Diagnosis of Metastatic Neoplasms: An Immunohistochemical Approach

Determination of the type and origin of metastatic tumors is an important and potentially challenging area in pathology. The site of origin is best determined by correlating clinical and pathologic findings; however, in the absence of a clinically known or suspected primary site, morphologic and immunohistochemical evaluations are key to determining the tumor lineage and origin.1 While routine microscopy may reveal characteristics that are diagnostic or suggestive of the lineage and/or origin (eg, mucin, melanin, keratin, clear cell change, bile production), absence of morphologically distinctive features often necessitates the use of immunohistochemistry, particularly in poorly differentiated malignancies. Even if a basic tumor lineage is apparent on routine hematoxylin-eosin stain, confirmation of a site of origin (eg, lung, colon) may be clinically important. Tumor characterization is often performed on limited tissue samples obtained by cytologic specimens or core biopsies, underscoring the need for a strategic approach to use of immunohistochemistry.2 

This review outlines the use of immunohistochemistry in the diagnosis of metastatic tumors, both in the context of confirming a clinically suspected site of origin as well as a tumor of unknown origin. It includes review of relevant current markers that may be used and of differential diagnostic considerations based on presence or absence of staining. It must be emphasized that no marker is entirely specific; thus, use of a carefully constructed panel of markers is important. Immunohistochemistry and other ancillary techniques should not diminish the role of clinical information and careful gross and routine histologic evaluation.

The first step in the evaluation of a metastatic tumor is the determination of tumor lineage (eg, epithelial, mesenchymal, melanocytic). Markers for lineage determination should include a panel with expected positive and negative staining in the different lineages under consideration. For example, to confirm the diagnosis of metastatic carcinoma, a reasonable panel may consist of epithelial markers such as keratins (expected positive) and melanocytic markers such as S100 and HMB-45 (expected negative).

Markers useful in confirming epithelial differentiation include low-molecular-weight cytokeratins recognized by CAM 5.2 and 35BH11 and broad spectrum cytokeratins (pankeratin) recognized by AE1/AE3 antibody. Cytokeratin (CK) expression may sometimes be seen in nonepithelial tumors such as melanomas and sarcomas, and CK alone does not distinguish a carcinoma from mesothelioma.3,4 This underscores the utility of a panel of immunostains. Conversely, absence of CK does not always exclude a carcinoma; for example, adrenal cortical carcinomas are often negative for CK, and hepatocellular carcinomas (HCCs) are often negative for pankeratin. Epithelial membrane antigen (EMA) may be used in conjunction with keratins; however, it is not specific to epithelial differentiation and may be expressed in normal and neoplastic hematolymphoid cells including plasma cells and anaplastic large cell lymphomas.5 Epithelial membrane antigen is usually absent in medullary thyroid carcinoma and adrenal cortical carcinoma. MOC-31 stains most adenocarcinomas, and CK5/6 stains most squamous cell carcinomas and mesotheliomas.

There is no reliable positive screening marker for confirming mesenchymal differentiation. While vimentin has been used to support a mesenchymal lineage in tumors, it is coexpressed with keratins in some carcinomas and mesothelioma, and it is also expressed in melanomas.6,7 Therefore, to confirm mesenchymal differentiation, it is important to exclude other lineages by incorporating appropriate markers in the panel together with vimentin.

S100 is the most sensitive screening marker for primary and metastatic melanomas (>95%).8,9 However, S100 is not specific for melanomas and can also be seen in some mesenchymal and epithelial tumors.10 Both nuclear and cytoplasmic staining should be present for the staining result to be interpreted as positive. To confirm the presence of melanoma, S100 should be combined with 1 or more markers with higher specificity, such as HMB-45, Melan-A/MART-1, tyrosinase, or microphthalmia transcription factor protein.11–15 Compared to S100, these markers are less sensitive for the diagnosis of melanomas and stain less than 10% of desmoplastic/spindle cell melanomas.16 Melan-A also stains adrenal cortical neoplasms and other steroid-producing tumors.17 Melan-A and HMB-45 commonly stain perivascular epithelioid cell tumors (PEComas), with less frequent staining for tyrosinase and microphthalmia transcription factor protein.18 

Distinguishing mesothelioma from metastatic adenocarcinoma is important and can be successfully achieved by using a panel of markers for both tumor types. There are no consensus-based guidelines on the choice of markers.

