Cytokeratin 20 specificity

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Synaptophysin was expressed in all (100%) of NE and lack of expression in SCC, AC, and NSCLC-NOS [Table 8] and [Figure 5].
Table 8: Synaptophysin specificity

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Figure 5: Neuroendocrine carcinoma immunohistochemical staining for synaptophysin (×10)

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Chromogranin-A was expressed in all (100%) of NE and one case of AC (3.70%). Chromogrannin was not expressed in SCC and NSCLC-NOS [Table 9] and [Figure 6].
Table 9: Chromogranin specificity

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Figure 6: Neuroendocrine carcinoma immunohistochemical staining for chromogranin A (×10)

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Nine cases of NSCLC-NOS were further analyzed for immunoreactivity to Napsin A. Two cases of these nine showed immunoreactivity to napsin A. This further analysis for the nine cases was done as Napsin A has more tissue specificity to primary lung ACs compared to metastatic lung ACs.[1]

 > Discussion Top

Precise histologic subtyping of lung cancers is essential to treat the patients with target-specific therapies. Biopsy samples provide limited tissue compared to surgical specimens, resulting in greater chances of misdiagnosis. IHC is now routinely employed on both biopsy and specimen samples to attain a more valid and correct diagnosis to initiate target therapy. Lung cancer samples are recommended by the international association to be tested with the immunohistochemical use of p63, CK 5/6, and TTF-1.

Sensitivity of the IHC markers was tabulated [Table 10].
Table 10: Sensitivity of the method-change in diagnosis after immunohistochemistry

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P63 is a member of the p53 family which plays a vital role in the pathogenesis of SCC's lung, head and neck, and cervix.[2] P63 utility is recommended as an optimal monoclonal antibody for identification of lung SCCs as its sensitivity ranges from 73% to 100% and a high specificity.[3] P63 is recommended for the differential diagnosis of SCC of the lung. Specificity percentage becomes higher when combined analysis is done along with CK 5/6.[4]

Role of p63 in lung cancers has been repeatedly studied. Argon et al. reported that p63 specificity is not high because it can be stained focal and/or weak in some ACs.[3] Intensity of the staining also varied based on diffuse and focal staining. AC diagnosis can be made despite p63 positivity in some cases, as in literature based on TTF-1 positivity and CK 5/6 negativity.[5]

In our study, p63 was expressed in all nine diagnosed SCCs as well as 3 (11.11%) of 27 diagnosed ACs.

Vollmer reported that the level of expression of p63 is inversely related to the degree of keratinization of lung SCC.[6]

Thyroid transcription factor-1

TTF-1 staining was not seen in any of the nine SCC cases, while it was seen in all 27 cases of AC. TTF-1 staining was positive in one case of small-cell carcinoma and as shown in a study by Stojsic et al. it is expressed in more than 90% of small-cell lung carcinomas.[7] TTF-1 is present in normal pneumocytes and bronchial epithelia and is sensitive in 62% and specific in 92% of ACs.[8] TTF-1 positivity in SCC was reported by some authors, the overall impression is that it should be absent in pulmonary SCC as seen in our study.[9],[10],[11]

Cytokeratin 5/6

CK 5/6 is expressed in the keratinizing epithelium and nonkeratinizing epithelium covering mucosal tissues. CK 5/6 is used mainly in differential diagnosis between SCC and AC. It is also used in optimal panel for differentiation between malignant mesothelioma and AC. Sensitivity (84%) and specificity (79%) of CK 5/6 are lower compared with p63. This lower sensitivity and specificity is based on the tumor differentiation. The percentage goes as the degree of differentiation decreases. CK 5/6 positivity was observed in 43 of 48 SCC cases in the study by Kim et al., but in all 32 cases in the study done by Nicholson et al. and in our study, it showed expression in seven of nine SCC cases.[12],[13] There are explanations for this wide range of sensitivity of CK 5/6 and this can be because some studies regard focal or weak staining as positive but others do not.

Cytokeratin 7

CK 7 is widely applied in the differential diagnosis of different histologic subtypes of NSCLCs (typical and atypical carcinoid and NSCLC with and without NE differentiation). CK 7 is widely expressed in ACs and also in SCCs and unclassified NSCL. The sensitivity (93%) and specificity (63%) remain highest in ACs in comparison to other lung tumor subtypes.[10],[14] In our study, CK 7 was expressed in 25 out of 27 (92.95%) AC cases.

