Skin tissue is composed of epidermal and adnexal components as well as mesenchymal dermal components. All epithelial cells in the epidermis, folliculosebaceous unit, and sweat glands reveal pan-keratin markers such as AE1/AE3 (Fig. 26.2a). Keratinized squamous cells and proliferative keratinocytes express cytokeratin (CK) 6/16, nonkeratinized squamous cells reacts with CK4/13, and basal keratinocytes exhibit reactivity for CK5/14/15 (Fig. 26.2b). Squamous cells in palm and sole are reactive for CK1/9/10 [1, 2]. Eccrine and apocrine glands comprise sweat structures of the skin. Normal eccrine glands show reactivity with CD7, CD20 (Fig. 26.2c, d), CEA, and S100, while apocrine glands exhibit immunostaining for CEA and GCDFP15 [3, 4]. Sebaceous glands exhibit reactivity for CK10 as well as EMA rimming cytoplasmic lipid vesicles (Fig. 26.2e) [5]. Normal melanocytes express S100, HMB45, and MART-1/melan-A but do not react with tyrosinase [6]. Langerhans cells are stained with CD1a (Fig. 26.2f), S100, langerin, and CD31 [7]. Displaying neurotactile differentiation, Merkel cells of normal skin are reactive for CK20, MOC-31, neurofilament, and CD56 [8–10]. Markers of the normal epidermal components are depicted in Fig. 26.3. The immunoprofile of normal skin components and respective cancers is summarized in Table 26.1.

Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are derived from the spinous layer and basal layer of the epidermis, respectively. Well-differentiated SCC expresses high molecular cytokeratin, while those with poor differentiation express low molecular cytokeratin. Cytokeratin, p63, and vimentin are present in the sarcomatoid variant of SCC [11]. EMA, one of the human milk fat globule proteins not expressed in normal keratinocytes, is expressed on malignant squamous cells. Basal cell carcinoma expresses BerEp4 (Fig. 26.4) but does not demonstrate reactivity with EMA and p63, distinguishing it from SCC [12].

Malignant eccrine tumors are distinct from benign eccrine tumors by displaying reactivity with EMA. Eccrine tumors display CEA, CD15, and p63 which are also common with apocrine tumors. Differentiating markers of apocrine tumors are TAG-72 (CA72.4) and GCDFP15 (Fig. 26.5) which are not expressed on eccrine tumors [4]. S100 is demonstrated in 50 % of eccrine tumors, but not in apocrine tumors. A remaining challenge is distinguishing primary eccrine carcinoma from metastatic carcinoma by immunoprofile of CK5/6 and p63 which are positive in eccrine carcinoma, but not in metastatic carcinoma [13]. Paget disease is an intraepidermal extension of neoplastic cells into the epidermis which shares similar histopathologic features with malignant melanoma and Bowen disease. Immunohistochemistry study can be a helpful method in differentiating these tumors as denoted in Table 26.2 [14]. CK20 and GCDFP-15 are useful markers in distinguishing primary and secondary perianal Paget diseases, respectively [15].

Tumors with trichilemmal differentiation display reaction with CK14/15/19, BerEP4, and p63 but do not react with EMA (except proliferating trichilemmal tumor), CEA, S100, CD15, CA72.4, HMB45, and GCDFP15 [3]. Trichilemmal carcinoma displays reactivity with CEA and S100, and proliferating trichilemmal carcinoma (malignant proliferating tumor) shows reactivity with EMA and CD34 [17]. Desmoplastic trichoepithelioma shares histopathologic similarities with infiltrating BCC and microcystic adnexal carcinoma. The immunoprofile of these tumors are demonstrated in Table 26.3.

Sebaceous tumors exhibit reactivity with CK5/14/15, CK8/18, EMA, CD15, anti-adipophilin (ADP) and androgen receptor. CK15 is positive in sebaceoma but does not exhibit reactivity with sebaceous carcinoma [21]. Sebaceous tumors do not express CEA, S100, CA72.4, and GCDFP-15 in comparison with sweat gland tumors, which are positive for these markers [4, 22]. Sebaceous carcinoma is differentiated from BCC by showing reactivity for EMA (Fig. 26.6) and negative reaction to BerEP4, vice versa of BCC [23]. Proliferating markers are good markers to differentiate sebaceous adenoma from sebaceous carcinoma (Table 26.4).