Positive markers that are useful for mesothelioma but not entirely specific include calretinin, CK5/6, and Wilms tumor gene product (WT1). Calretinin is a sensitive marker, frequently expressed (>80%) in epithelial and sarcomatoid subtypes.19 Staining is both nuclear and cytoplasmic. Other tumor types may also be immunoreactive for calretinin, including sex cord–stromal tumors, adrenal cortical tumors, and a minority of adenocarcinomas and squamous cell carcinomas.19 Cytokeratin 5/6 and WT1 both stain more than 75% of epithelial mesotheliomas but are frequently absent in sarcomatoid mesotheliomas.20 Cytokeratin 5/6 also stains most squamous cell carcinomas, and WT1 stains most ovarian serous carcinomas, Wilms tumors, and desmoplastic small round cell tumors. Other positive markers that can be used for mesothelioma include HBME-1, thrombomodulin, mesothelin, D2-40, and podoplanin.21,22 

Markers for adenocarcinoma include carcinoembryonic antigen (CEA), MOC-31, thyroid transcription factor 1 (TTF-1), Ber-EP4, B72.3, and CD15 (Leu-M1). These markers have a high specificity for adenocarcinomas relative to mesotheliomas but may also variably stain squamous cell carcinomas.19 Thyroid transcription factor 1 is highly specific for adenocarcinoma of lung if thyroid carcinoma is excluded, and both CD15 and TTF-1 have lower sensitivity compared to the other markers.

Leukocyte common antigen (CD45) is a useful screening marker for hematolymphoid neoplasms, with a sensitivity and specificity greater than 95%. Rare examples of undifferentiated or neuroendocrine tumors have been reported to be positive for CD45. Additional markers that could be used include terminal deoxynucleotidyl transferase, CD3, CD20, CD30, CD43, CD21, or CD23 and myeloperoxidase, depending on the morphologic features.23,24 Subclassification of metastatic hematolymphoid malignancies is beyond the scope of this review.

Placental alkaline phosphatase is a membrane-bound isoenzyme that is produced by placental syncytiotrophoblasts and many neoplasms.25–27 Among germ cell tumors, it is expressed in nearly all (>95%) seminomas and embryonal carcinomas, most yolk sac tumors, and variably in choriocarcinomas. Placental alkaline phosphatase is also expressed in some nongerm cell carcinomas, including serous carcinomas, and is therefore not specific for germ cell tumors.27,28 

α-Fetoprotein is an oncofetal glycoprotein that is a useful marker for yolk sac tumors and hepatocellular carcinomas. Most yolk sac tumors are positive with this marker, although staining may be patchy.27 Pure seminomas are negative for α-fetoprotein, and positivity in mixed germ cell tumors identifies yolk sac differentiation.

Human chorionic gonadotropin is a glycoprotein composed of α and β subunits, the latter produced by syncytiotrophoblasts.29 It is therefore a useful marker for choriocarcinoma and also identifies multinucleated syncytiotrophoblastic cells in seminomas, embryonal carcinomas, and yolk sac tumors.

Other markers that are useful include OCT3/4 and CD30. OCT3/4 is a sensitive and specific marker for seminoma and embryonal carcinoma.30 CD30 stains most embryonal carcinomas and can be used as part of a panel of markers to distinguish metastatic embryonal carcinoma from somatic carcinoma.31 

Determination of site of origin of carcinomas is often possible and necessary for adequate clinical management of patients. After a metastasis is determined to be a carcinoma on the basis of screening immunostains, a panel of tissue- or organ-specific markers can be used in an attempt to determine or suggest the origin. Most “tumor or organ-specific” markers may variably react with other tumor types also; hence, for metastatic tumors of unknown origin, the use of a panel of markers is strongly encouraged. For example, to determine whether an adenocarcinoma is of lung or colonic origin, a combination of CK7, CK20, TTF-1, and CDX-2 markers is likely to establish a higher degree of certainty than TTF-1 alone. Table 1 summarizes the key markers for commonly encountered carcinomas and mesothelioma.