Recent studies have proposed that the combined use of antibodies for AC (TTF-1 and CK 7) and SCC (p63 and CK 5/6) provides very reliable distinction between the 2 NSCLC subtypes.[15],[16] Our study shows that CK 5/6 was positive in 77.77% of SCC and CK 7 was positive in 92.59% of ACs. This statistical relevance of combined use of antibodies being higher in ACs compared to SCCs is due to the limited number of SCCs in our study. CK 7 showed expression in 7 (77.77%) cases of SCC, 7 (70%) cases of NSCLC-NOS, and 2 (50%) cases of NE. Hence, when used alone, the specificity is low. CK 5/6 was expressed in one case (3.70%) of AC. Similar to prior studies, p63 and CK 5/6 were excellent markers for SCC in our study.[15],[17],[18],[19]

Cytokeratin 20

CK7/CK20 combination is helpful with other markers such as TTF-1 to differentiate between primary and metastatic carcinomas. CK 7+/CK 20− profile is not restricted to lung cancers as breast carcinomas, endometrial ACs, and nonmucinous ovarian carcinomas are also classically CK 7+/CK 20−.[10]

Synaptophysin and Chromogranin A

Morphological NE tumors are identified by the presence of organoid distribution of tumor cells. The WHO recommends that, in addition to the morphologic diagnosis, the diagnostic criteria of NSCLCs-NE to include the presence of at least one NE marker. Synatophysin, Chromogranin A, and CD56/NCAM are NE markers used in the diagnosis of this tumor type.[7],[14],[20] In our study, synaptophysin and chromogranin A were expressed in all four (100%) cases. One case of AC showed chromogranin A expression.

Staining intensity of monoclonal antibodies varies based on the lung tumor. p63 is usually diffuse and strongly positive in SCC in contrast to AC where the expression is focal and weak. P63 is expressed in the bronchial reserve cells, which should not be mistaken for tumor cells showing positivity.[21] p63 improves the diagnostic accuracy for SCC as shown in our study. TTF-1 showed stronger staining intensity in AC compared to NE.

Our study demonstrated a slight increase in attaining a final diagnosis in cases which were morphological diagnosed as poorly differentiated carcinomas. However, despite the use of ancillary studies, a few cases remained classified as NSCLC-NOS. NSCLC-NOS subtyping was included in our study in contrast to other studies which discourage the use of this generic term. There are possible explanations for this observation; one is that poorly differentiated carcinomas can be difficult to classify even on large resection specimens. The heterogeneity of immunostaining of lung carcinomas on resection specimens, particularly AC, is well known. Pathologists may be faced with even greater uncertainties with small specimens. Many poorly differentiated tumors may not show specific differentiation or demonstrate specific tumor marker expression, and therefore, it is likely that hypocellular specimens from these tumors would remain unclassified despite ancillary studies. Hence, the question of using immunohistochemical stains on such cases is still unclear.[22]

From the perspective of treatment of NSCLC, the distinction between AC and SCC is most important for our clinical colleagues. Hence, information influencing therapeutic decision-making may be obtained by other methods. Further studies are needed to determine whether ancillary studies for precise classification are more or less important than other potential molecular studies for prognostication of therapeutic response. Furthermore, ancillary studies should not be used for additional diagnostic reassurance in cases, in which morphologic studies are sufficient for NSCLC subtyping.

 > Conclusion Top

IHC provides a valuable role in identification and diagnosis of different histological types of lung cancer. A comprehensive knowledge on its applications and limitations are essential to avoid inappropriate diagnostic outcomes. This further allows to categorize lung cancer patients to avail specific treatment, identify aggressive tumors, prevent the risk of recurrence and further reduce mortality rates.


The authors wish to thank Mrs Maggie Mootosingh (Histotechnician), The University of the West Indies, Department of Histopathology and Department of Clinical Surgical Sciences at the Eric Williams Medical Sciences Complex, North Central Regional Health Authority for assisting in data collection, block retrieval and histopathological report collection.

Financial support and sponsorship

This study was funded by the grant CRP.3MAR14.10 from the University of the West Indies, Trinidad.

Conflicts of interest

There are no conflicts of interest.