Being a sensitive but a nonspecific marker of melanoma, S100 is a calcium-binding protein given its name because of solubility in 100 % saturated ammonium sulfate solution. Other S100-positive tumors include undifferentiated carcinoma, nerve sheath and glial tumors, adipose tumors, and histiocytic and Langerhans cell proliferations [26, 27]. Considering as highly specific marker of melanocytes, the gp100 group includes HMB-45 and MART-1/melan-A with 60 and 80 % sensitivity, respectively. Melanoma antigen recognized by T-cells-1 (MART-1) is a protein which serves as a potential target for cytotoxic T lymphocytes recognized by two monoclonal antibodies (mAbs), A103 and melan-A [28]. Desmoplastic/spindle cell variant of melanomas does not show reactivity with HMB45 and MART/melan-A. Instead, these melanomas are more reactive with S100, p75-NGF-R, and tyrosinase [29]. Small cell melanoma is another variant of the melanoma which could be distinguished from other small cell undifferentiated tumors of the skin and subcutaneous tissue by Abs panel (Fig. 26.7). The immunoprofiles of these tumors are summarized in Table 26.5.

Detection of BRAF p.V600E mutation by immunohistochemistry in melanomas could be used as a first step to identify patients with melanoma as candidates for BRAF inhibitors. Displaying by immunohistochemistry, melanoma progression is correlated with MERTK expression: highest in metastatic melanomas, followed by primary melanomas and nevi [32, 33]. Other prognostic markers correlated with melanoma progression and prognosis include MIB-1 (Ki-67), Bcl2, p53, p16, cyclin-D1, cyclin-D3, osteopontin, NM23, E-cadherin, beta-catenin, Wnt5a/frizzled, Cdc42, and CXCR4 [34–40].

Mesenchymal tumors are discussed in soft tissue tumors, but some tumors which are more seen in skin are discussed here. Kaposi sarcoma which originates from endothelial cells is an intermediate malignant potential vascular tumor of the skin positive for a highly sensitive and specific Ab called HHV8 latent nuclear antigen-1 [41]. Dermatofibrosarcoma protuberance is an intermediate tumor of fibrohistiocytic cell origin which is diffusely positive for CD34 (Fig. 26.8) and negative for factor XIIIa separate from dermatofibroma which is in reverse of DFSP (CD34−, factor XIIIa+) [42]. Considering it as a superficial variant of malignant fibrous histiocytoma, atypical fibroxanthoma is a fibrohistiocytic tumor exhibiting reactivity with vimentin, CD10, and CD99 (Fig. 26.9) [43]. Among tumors with smooth muscle differentiation, leiomyoma and leiomyosarcoma are reactive for SMA, desmin, and caldesmon similar to extracutaneous equivalents [44, 45]. Neurothekeoma (NTKs) is a distinctive neoplasm of the skin showing schwannian and neuroectodermal differentiation which typically labels with S100 (conventional variant), CD99, and NKI-C3 (cellular variant) [46].

Tumors of the nose and paranasal sinuses can be categorized in two groups of small cell carcinomas and undifferentiated carcinomas. Small cell carcinomas of the nasal cavity and paranasal sinuses include olfactory neuroblastoma (ONB), melanoma, lymphoma, rhabdomyosarcoma, small cell neuroendocrine carcinoma, and ES/PNET (Table 26.6). Undifferentiated carcinomas include sinonasal undifferentiated carcinoma, undifferentiated nasopharyngeal carcinoma (Fig. 26.10), and undifferentiated neuroendocrine carcinoma (Fig. 26.11) [47, 48]. All poorly differentiated and undifferentiated carcinomas express cytokeratin [49]. Undifferentiated nasopharyngeal carcinoma reacts with EBV, and undifferentiated neuroendocrine carcinoma is positive for neuroendocrine markers and S100 [50]. NUT midline carcinoma (NMC) is an aggressive tumor with translocation of the NUT (nuclear protein in testis) gene resulting in the formation of BRD4–NUT fusion gene. Recently, new mAbs against the NUT Ag have been designed which will improve the diagnosis of NMC [51]. Immunohistochemistry of poorly differentiated and undifferentiated carcinomas are denoted in the Table 26.7.