A combination of CK7 and CK20 has been shown to provide discrimination between subsets of carcinomas and is often used as part of a panel to establish a primary site (Table 1). Cytokeratins 7 and 19 may be used to distinguish between cholangiocarcinoma (CK7⁺/CK19⁺) and hepatocellular carcinoma (CK7⁻/CK19⁻). AE1/AE3 stains most carcinomas; however, hepatocellular carcinoma is commonly negative, and AE1/AE3 may be used as part of a panel to distinguish between HCC and a metastatic carcinoma. Cytokeratin 5/6 is a useful marker for squamous cell carcinomas, epithelioid mesotheliomas, and urothelial carcinomas. Because of overlap in expression, cytokeratin stains should always be combined with more specific markers if available.

Thyroid transcription factor 1 is a nuclear transcription factor that regulates gene expression in the thyroid, lungs, and diencephalon during embryogenesis. It is expressed by most adenocarcinomas (72%), large cell neuroendocrine carcinomas (75%), and small cell carcinomas of the lung (>90%) (Figure 1, A and B). Squamous cell and large cell carcinomas are less frequently positive for TTF-1, in 7% and 20% of cases, respectively.32 While most pulmonary small cell carcinomas are positive for this marker, TTF-1 may also stain small cell carcinomas from other sites, including those arising from prostate, urinary bladder, and uterine cervix.33 

Other markers for lung primary tumors include napsin A, which stains most pulmonary adenocarcinomas, and a novel marker, ES1.32 These markers are currently not widely used.

Thyroglobulin is a marker that is highly sensitive and specific for follicular and papillary thyroid carcinomas, staining more than 95% of these tumors. Immunoreactivity is also maintained in these tumors at metastatic sites.34 

Thyroid transcription factor 1 is also a useful marker for thyroid tumors, being expressed in more than 90% of follicular cell–derived tumors and medullary carcinomas.35 As described above, TTF-1 also stains carcinomas of lung origin, and rarely, carcinomas from other sites. It is therefore a reliable marker for thyroid differentiation when a lung primary tumor can be excluded, or when used in conjunction with thyroglobulin. Poorly differentiated (insular) carcinomas are focally and/or weakly positive for both thyroglobulin and TTF-1. Anaplastic thyroid carcinomas are usually negative for both markers but commonly coexpress keratin and vimentin.

Calcitonin is a protein secreted by thyroid C cells and is a useful marker for medullary thyroid carcinomas (MTCs). It can also be used to evaluate for C-cell hyperplasia associated with familial MTC.36 Although highly specific, calcitonin staining may be patchy in medullary carcinomas and 5% of these tumors may be negative for this marker.37 In tumors with no staining and still suspected to be MTC, calcitonin and calcitonin gene–related peptide mRNA can be demonstrated by in situ hybridization.38 Carcinoembryonic antigen, chromogranin, and synaptophysin are also expressed by most MTCs, while follicular and papillary carcinomas are negative for these markers. However, they are not specific to MTC and may also stain other neuroendocrine tumors, and thus are less helpful in the setting of metastases. A reasonable panel to confirm metastatic MTC would be chromogranin, synaptophysin, TTF-1, and calcitonin (Figure 2, A through D).

Hepatocyte paraffin 1 (Hep Par 1) stains a cytoplasmic antigen and is highly sensitive for hepatocyte differentiation, thus is useful in the diagnosis of hepatocellular carcinoma (Figure 3, A and B). Expression is particularly strong and diffuse in well-differentiated HCC but decreases in higher-grade tumors. However, Hep Par 1 is not entirely specific for hepatocyte differentiation and may also be seen in other carcinomas, such as gastric signet ring cell carcinomas.39 

Canalicular staining with polyclonal CEA (pCEA) can be used as a specific marker for the diagnosis of hepatocellular carcinomas. This pattern results from cross-reactivity with biliary glycoprotein I and is demonstrable in 90% of HCCs and is not seen with monoclonal CEA.40 Similar to pCEA, villin and CD10 also show a canalicular pattern of staining. Another marker that is specific for hepatocyte differentiation is albumin, which is produced exclusively by hepatocytes. Immunohistochemical staining for albumin is difficult to interpret because of the abundance of this protein in the serum; however, albumin mRNA can be demonstrated by using in situ hybridization. Both canalicular pCEA and albumin are more specific for HCC than Hep Par 1, but like the latter, tumors arising at other sites with hepatoid differentiation (such as hepatoid gastric adenocarcinomas) may also be positive for these markers.41 

A specific marker for cholangiocarcinoma is currently not available, and a diagnosis is established by exclusion of HCC and metastatic carcinoma. This can be achieved by the presence of appropriate clinical and radiographic features and the use of markers that exclude carcinomas from other sites (eg, lung and breast).