 > References Top

Zhang P, Han YP, Huang L, Li Q, Ma DL. Value of napsin A and thyroid transcription factor-1 in the identification of primary lung adenocarcinoma. Oncol Lett 2010;1:899-903.  Back to cited text no. 1
Au NH, Gown AM, Cheang M, Huntsman D, Yorida E, Elliott WM, et al. P63 expression in lung carcinoma: A tissue microarray study of 408 cases. Appl Immunohistochem Mol Morphol 2004;12:240-7.  Back to cited text no. 2
Argon A, Nart D, Veral A. The value of cytokeratin 5/6, p63 and thyroid transcription factor-1 in adenocarcinoma, squamous cell carcinoma and non-small-cell lung cancer of the lung. Turk Patoloji Derg 2015;31:81-8.  Back to cited text no. 3
Kaufmann O, Fietze E, Mengs J, Dietel M. Value of p63 and cytokeratin 5/6 as immunohistochemical markers for the differential diagnosis of poorly differentiated and undifferentiated carcinomas. Am J Clin Pathol 2001;116:823-30.  Back to cited text no. 4
Conde E, Angulo B, Redondo P, Toldos O, García-García E, Suárez-Gauthier A, et al. The use of P63 immunohistochemistry for the identification of squamous cell carcinoma of the lung. PLoS One 2010;5:e12209.  Back to cited text no. 5
Vollmer RT. Primary lung cancer versus metastatic breast cancer: A probabilistic approach. Am J Clin Pathol 2009;132:391-5.  Back to cited text no. 6
Stojsic J, Jovanic I, Markovic J, Gajic M. Contribution of immunohistochemistry in the differential diagnosis of non-small cell lung carcinomas on small biopsy samples. J BUON 2013;18:176-87.  Back to cited text no. 7
Travis WD, Brambilla E, Noguchi M, Nicholson AG, Geisinger K, Yatabe Y, et al. Diagnosis of lung cancer in small biopsies and cytology: Implications of the 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification. Arch Pathol Lab Med 2013;137:668-84.  Back to cited text no. 8
Pelosi G, Fraggetta F, Pasini F, Maisonneuve P, Sonzogni A, Iannucci A, et al. Immunoreactivity for thyroid transcription factor-1 in stage I non-small cell carcinomas of the lung. Am J Surg Pathol 2001;25:363-72.  Back to cited text no. 9
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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.  Back to cited text no. 12
Nicholson AG, Gonzalez D, Shah P, Pynegar MJ, Deshmukh M, Rice A, et al. Refining the diagnosis and EGFR status of non-small 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.  Back to cited text no. 13
Mukhopadhyay S, Katzenstein AL. Subclassification of non-small cell lung carcinomas lacking morphologic differentiation on biopsy specimens: Utility of an immunohistochemical panel containing TTF-1, napsin A, p63, and CK5/6. Am J Surg Pathol 2011;35:15-25.  Back to cited text no. 14
Rebecca O, Naobumi T, Paul ON, Sanja D. Usefulness of immunohistochemical and histochemical studies in the classification of lung adenocarcinoma and squamous cell carcinoma in cytologic specimens. Am J Clin Pathol 2011;136:81-7.  Back to cited text no. 15
Rossi G, Marchioni A, Milani M, Scotti R, Foroni M, Cesinaro A, et al. TTF-1, cytokeratin 7, 34βE12 and CD56/NCAM immunostaining in the subclassification of large cell carcinomas of the lung. Am J Clin Pathol 2004;122:884-93.  Back to cited text no. 16
Kargi A, Gurel D, Tuna B. The diagnostic value of TTF-1, CK 5/6, and p63 immunostaining in classification of lung carcinomas. Appl Immunohistochem Mol Morphol 2007;15:415-20.  Back to cited text no. 17
Khayyata S, Yun S, Pasha T, Jian B, McGrath C, Yu G, et al. Value of P63 and CK5/6 in distinguishing squamous cell carcinoma from adenocarcinoma in lung fine-needle aspiration specimens. Diagn Cytopathol 2009;37:178-83.  Back to cited text no. 18
Wu M, Wang B, Gil J, Sabo E, Miller L, Gan L, et al. p63 and TTF-1 immunostaining. A useful marker panel for distinguishing small cell carcinoma of lung from poorly differentiated squamous cell carcinoma of lung. Am J Clin Pathol 2003;119:696-702.  Back to cited text no. 19
Jafarian AH, Gharib M, Mohammadian Roshan N, Sherafatnia S, Omidi AA, Bagheri S. The diagnostic value of TTF-1, P63, HMWK, CK7, and CD56 immunostaining in the classification of lung carcinoma. Iran J Pathol 2017;12:195-201.  Back to cited text no. 20
Wang BY, Gil J, Kaufman D, Gan L, Kohtz DS, Burstein DE. P63 in pulmonary epithelium, pulmonary squamous neoplasms, and other pulmonary tumors. Hum Pathol 2002;33:921-6.  Back to cited text no. 21
Coughlin SS, Matthews-Juarez P, Juarez PD, Melton CE, King M. Opportunities to address lung cancer disparities among African Americans. Cancer Med 2014;3:1467-76.  Back to cited text no. 22


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10]


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