Squamous cell carcinoma (SCC) is the most common malignancy in the head and neck. Typically, head and neck SCCs are positive for cytokeratin cocktails, AE1/AE3, and pan-cytokeratin. Human papilloma virus (HPV) is detected in some SCCs of the oropharynx and known as a risk factor of head and neck SCCs [60, 61]. Being as a variant of SCC, basaloid squamous cell carcinoma (BSCC) is another tumor with predominance of basaloid components. Basaloid squamous cell carcinomas express p63 which is relatively specific but also found in other squamous tumors (Fig. 26.12). Neuroendocrine markers are negative in BSCC [62]. Spindle squamous cell carcinoma (SSCC) is a cytokeratin-negative SCC in which spindle cell component is uniformly and strongly positive for vimentin [63]. Undifferentiated nasopharyngeal carcinoma shows reactivity to EBV immunostaining as well as some SCCs and BSCCs [64, 65].

Salivary glands are tubuloacinar exocrine glands having two-layered epithelium which comprise of luminal (acinar and ductal cells) and abluminal (myoepithelial and basal cells). Luminal cells are positive for low molecular cytokeratin, whereas myoepithelial and basal cells react with high molecular cytokeratin and myoepithelial markers. The majority of salivary gland carcinomas can be diagnosed by routine hematoxylin and eosin (H&E)-stained slides, and immunohistochemical (IHC) staining has only a limited role in the diagnosis of salivary gland tumors [47, 67]. Figure 26.13 summarized the various components of the normal salivary glands with an emphasis on the immunohistochemistry Abs.

The most common malignant tumors of salivary glands consist of acinic cell carcinoma, adenoid cystic carcinoma (Fig. 26.14), basal cell adenocarcinoma, epithelial-myoepithelial carcinoma, mucoepidermoid carcinoma (Fig. 26.15), myoepithelial carcinoma, polymorphous low-grade adenocarcinoma, and salivary duct carcinoma. All tumors are cytokeratin positive; however, different immunoprofile patterns exist [68]. C-kit (CD117) is positive in acinic cell carcinoma and adenoid cystic carcinoma [69, 70]. Acinic cell tumor and mucoepidermoid carcinoma demonstrate reactivity with membrane-bound mucin (MUC) [71, 72]. Myoepithelial carcinomas are positive for both epithelial and myoepithelial markers but do not exhibit reaction with EMA and CEA [73]. Malignant monophasic salivary gland tumors include acinic cell carcinoma, myoepithelial carcinoma, mucoepidermoid carcinoma, and polymorphous low-grade adenocarcinoma. Immunophenotype profiles of monophasic and biphasic tumors are denoted in Tables 26.8 and 26.9. Application of CK7 and CK20 is a useful panel in distinguishing primary salivary gland carcinoma (CK7⁺, CK20⁻) from metastatic carcinoma (CK7⁻, CK20⁺) [74].

The functional unit of thyroid is the follicle which is composed of follicular cells and C cells. Follicular cells exhibit reactivity with thyroglobulin, TTF1, PAX8, AE1/AE3, EMA, and CK7 and CK8/18/19, whereas C cells are positive for calcitonin, TTF1, CK7, synaptophysin, and chromogranin. Being as a nuclear transcription factor, TTF1 is expressed on follicular and C cells. A follicular cell-specific marker is thyroglobulin which does not react with C cells (Fig. 26.16). As a member of the paired box (PAX) gene family, PAX8 is a sensitive marker of thyroid tumors similar to TTF1. Among intermediate filaments, CK19 is more expressed in papillary carcinoma than other tumors. Parathyroid hormone (PTH) and parafibromin are markers of parathyroid tumors. Parafibromin is uniformly expressed in parathyroid adenomas, whereas its expression is often reduced in parathyroid carcinomas. Table 26.10 shows an immunopanel of thyroid and parathyroid tumors. Figure 26.17 depicts thyroid medullary carcinoma.