CDX-2 is a nuclear transcription factor that is used as a marker for gastrointestinal tract adenocarcinomas and is expressed in most (>95%) colorectal and duodenal adenocarcinomas, with nuclear staining. Variable expression is also seen in adenocarcinomas arising in the stomach, esophagus, pancreas, and biliary tract. Extraintestinal carcinomas that are frequently positive for CDX-2 include urachal adenocarcinomas arising in the urinary bladder and ovarian mucinous carcinomas.42 CDX-2 staining may also be seen in a minority of gastrointestinal and extragastrointestinal neuroendocrine tumors and is therefore less specific in this setting than it is for adenocarcinomas.43 

Villin is a cytoskeletal protein associated with microvilli of the intestine and proximal renal tubular epithelium. It is a marker of gastrointestinal adenocarcinomas, stains most (82%–100%) primary and metastatic colonic adenocarcinomas, and does not appear to be decreased in metastases.23 The specificity of villin is limited by its expression in extraintestinal adenocarcinomas including those arising in lung, ovary, endometrium, kidney, and bladder.44 Like pCEA, villin may also stain HCC with a canalicular pattern.40 

As with primary hepatobiliary adenocarcinomas, a specific marker is not currently available for pancreatic ductal adenocarcinoma. The rare acinar cell carcinoma of the pancreas can be confirmed with stains for pancreatic enzymes such as trypsin, chymotrypsin, lipase, and elastase. It is also commonly negative for CEA, unlike ductal adenocarcinomas.45 

Gross cystic disease fluid protein 15 (GCDFP-15; BRST-2), also known as prolactin-inducing protein, is a glycoprotein present in various body fluids including saliva, milk, and seminal fluid.46,47 It is expressed in cells with apocrine features including those present in breast, axillary and anogenital skin apocrine glands, salivary glands, and Paget disease of skin.48 For a metastatic carcinoma, GCDFP-15 has a high (>95%) specificity for breast primary tumor if the other mentioned sites are clinically excluded. The utility of this marker, however, is somewhat limited by a lower (50%–74%) sensitivity. Therefore, absence of staining does not exclude a breast primary tumor.

Mammaglobin is a marker that is overexpressed in 48% to 84% of breast carcinomas. It is more sensitive but less specific than GCDFP-15 for diagnosis of a breast primary tumor.23,49 

Estrogen and progesterone receptors are not specific to breast and cannot be used as primary markers to support evidence of a breast primary tumor, but may be used in conjunction with GCDFP-15 and mammaglobin.

Prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) are 2 commonly used markers for prostatic carcinoma. Essentially, all low- to intermediate-grade prostatic adenocarcinomas stain for both markers, with reactivity decreasing by 10% to 20% in high-grade carcinomas.50 A combination of PSA and PAP detects most metastatic prostatic carcinomas. Prostate-specific antigen is more specific as a marker for prostate than PAP, although both have also been reported in extraprostatic sites.51,52 Prostate-specific membrane antigen is another marker that is less widely used but demonstrates high specificity for prostate carcinoma and better sensitivity for higher-grade carcinomas than PSA and PAP.53 While α-methylacyl coenzyme A racemase is useful for the diagnosis of carcinoma in the prostate, it is not useful as a specific marker for prostatic carcinoma, as it is expressed in many normal tissues and nonprostatic tumors.

Potential future markers for prostatic carcinoma include NKX3.1 and prostein. NKX3.1 is a nuclear marker that more frequently stains high-grade carcinoma than PSA. It may also be expressed in some other tissues, such as testicular germ cells and lobular carcinoma of breast. Prostein is a transmembrane protein with a high specificity for benign and malignant prostatic epithelium.54 

Renal cell carcinoma (RCC) marker antibody is directed against a normal proximal tubular brush border glycoprotein. It stains most clear cell and papillary renal cell carcinomas, but the sensitivity decreases in high-grade carcinomas and metastatic carcinomas. Only a minority of sarcomatoid renal cell carcinomas are positive for RCC marker.23 

Staining has also been reported in some breast carcinomas and embryonal carcinoma.55 Among the subtypes of RCC, α-methylacyl coenzyme A racemase is a marker for papillary RCC.56 

Renal cell carcinomas are also commonly positive for CD10 and vimentin and negative for CEA; however, these markers are not specific and should always be used in conjunction with other markers.

