A majority of squamous cell carcinomas do not pose a diagnostic problem, but poorly differentiated tumors can be challenging since intercellular bridges and keratinization, hallmark features of these tumors, may not be identifiable. Pulmonary squamous cell carcinoma frequently expresses high molecular weight cytokeratins (HMW-CKs) and p63, both of which are generally regarded as markers for stratified epithelium. There are a variety of commercially available HMW-CKs. In lung tumor pathology, the most frequently investigated and commonly used markers are 34βE12 and CK5/6, and less frequently CK14.

34βE12 is a monoclonal antibody that reacts with HMW-CKs 1, 5, 10, and 14. It is a sensitive marker for squamous cell carcinoma (88–100 %), with homogeneously strong and diffuse staining expected in the vast majority of tumors, but its specificity is variable (33–94 %) [2, 4–6, 31, 50, 52, 55]. In adenocarcinomas, diffuse 34βE12 staining is also common (up to 82 %); therefore, it is not recommended as the first line of immunohistochemistry to distinguish between squamous cell carcinoma and adenocarcinoma [5].

34βE12 may be useful to distinguish squamous cell carcinoma from small cell carcinoma since the latter is negative for this marker, while p63 expression is not uncommonly seen in small cell carcinoma (reported incidence as high as 76.9 %) [8] though generally weak and focal staining [66]. It should be noted that p40 is negative in small cell carcinoma [66].

CK5 and CK14 are expressed in the basal layer of the stratified squamous epithelia; therefore, they are often used as markers for squamous cell carcinoma [67].

Anti-CK5/6 antibody reacts with CK5 and CK6 and, hence, recognizes basal cells of squamous epithelia and mesothelium, but is not reactive with simple epithelium or their tumors such as adenocarcinoma. As a marker for squamous cell carcinoma, CK5/6 is slightly less sensitive than or comparable to 34βE12, with reported sensitivity ranging from 53 to 100 % (Fig. 5.1) [2, 4–6, 26, 28, 31, 39, 40, 43, 46, 55, 59, 68], and the extent of its expression can be focal or weak in approximately 10 % of squamous cell carcinomas [55]. An inverse association is generally found between tumor grade and CK5/6 expression; hence, higher-grade tumors show decreased CK5/6 expression [69]. It has been reported that 20 % of poorly differentiated tumors had less than 10 % of CK5/6 reactivity [5]. Approximately 20 % of adenocarcinomas express CK5/6 (reported frequency from 0 to 27 %), but its expression is usually focal and weak [2, 5, 6, 22, 26, 28, 31, 46, 55, 59].

Of note, CK5/6 is expressed by approximately 80 % of malignant mesotheliomas and useful to distinguish between adenocarcinoma and mesothelioma when used in a panel of immunohistochemistry markers [68, 70].

The utility of CK14 for the diagnosis of pulmonary squamous cell carcinoma has not been fully explored. The majority of squamous cell carcinoma is positive for CK14, with reported sensitivity from 77 to 92 % [14, 17, 44, 61]. However, a subset of adenocarcinoma (0–25 %) also expresses CK14 [14, 44, 61, 71]. One study reported that reactivity was similar regardless of tumor grade [14].

Desmosomes, which are intercellular junctions whose primary function is cell adhesion, contain three major component protein groups, including the desmosomal cadherins which comprise desmogleins (DSG1–4) and desmocollins (DSC1–3).

Desmocollin 3 (DSC3) is a component of the transmembrane core of desmosomes, and it is present in the lower portion of squamous epithelium [72]. It shows membranous staining. DSC3 has been described as a squamous marker with high sensitivity and specificity [35, 58]. DSC3 is expressed in 72–84 % of squamous cell carcinoma, while its expression is rarely seen in adenocarcinoma with reported incidence being 0–0.75 %; hence, it provides high specificity [35, 55, 71]. However, its sensitivity to poorly differentiated tumors drops to 52 % [55]. Overall, p63 and CK5/6 provide better sensitivity, and DSC3 is not likely to have an additional effect on sensitivity over CK5/6 or p63 in the vast majority of cases.

Desmoglein 3 (DSG3) is a calcium-binding transmembrane glycoprotein component of desmosomes, similar to DSC3, and positive immunoreaction occurs as a membranous staining.