The most frequent IHC pattern observed in lung tumors is positivity for CK7, TTF1, and Napsin A, along with negative staining for CK20, CDX2, and MUC2. It is highly advocated to consider the fact that there are recently increasing reports of primary pulmonary adenocarcinomas with intestinal differentiation which are CK7 and TTF1 negative but CK20 positive which can be highly misinterpreted as metastatic colorectal adenocarcinomas. Therefore, the importance of physical examination and imaging studies is highlighted. It should be noted that neuroendocrine markers including chromogranin, synaptophysin, NSE, and Leu7 (CD57) can be positive in lung non-neuroendocrine carcinomas such as adenocarcinomas and SCC. Recent studies have shown EGFR, Her2, and BRAF mutations in lung cancers which can increase the chance for targeted therapies in these cancers [103–106].

Neoplasms of the pleura are very rare, and most tumors in this area are usually metastatic lesions. One of the most important applications of IHC is to assist pathologists in differentiating mesotheliomas from lung adenocarcinomas [107–109]. Table 26.11 shows the most frequent markers stained by IHC staining in mesothelioma compared with pulmonary adenocarcinoma (Fig. 26.18).

The most common primary hepatic cancer is hepatocellular carcinoma which is well known to have a wide spectrum of histologic differentiation and a great diversity of appearances. It necessitates the application of IHC as an ancillary aid for better diagnosis of the lesion. It is important to reiterate that IHC is after all an ancillary aid. A significant clinicopathologic correlation seems mandatory for the final diagnosis. If a definitive diagnosis cannot be clinched, at the least, certain differential diagnoses can be excluded [114–118]. Immunophenotype of normal liver is summarized in Table 26.12 (Figs. 26.19 and 26.20).

Cholangiocarcinoma is a malignant tumor with characteristics mostly similar to other types of adenocarcinomas. The tumor is usually positive for CK7, CK19, CAM5.2, CK AE1/AE3, pCEA, mCEA (noncanalicular pattern), and MOC31. MUC4, MUC5AC, and MUC6 can also be useful not in diagnosis but in classification and predicting the prognosis. Additionally, CD56 which is positive in benign bile ductular proliferations and negative in cholangiocarcinomas can be useful in differentiating malignant lesions from benign proliferation. The exception for this rule is clear cell cholangiocarcinoma which is positive for CD56. Staining for CK7 and CK19 in cholangiocarcinoma can help to differentiate this tumor from HCC, which is negative for the mentioned markers [119, 120]. Table 26.13 indicates the immunophenotypes of hepatocellular carcinoma and cholangiocarcinoma.

The most common esophageal cancers are adenocarcinomas and SCC. Adenocarcinoma of the esophagus is immunophenotypically similar to gastric adenocarcinomas, and there is no IHC panel to distinguish these two. Esophageal SCC is usually positive for most CK markers including CK AE1/AE3, CK 34bE12, CK5/6, CK19 (positivity increases with tumor grade whereas benign squamous lesions are negative for this marker), and p63. Additionally, most SCCs are negative for CK7 and CK20 which can be useful in distinguishing poorly differentiated SCCs from poorly differentiated adenocarcinomas positive for these two CK markers [121–123].

Stomach glandular epithelium expresses CK20 and less commonly CK7 (CK7+, CK20+) and MUC5AC, distinguishing it from small intestine and colorectal epithelium. Immunoprofile of normal gastrointestinal mucosa is denoted in Table 26.14. Gastric adenocarcinoma has many histologic variants, but they have almost similar immunophenotyping. It should be mentioned that synaptophysin and chromogranin as neuroendocrine markers can be positive in gastric adenocarcinomas; therefore, positive staining with these markers is not sufficient for the diagnosis of neuroendocrine carcinoma [124–126]. Immunoprofile of gastric adenocarcinoma is demonstrated in Table 26.15 (Fig. 26.21).

Immunophenotyping of adenocarcinoma is also valuable in neuroendocrine tumors (NET) [127–129]. Tables 26.14 and 26.15 summarize the immunoprofile of normal small intestine, its adenocarcinoma, as well as their comparison with stomach and colon adenocarcinoma.

In contrast to older studies which have discussed colon cancers generally, recent studies reveal that colon cancers arise from two different pathways (chromosomal instability of APC gene vs. microsatellite instability (MSI) pathway) with different immunophenotypic features [116, 130–136]. Immunoprofile of normal and colon adenocarcinoma is denoted in Tables 26.14, 26.15, and 26.16.

