Immunohistochemistry involves the detection and localization of antigens or proteins in tissue sections by the use of antibodies that bind specifically to the antigen of interest. The antibodies are coupled to a detection system which allows them to be visualized in tissue sections. IHC has a range of applications in the practice of pathology and is commonly used by pathologists to help in distinguishing cell types or their origin, using markers that are expressed differentially between different cell types and organs. Additionally, IHC enables one to observe or determine the localization and distribution of various antigens or proteins within the tissue.
However, it needs to be recognized that IHC is neither 100% specific nor 100% sensitive. For example, thyroid transcription factor-1 (TTF-1) is widely known as a marker for pulmonary adenocarcinoma but is also highly expressed in thyroid tumors and may uncommonly also be expressed in carcinomas originating from other primary sites, including, for example, colorectal carcinoma [3
]. Morphologic features on H&E sections remain the basis for diagnosis, and ancillary tests need to be interpreted in the context of the histomorphologic findings. Despite these limitations, it has been shown that a high degree of accuracy in the subtyping of NSCLC can be achieved by applying a simple panel of immunohistochemical markers including TTF-1, tumor protein 63 (P63), cytokeratin (CK) 7, and CK5/6 [4
]. Other antibodies such as desmocollin-3 and napsin A have also been recently described as helpful in further refining the subtypes in difficult cases [6
CKs are intermediate filament proteins that provide structural support within the cytoplasm of epithelial cells. Fifty-four CK genes have been identified, and the corresponding keratin proteins are classified by molecular weight and isoelectric pH [8
]. In the most general terms, tumors of epithelial origin (termed carcinomas or adenocarcinomas, if gland forming) express CKs, which differentiates them from tumors of mesenchymal origin (sarcomas), hematopoietic origin (lymphomas/leukemias), and melanoma. The various epithelial tissues of the human body also express different CKs, thus different epithelial tumors may often be distinguished based on their unique cytokeratin expression profile.
TTF-1 plays an important role in the embryogenesis of lung, and its expression remains high in type II pneumocytes and Clara cells [9
]. TTF-1 has been used as an immunohistochemical marker for primary lung adenocarcinoma, despite recent reports of occasional aberrant TTF-1 staining in tumors from other primary sites (e.g., [3
]). TTF-1 is known to regulate the expression of several lung-specific proteins including napsin A, surfactant proteins, and others. Antibodies to napsin A in combination with TTF-1 have been proposed as additional evidence of pulmonary origin of a tumor.
2.1. Squamous Cell Carcinoma
In H&E stained sections, squamous differentiation is identified by keratinisation and/or formation of intercellular bridges. Both features are specific for squamous cell differentiation and are not seen in other tumor types. While these features are readily observed in well-differentiated tumors, they may be difficult to appreciate or absent in poorly differentiated tumors, especially in small biopsy samples or fine needle aspirate cytology specimens. In such cases, an IHC panel including P63, CK5/6, TTF-1, and CK7 may be helpful, with positive staining for P63 and CK5/6 and concurrent lack of staining for TTF-1 and CK7 supporting squamous differentiation () [5
Immunohistochemical profiles of squamous cell carcinoma and pulmonary adenocarcinoma.
From a clinical standpoint, it is important to note that squamous differentiation is not evidence of the tumor's site of origin. Metastatic squamous cell tumors to the lung are histologically and immunohistochemically identical to primary lung squamous tumors. At present, there are no immunohistochemical or molecular markers in routine use that reliably differentiate primary pulmonary from metastatic squamous cell carcinomas, as TTF-1 typically is not expressed in pulmonary squamous cell carcinomas. Therefore, clinical history is crucial to determining the site of origin.
The accurate diagnosis of squamous cell histology has important therapeutic implications. Certain systemic therapy agents are not used in patients with squamous histology for safety or efficacy concerns. Bevacizumab, a monoclonal antibody directed against vascular endothelial growth factor (VEGF) is associated with an increased risk of life threatening pulmonary hemorrhage in patients with squamous cell histology [10
]. The association between bleeding complications and this histological type has been seen with other VEGF inhibitors although not all [11
]. Also, pemetrexed, a chemotherapeutic agent has been associated with inferior outcomes compared with docetaxel chemotherapy. By contrast, pemetrexed use in the first-, second-line, and maintenance settings has been associated with superior outcomes in patients with nonsquamous histology [13
]. These studies confirm that accurate classification of NSCLC has an important role in patient management and outcomes.