Using gene expression profiling, DSG3 was found to be highly expressed in squamous cell carcinomas compared with adenocarcinomas [73]. The study also reported that DSG3 was more frequently expressed by immunohistochemistry in squamous cell carcinoma (79.5 %) than adenocarcinoma (54.8 %), but DSG3 was also expressed in typical carcinoid (93 %), atypical carcinoid (82.8 %), large cell neuroendocrine carcinoma (56 %), and small cell carcinoma (32.3 %). However, other studies demonstrated higher sensitivity with fairly high specificity. DSG3 is expressed in the majority of squamous cell carcinomas (92.8–98.5 %) [74, 75]. It is rarely expressed in adenocarcinomas (2–7 %) of the lung or other types including carcinoid tumors, large cell neuroendocrine carcinoma, or small cell carcinoma [71, 74, 75]. Hence, it seems to have high sensitivity and specificity comparable to DSC3 although further studies are needed.

p63: The human p63 gene, a homolog of the p53 tumor suppressor gene, contains two promoters that generate two classes of proteins: the full-length protein TAp63, which contains the N-terminal transactivation (TA) domain, and N-terminal truncated protein isoform ΔNp63, which lacks this transactivation domain [76]. The ΔNp63 is thought to function as a transcription factor for the stratified epithelium. The commonly used clone 4A4 recognizes both TAp63 and ΔNp63, while p40 only recognizes ΔNp63 [41, 77]. p63 expression using clone 4A4 is seen in basal cells and myoepithelial cells of glands of various organs as well as cells of stratified epithelium such as squamous, transitional, and thymic epithelium, and in the squamous and transitional epithelium, more intense expression is observed in the lower portion of the epithelium, particularly basal and parabasal cells [78]. Likewise p63 is expressed in squamous cell carcinomas, urothelial carcinomas, and thymomas as well as basal cell components of a number of benign and malignant tumors including basal cell carcinoma of the skin, various skin adnexal tumors [79], and salivary gland-type tumors such as adenoid cystic carcinoma, mucoepidermoid carcinoma, and myoepithelioma [78, 79]. In addition to the tumors with squamous, urothelial, and basal cell elements, p63 expression, by using 4A4, has been reported in a number of other tumors, including adenocarcinomas of the lung, colon, breast, and ovary, epithelioid trophoblastic tumor, choriocarcinoma, rhabdomyosarcoma, and B cell lymphomas [78]. It is believed that p63 expression in these tumors using clone 4A4 is most likely attributed to TA isoform expression [41].

p63 expression is seen in the vast majority of squamous cell carcinomas, with reported sensitivity ranging from 82 to 100 % (Fig. 5.2) [2, 4–6, 8, 9, 12, 26, 28, 31, 39–41, 46, 49, 55, 59–61], and it is a highly stable marker regardless of the grade of differentiation [5]. p63 expression can be seen in a subset of lung adenocarcinoma with reported frequency ranging from 0 to 48.4 % (Fig. 5.3) [2, 5, 8, 9, 12, 41, 46, 49, 55, 59]. p63 reaction has also been reported in small cell carcinoma, large cell neuroendocrine carcinoma, atypical carcinoid tumor, and typical carcinoid tumor [8]. Its expression in adenocarcinoma is generally focal (usually 25–30 % of the tumor) and weak in resected tumors, and diffuse and strong p63 expression at the level typical for squamous cell carcinoma is an exceptional occurrence in adenocarcinoma. One study showed that the positive predictive value of p63 staining alone for diagnosing squamous cell carcinoma was only 53 % when no quantitative thresholds were placed, but it went to up to 100 % when positive staining was defined as labeling of at least 40 % of tumor cells in the context of negative expression of TTF-1 and Napsin A [9]. However, when dealing with small tumor volume in biopsy or cytology samples, focality of p63 expression may not be appreciated, and any positive reaction can be potentially interpreted as evidence of squamous differentiation. In this scenario, any TTF-1 or Napsin A reaction allows poorly differentiated tumors to be diagnosed as adenocarcinoma or adenosquamous carcinoma [4].