The most frequent anal cancers are SCC and adenocarcinoma. Anal SCC is almost similar to SCC of other origins; nonetheless, the role of HPV is highlighted. Adenocarcinomas of the anus are usually positive for CK7 and negative for CK20, CDX2, and CK5/6 which helps to differentiate them from adenocarcinomas of colon origin [135, 137, 138].

Mucinous adenocarcinomas of appendix origin can be distinguished from mucinous colorectal carcinomas with immunostaining for CK7 and MUC markers [139–141].

Pancreas is composed of glandular/ductal, acinal epithelium, and endocrine cells. Pancreatic neoplasms can be roughly divided into two categories of exocrine and endocrine system neoplasms. This part mostly discusses the exocrine system and mostly adenocarcinomas of this area. Additionally, tumor suppressor genes including DPC4 and SMAD4 are inactivated in about 50–60 % of the adenocarcinomas of this site [116, 142, 143]. Immunoprofile of normal pancreas and some pancreatic tumors are summarized in Tables 26.17 and 26.18. Figure 26.22 depicts solid pseudopapillary neoplasm.

Gastrointestinal stromal tumor (GIST) is a soft tissue tumor of the GI wall which is in the differential diagnosis of leiomyoma and fibromatosis. Most GISTs express c-kit (>95 %), CD34, and CD99 (Fig. 26.23). Sometimes weak positivity for S100, SMA, desmin, and synaptophysin (but not chromogranin) can also be found [135, 144, 145].

Neuroendocrine tumors arise from different organs. Most have similar morphology and tumor marker expression, and the most important diagnostic clues are histologic features, as well as immunostaining for synaptophysin, chromogranin, and NSE (Fig. 26.24). In addition to the mentioned markers, most of neuroendocrine tumors can express the tissue markers in which they originated which help to diagnose the origin of metastatic neuroendocrine tumors [143, 146–148].

Renal cell carcinoma (RCC) is the most common tumor of the kidney with variants of clear renal cell carcinoma (CRCC), papillary renal cell carcinoma (PRCC), and chromophobe carcinoma (CC). Commonly used immunohistochemical Abs in the urinary system are summarized in Table 26.19. Immunohistochemistry is an ancillary test used to distinguish variants of RCC as well as tumors with histopathologic similarities including collecting duct carcinoma and urothelial carcinoma of the renal pelvis. Carcinomas with clear cell feature include CRCC (Fig. 26.25), papillary renal cell carcinoma, and transitional (urothelial) cell carcinoma of the renal pelvis. Differential diagnoses of carcinoma with oncocytic appearance are chromophobe carcinoma, oncocytoma, and oncocytic papillary RCC (Fig. 26.26) [149–154]. The immunophenotype of collecting duct carcinoma is 34βE12⁺, CD10⁻, and AMACR⁻, in contrast to PRCC which is 34βE12⁻, CD10⁺, and AMACR⁺ [150, 155]. Considering the histopathologic pattern, the following immunopanels (Tables 26.20 and 26.21) compare the immunohistochemical Abs in these tumors.

Normal urothelium exhibits a unique pattern of cytokeratin expression characterized by coexpression of simple epithelium cytokeratin (CK7, CK20, and CAM5.2) and HMWCK (CK5/6 and 34βE12). While CK20 is expressed in umbrella cells of the normal urothelium, in dysplastic urothelium and carcinoma in situ, it is expressed in all layers of the urothelium [150–154, 168, 169]. CD44 is expressed in the basal layer of normal urothelium and shows focal staining of basal layers of the dysplastic urothelium [170]. Urothelial carcinomas are divided into (1) noninvasive papillary carcinoma and (2) invasive carcinoma which can appear as papillary or non-papillary itself (Fig. 26.27). Immunohistochemistry can be helpful to differentiate urothelial carcinoma from direct extension of an adjacent primary carcinoma (prostate, colorectal, cervix, and uterine) as well as metastasis and also to distinguish variants of urothelial carcinoma. Common immunohistochemistry Abs in normal urothelium, urothelial hyperplasia, urothelial dysplasia, and urothelial carcinoma are summarized in Table 26.22.