The WHO classification [1
] of lung tumors has long divided adenocarcinomas primarily into acinar, papillary, solid, bronchioloalveolar (BAC), or mixed subtypes based on histomorphologic features. Classically, adenocarcinomas display gland formation on H&E, although subtypes with bronchioloalveolar or solid patterns of growth may lack well-defined glandular structures. The BAC subtype (or adenocarcinoma in situ (AIS) in the new IASLC/ATS/ERS classification [2
]) is characterized by exclusively “lepidic” growth pattern, in which neoplastic cells grow along the surfaces of preexisting alveolar structures, without evidence of invasion. Two variants of BAC have been recognized classically, mucinous and nonmucinous. The mucinous variant (mucinous AIS or invasive mucinous adenocarcinoma in the IASLC/ATS/ERS classification [2
]) is composed of tall columnar cells with abundant pale cytoplasm that stains positively with histochemical stains for mucin, such as mucicarmine or periodic acid Schiff (PAS). Pulmonary adenocarcinoma is differentiated from squamous cell carcinoma by being typically positive for CK7 and TTF-1, and negative for p63 and CK5/6 (). Although most pulmonary adenocarcinomas are positive for TTF-1, a significant subset is negative (15–30%), especially the mucinous BAC subtype, or those originating in more central locations [15
Figure 1 Immunohistochemistry stains in squamous cell carcinoma and adenocarcinoma of lung. H&E: hematoxylin and eosin; CK: cytokeratin; TTF-1: thyroid transcription factor 1. Squamous carcinomas are typically positive for CK5/6 and P63, and negative for (more ...)
Primary lung adenocarcinoma must also be differentiated from adenocarcinoma that has metastasized to lung. Clinical history and IHC can be invaluable in this regard. In particular, the differential expression of CK7 and CK20 may be very useful in characterizing the origin of epithelial neoplasms. Pulmonary adenocarcinoma is typically CK7+/CK20− by IHC although this cytokeratin profile is not specific to adenocarcinomas of lung and may be seen also in tumors arising from the breast, thyroid, upper gastrointestinal and pancreaticobiliary tracts, and gynecologic tract. Tumors demonstrating CK7−/CK20+ staining profile include colorectal and Merkel cell carcinomas. CK20 staining may occasionally be seen in pulmonary adenocarcinomas, but CK7 typically is also positive in such cases. While an exhaustive discussion is beyond the scope of this manuscript, suffice to indicate that most primary lung adenocarcinomas are CK7 and TTF-1 positive, although as previously noted, TTF-1 staining may also be seen in thyroid tumors and infrequently in tumors originating from other body sites. Lack of TTF-1 expression does not exclude pulmonary origin for a CK7-positive adenocarcinoma in the lung; however, in this situation, metastatic carcinoma arising from other body sites that demonstrate a CK7+/CK20− cytokeratin profile would need to be excluded clinically.
Napsin A, an aspartic acid protease whose expression in the lung is regulated by TTF-1, has also shown promise in helping to differentiate primary lung from metastatic adenocarcinomas. While napsin A expression may also be seen in normal kidney and in a proportion of renal tumors, positivity for both TTF-1 and napsin A is a strong indication that an adenocarcinoma originated from lung [6
2.3. Neuroendocrine Tumors
Tumors with neuroendocrine (NE) differentiation include small cell carcinoma, large cell neuroendocrine carcinoma, and typical and atypical carcinoid tumors. NE tumors are defined ultrastructurally by having neurosecretory granules and immunohistochemically by positivity for NE markers (). According to a recent international workshop on pulmonary NE tumors, CD56 has a sensitivity of 95% and a specificity of 97% for detecting cells showing NE differentiation. The sensitivity for synaptophysin and chromogranin is 80–85%, and the specificity for both is 97% [17
]. Using a panel of two or three NE markers ensures a high degree of accuracy for detecting NE differentiation.
Immunohistochemical profile of neuroendocrine tumors.
Differentiating between the different types of NE tumors may be very difficult in small biopsy specimens because the small sample may not be entirely representative of the overall tumor and is subject to crush artefact because of the relative fragility of these cells. In some cases, the evaluation of Ki-67 may be useful in this regard. Ki-67 is a marker of cell proliferation, and a recent review has concluded that the Ki-67 proliferation index of typical carcinoid (TC) is less than 2%, while atypical carcinoid (AC) is less than 20% (typically around 10%) [18
]. Small cell carcinomas often have a Ki-67 proliferation index of greater than 60%. It has been suggested that a Ki-67 index of less than 25% excludes small cell carcinoma [19
Differentiating primary NE tumors of the lung from NE tumors from other body sites may also be a challenge, as NE tumors arising from other body sites may demonstrate identical morphology. In this regard, expression of TTF-1 may be helpful to identify a carcinoid tumor of lung origin, as carcinoid tumors from other sites rarely express this marker [20
]. However, small cell carcinomas arising from the lung frequently express TTF-1 while those arising from other sites may occasionally also express TTF-1. In the case of small cell carcinoma with TTF-1 positivity, lung origin is favored, but this is not entirely definitive [21
2.4. Other IHC Markers
P53 functions as a tumor suppressor protein by playing a key role in cell cycle progression, apoptosis, and DNA repair [22
]. Overexpression of P53 in NSCLC has been reported to be both a prognostic marker, associated with increased tumor aggressiveness and shorter overall survival, and a potential predictive marker, associated with a favorable response to platinum-based adjuvant chemotherapy, resulting in a survival benefit [23
]. However, this needs additional validation.
From an embryologic perspective, there is now evidence that the transcription factor achaete-scute homologue-1 (ASH1) is pivotal for NE cell differentiation and may be necessary for transformation to NE lung carcinoma [24
]. Immunohistochemical analysis of ASH1 is available but not routinely used outside of the research setting at present. It may potentially help to identify early progenitor cells that are committed to NE differentiation and better define subsets of lung cancers demonstrating NE differentiation [24