According to a recent study, ALK mutated adenocarcinoma often shows p63 expression [64]. In the study, nine out of ten ALK mutated adenocarcinomas co-expressed p63 and TTF-1.

p40: Recent studies have demonstrated p40 provides very high sensitivity and specificity for squamous differentiation [41, 77, 80]. It is available as both monoclonal and polyclonal antibodies. All the published studies utilized polyclonal antibodies since the monoclonal antibody has become available only recently, which offers less technical problems. Reported sensitivity of p40 for the diagnosis of squamous cell carcinoma is 100 % and specificity ranges from 83 to 100 % (Fig. 5.4) [41, 49, 77, 80].

p40 expression is generally absent in adenocarcinoma. Rare cases of adenocarcinoma (up to 3 %) may contain scattered p40 positive tumor cells [77]. One study demonstrated that if reactivity of <5 % was disregarded, the specificity became 100 % for p40.

A recent study using TMAs showed p40 expression in 36/317 adenocarcinomas [71], which is markedly skewed from data from the majority of studies published in the literature.

As mentioned in the p63 section, ALK mutated adenocarcinoma often shows p63 expression, but in tumors with features of morphologically unequivocal adenocarcinoma, p40 expression was negative [71].

Well-differentiated adenocarcinoma generally does not require immunohistochemistry and special stains to confirm the diagnosis. Immunohistochemistry is often required in poorly differentiated tumors, particularly those with solid growth, so-called squamoid morphology. Additionally immunohistochemistry may be needed for confirmation of lung origin. A number of studies have confirmed the efficacy of TTF-1 and Napsin A in the diagnosis of pulmonary adenocarcinoma over other antibodies [81, 82].

TTF-1, thyroid transcription factor-1, also known as Nkx2.1 or thyroid-specific enhancer binding protein, is a homeobox transcription factor of the NK-2 gene family, and its locus is found on chromosome 14q13. TTF-1 is expressed in the lungs, thyroid gland, and ventral forebrain [83]. In the lungs, TTF-1 regulates surfactant apoproteins A, B, and C and Clara cell secretory protein in type II pneumocytes and Clara cells; therefore, these cells are positive for TTF-1 [84]. The majority of adenocarcinomas show differentiation toward these cells, and approximately 75–80 % of pulmonary adenocarcinomas express TTF-1 (Fig. 5.5). 8G7G3/1 and SPT24 monoclonal antibodies are commonly used. Both antibodies show similar sensitivity; however, the specificity of SPT24 is reportedly lower. The majority of pulmonary adenocarcinomas are positive for TTF-1 by using both clones. One study compared both clones and reported sensitivity being 72.4 % for SPT24 and 65.4 % for 8G7G3/1 [34]. The SPT24 clone, however, detects more primary lung tumors of various histologic subtypes, including squamous cell carcinomas, large cell carcinoma, and carcinoid tumors, than clone 8G7G3/1 [34]. A recent study using SPT24 has shown statistical differences between tumor differentiation and positivity: 98 % in well differentiated, 94.2 % in moderately differentiated, and 72.4 % in poorly differentiated [55]. A small proportion of carcinomas from prostate, stomach, salivary gland, and colon are positive for both antibodies, with similar intensity and distribution [34]. It should be noted that the majority of pulmonary and extrapulmonary small cell carcinoma expresses TTF-1. Likewise TTF-1 expression is not uncommonly seen in pulmonary or extrapulmonary large cell neuroendocrine carcinoma [82].

Napsin A, also known as aspartyl protease 4, is predominantly expressed in alveolar type II pneumocytes where it is involved in the processing of the prosurfactant B protein [85]. Napsin A expression is also commonly seen in intra-alveolar macrophages, which is believed to be a result of phagocytosis [81]. Localization of the positive reaction is cytoplasm with granular staining quality.

For Napsin A, both polyclonal and monoclonal (TMU-Ad02, IP64, KCG1.1, MRQ60) antibodies are available. It has been reported that the monoclonal antibody shows greater specificity for lung adenocarcinoma. Napsin A expression is seen in 58–91 % of lung adenocarcinoma by monoclonal antibody (Fig. 5.6) and 81 % by polyclonal antibody [36, 81]. In a combined review of 11 studies, 627 (75 %) of 836 lung adenocarcinomas were reported to be Napsin A positive, whereas 623 (74.4 %) of 837 exhibited TTF-1 positivity [81]. These results indicate that the sensitivity of Napsin A for lung adenocarcinomas is comparable with that of TTF-1. This marker is negative in small cell carcinoma and carcinoid tumor contrary to TTF-1. There are rare adenocarcinomas where TTF-1 is positive but Napsin A negative or vice versa, which may indicate the importance of including the two markers for the diagnosis of pulmonary adenocarcinoma in difficult cases [51, 62].