The most important and also frequent cervix cancers are cervix SCCs and adenocarcinomas. Cervix SCC markers are similar to those seen in SCCs of other origins. p16 is a unique marker expressed in tumors of the cervix which can help in differentiating this lesion from the same counterparts from uterine or other origins. Adenocarcinomas of the cervix also express most adenocarcinoma markers. One of the advantages of IHC is to differentiate adenocarcinomas of the cervix from the endometrium. Cervix adenocarcinomas usually express p16 and CEA, and are negative for vimentin and ER, whereas endometrium adenocarcinomas have a reverse expression pattern [176–181].

As other organs, various malignancies can occur in these two organs, but similar to cervix, the most common cancer of these two sites is SCC, with IHC marker expression similar to cervix counterparts [182, 183].

Uterine tumors are of myometrium or endometrium origin. The myometrial tumors are usually sarcomas and were discussed in the sarcoma section. The endometrium may develop various cancers, but the most frequent one is endometrial adenocarcinoma. Endometrial adenocarcinoma has some variants in which endometrioid adenocarcinoma is the most frequent one. Endometrioid adenocarcinoma usually expresses CK7, CA125, ER, PR, and vimentin but is negative for CEA, CK20, and p16. Some endometrial carcinomas express Her2/neu marker, which along with ER and PR markers can be used in targeted therapies [176–181, 184–187].

Except the intestinal type of mucinous adenocarcinoma, all primary ovarian carcinomas are CK7 positive and CK20 negative (Fig. 26.28). This can be used in differentiating primary ovarian carcinoma from metastatic tumors [139–141, 180, 188–191]. The immunophenotype of primary ovarian tumors is described in Table 26.23.

Breast cancer is one of the most common malignancies with various histopathological types; however, adenocarcinomas and its two subtypes including invasive ductal (IDC) and lobular carcinomas (ILC) comprise the majority. Most breast cancers including IDC and ILC are positive for mammaglobin, GCDFP15, ER, and PR, and some are positive for Her2/neu markers. Additionally, epithelial tumor markers, CK (especially CK7) and EMA, are also positive in these tumors [192–197]. The lack of reaction with myoepithelial markers is in favor of an invasive carcinoma. Both normal (Fig. 26.29) and proliferative glands (Fig. 26.30) and ductal carcinoma in situ (Fig. 26.31) exhibit reactivity with myoepithelial markers. Application of p63 and calponin or p63 and SMA is a good way to evaluate the presence of myoepithelial cells [192, 198]. Immunoprofile of normal breast glands and breast cancers are summarized in Tables 26.24 and 26.25 (Figs. 26.32 and 26.33).

Prostate gland is composed of two layers, epithelium and basal cell layer. Normal prostate epithelium exhibits immunoreactivity with prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), prostate-specific acid phosphatase (PSAP), prostein (P501S), and α-methylacyl-coenzyme-A racemase (AMACR) enzyme, whereas prostate basal cells display immunostaining with HMWCK (34βE12), p63, and S100A6 (Fig. 26.34) [149–154]. Immunolabeling for basal cell markers is usually used in a mode of “negative” diagnostic marker in order to show the absence of basal cells in prostate carcinoma (Fig. 26.35). Basal cell cocktail is a mixture of basal cell markers (HMWCH and p63 or CK5/6 and p63) used to highlight the presence of basal cells in normal glands which differentiates benign lesions from prostate intraepithelial neoplasia (PIN) and prostate adenocarcinoma [201]. Metastatic carcinoma of prostate origin exhibits reactivity to CK 7 and CK20 as well as PSA (Fig. 26.36). Table 26.26 summarized the immunoprofile of normal prostate glands as compared with PIN and adenocarcinoma.

Testicular tumors are classified into germ cell tumors and sex cord stromal tumors. Germ cell tumors are the most common type with classic seminoma subtype comprising the majority. The definite diagnosis of these tumors is dependent on proper application of the immunohistochemistric markers and histopathologic evaluation of the biopsy (Figs. 26.37, 26.38, and 26.39). Table 26.27 summarized the immunophenotype of testicular tumors.