Napsin A expression is also commonly seen in carcinomas of the kidney (45 and 67 % each by monoclonal and polyclonal) and thyroid gland (50 % each by monoclonal and polyclonal). While its expression is only rarely seen in carcinomas of other organs such as liver and endometrium by monoclonal antibody, Napsin A positive reaction using the polyclonal antibody is seen in a variety of adenocarcinomas, such as those from the colon, pancreas, esophagus, and stomach, as well as urothelial carcinomas [36, 81]. Napsin A is not expressed in pulmonary squamous cell carcinoma. Alleged Napsin A expression in squamous cell carcinoma is believed to represent expression in hyperplastic type II pneumocytes and intra-alveolar macrophages, both of which are sometimes seen entrapped within the tumor (Fig. 5.7) [81].

CK7 is an intermediate-sized and basic keratin and expressed in bronchial and alveolar epithelium, while CK20 is not expressed in respiratory epithelium. Lung adenocarcinoma typical shows CK7+/CK20− pattern. This may be helpful for supporting a diagnosis of primary lung malignancies, especially in cases with negative TTF-1. However, a subset of the tumors show an aberrant expression pattern. CK7 status has been thought to be useful to distinguish between lung adenocarcinoma and squamous cell carcinoma since CK7 is more frequently expressed in adenocarcinomas than in squamous cell carcinomas. In a typical scenario, CK7 is positive in adenocarcinoma and negative in squamous cell carcinoma. This finding might be helpful when dealing with TTF-1 negative poorly differentiated carcinoma since CK7 reaction tends to be retained in poorly differentiated adenocarcinoma, which is occasionally TTF-1 negative. However, this marker solely is not specific enough to stand as an adenocarcinoma marker. Indeed, 95 % of adenocarcinoma is positive for CK7, and 20 % of lung squamous cell carcinoma is also positive for CK7 [58]. These findings lead to the conclusion that CK7 should not be used to differentiate between adenocarcinoma and squamous cell carcinoma [4].

There are several antibodies specific for Clara cells and type II pneumocytes, which have been tested in pulmonary adenocarcinomas. As expected, they are expressed predominantly in well-differentiated adenocarcinoma but not in poorly differentiated carcinoma; therefore, they are not particularly useful in the setting of poorly differentiated tumors.

Surfactant protein-A (Sp-A) is expressed in Clara cells and type II pneumocytes of the lung parenchyma, and its gene expression is regulated by TTF-1 [86]. It is a marker for tumors differentiating into cells of such lineage and has high specificity, but sensitivity is low, and as expected it is usually negative in poorly differentiated adenocarcinomas. The monoclonal antibody PE10 detected Sp-A in 24–74 % of lung adenocarcinomas by using clone PE10, which is now out of production [62, 87–89]. No expression was seen in squamous cell carcinoma by using clone PE10 [62] or clone 32E12 [71].

Surfactant protein-B (Sp-B) is also detected in type II pneumocytes and Clara cells. Likewise, Sp-B is generally expressed by well-differentiated tumors but much more infrequently by poorly differentiated tumors. The reported specificity ranges from 80 to100%, but its sensitivity is 38–61 % [27, 29, 56].

DC-LAMP (CD208) and CC-10 (Clara cell protein 10) also stain differentiated pulmonary adenocarcinomas with Clara cell differentiation, but they are usually negative in poorly differentiated adenocarcinomas where immunohistochemistry is usually needed for histotyping; therefore, the usage of these markers is limited to more academic purposes [89].

The entity represents poorly differentiated carcinoma where the tumor lacks morphological features of squamous cell carcinoma, adenocarcinoma, or small cell carcinoma. As such, the diagnosis requires thorough sampling of the resected tumor, and its definitive diagnosis is not made based on a small biopsy or cytologic material. It is a morphologically defined entity; however, it is known that a proportion of large cell carcinoma, not otherwise specified, expresses lineage markers for either squamous cell or adenocarcinoma [35]. A recent study showed that approximately 80 % of this group of tumor can be classified as variants of adenocarcinoma (60 %) or squamous cell carcinoma (20 %), when defined by TTF-1+/p40− for adenocarcinoma variant, TTF-1-/p40+ for squamous cell carcinoma variant, TTF-1+/p40+ for adenosquamous carcinoma variant, and TTF-1-/p40− for null type [90]. Interestingly, molecular alterations characteristic of adenocarcinoma occurred in tumors with immunoprofiles of adenocarcinoma or marker null but not in tumors with squamous immunoprofiles. Whether such tumors should be classified to adenocarcinoma or squamous cell carcinoma solely by immunohistochemistry is arguable from the point of histology-based classification [91], but the abovementioned molecular findings support some therapeutic relevance for immunohistochemistry-based classifications.

Basaloid carcinoma expresses squamous-lineage markers such as HMW-CKs and p63 [44, 52]. Basaloid carcinoma shares morphological features with small cell carcinoma and large cell neuroendocrine carcinoma, and this may cause great diagnostic difficulties, particularly when dealing with a small biopsy or fine-needle biopsy specimens. Focal expression of neuroendocrine markers further complicates the diagnostic dilemma [92]. Basaloid carcinoma is negative for TTF-1 and almost invariably positive for 34βE12, while high-grade neuroendocrine carcinoma often expresses TTF-1 but it is negative for 34βE12.

Lymphoepithelioma-like carcinoma is a distinct clinicopathologic entity with characteristic morphological features, and similar to the more familiar nasopharyngeal counter, most of the cases reported in Asians are associated with Epstein-Barr virus (EBV) [93]. Evidence of EBV can be demonstrated by both immunohistochemistry and in situ hybridization. Detection of EBER-1 RNA by in situ hybridization method is very sensitive. Immunohistochemistry with antibodies to latent membrane protein 1 (LMP1) is less sensitive and often results in heterogeneous staining reaction. The tumors in Caucasians are less frequently associated with EBV [93].

Multiple keratin antibodies can demonstrate the epithelial lineage of spindle and pleomorphic cell components in many of the cases. AE1/3, CAM 5.2, CK18, and CK7 are positive more frequently than epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA), CD15, and Ber-EP4. Keratin antibodies may also highlight the epithelial component of carcinosarcoma. Positive epithelial markers are not required for diagnosis if components of differentiated carcinoma such as adenocarcinoma or squamous cell carcinoma are present. Sarcomatoid carcinoma that is negative for any of the epithelial markers is difficult to separate from sarcoma. Alternatively, expression of epithelial markers, particularly, low molecular weight cytokeratins in the sarcoma, has a weak and focal staining quality [94]. Conversely, a true strong CK expression is also seen in certain sarcomas with epithelioid morphology, such as synovial sarcoma, epithelioid hemangioendothelioma, and epithelioid sarcoma.

Vimentin is usually diffusely positive in sarcomatoid carcinoma, and it may support the morphological interpretation of sarcomatoid carcinoma since it is infrequently expressed in squamous cell carcinoma and adenocarcinoma, with usually focal staining extent [46].

Muscle markers and S100 protein stain rhabdomyosarcoma and chondrosarcoma, respectively, when present in either carcinosarcoma or pulmonary blastoma.

CK7 is positive in approximately 60–70 % of spindle or giant cell component of sarcomatoid carcinoma. The frequency is higher in sarcomatoid carcinoma associated with adenocarcinoma than tumors associated with squamous cell carcinoma (80 % vs. 16 %) [95]. Only a subset of sarcomatoid carcinoma expresses TTF-1 (55 %), which is useful in supporting the pulmonary origin of these tumors, and its expression is usually focal [95]. Approximately 80 % of sarcomatoid carcinoma is accompanied by better differentiated carcinoma component, such as squamous cell carcinoma or adenocarcinoma [95]. If a tumor presents as a solitary lung tumor with no history of tumors in other anatomic location, and a tumor expresses epithelial markers such as cytokeratins, or a tumor is associated with better differentiated component, the diagnosis of sarcomatoid carcinoma can be rendered with confidence, but in the absence of these features, the diagnosis can be challenging, with differential diagnosis lying between sarcomatoid carcinoma and sarcoma, either primary or metastatic, or when pleura is diffusely involved, sarcomatoid mesothelioma. Synovial sarcoma, which is the most common primary lung sarcoma, can be ruled out by examining the characteristic translocation t(X;18)(p11;q11) by FISH or RT-PCR. However, for the majority of cases, solid diagnosis cannot be rendered by histology and immunohistochemistry alone, and clinical and radiographical correlations are necessary for disease management.