Uroplakins (Ia, Ib, II, and III) are transmembrane proteins that are specific to urothelial cells. A monoclonal antibody for uroplakin III is highly specific for urothelial differentiation and expressed in urothelial carcinomas and Brenner tumors.57 Use of this marker, however, is limited by low sensitivity, staining up to 60% of urothelial carcinomas at primary sites. Metastatic urothelial carcinomas are less frequently positive for this marker.

Thrombomodulin is a marker that is expressed by most urothelial carcinomas; however, it lacks specificity and therefore should not be used alone as confirmatory evidence for urothelial differentiation. It is also expressed by squamous cell carcinomas, endothelial tumors, and mesothelioma.58,59 

A specific marker for adrenal cortical carcinoma (ACC) is not commercially available. However, ACCs show a relatively specific immunoprofile when a combination of markers are used. They are commonly positive for vimentin, Melan-A, inhibin, and calretinin and negative or weakly positive for keratin.60,61 Adrenal cortical carcinomas may be positive for synaptophysin but are negative for chromogranin. When included as part of a panel, these markers are effective in distinguishing ACC from the usual differential diagnostic considerations, such as pheochromocytoma, hepatocellular carcinoma, and RCC. Melan-A has been primarily used in the diagnosis of melanoma but also reacts with steroid-producing cells of the adrenal cortex. Inhibin A identifies steroid-producing cells, and thus is a sensitive marker for ACC, ovarian granulosa cell tumor, and testicular Sertoli-Leydig cell tumor. Calretinin is a calcium-binding protein that is also commonly used as a marker for mesothelioma and ovarian sex cord–stromal tumors.61 

Carcinomas arising in the female genital tract have overlapping morphologic and staining characteristics, and reliable site-specific markers are not available. A combination of markers may be used to suggest origin in the female genital tract, and differential expression of markers may suggest a site of origin or tumor type. Markers for germ cell tumors are discussed separately in this review.

Endocervical adenocarcinomas are usually positive for CEA but negative for vimentin and for estrogen and progesterone receptors, in contrast to endometrial adenocarcinomas of endometrioid type.62 Use of p16 as a surrogate marker for high-risk human papillomavirus infection to prove endocervical origin has been controversial, as it may also be expressed in endometrial carcinomas, especially endometrial serous carcinomas.63 Distinction between high-grade endometrioid and serous carcinomas is not reliably achieved with the use of markers because of significant overlap.64 

Ovarian nonmucinous carcinomas are positive for CK7 but usually negative for CK20 and monoclonal CEA. Mucinous carcinomas are positive for CK7, CK20, and monoclonal CEA.65,66 Inhibin and calretinin are markers for sex cord–stromal tumors, and as mentioned above, also stain adrenal cortical tumors. Wilms tumor gene product (WT1) stains most serous ovarian carcinomas but usually not breast, gastrointestinal tract, and pancreaticobiliary carcinomas.67 It also commonly stains mesotheliomas and therefore should be used as part of a panel of markers. Other tumors that are positive for this marker include Wilms tumor and desmoplastic small round cell tumor.68,69 Metastatic choriocarcinomas can be confirmed with positive staining for human chorionic gonadotropin.

Sarcomas are composed of a complex and heterogeneous group of tumors that are classified by cell lineage. The first step in the approach to diagnosis of these tumors is to confirm the mesenchymal lineage, followed by an attempt to define the subtype. Subtyping will require a panel of markers and may not be achieved with the currently available markers in all cases. The importance of precise subtyping will also depend on its clinical relevance and available therapeutic options, such as therapy with imatinib mesylate for gastrointestinal stromal tumors (Figure 4, A and B).70 Metastatic sarcomas are commonly high grade, variable in histomorphology, and include tumors with epithelioid, spindle cell, pleomorphic, and small round cell appearance. The immunohistochemical characteristics of a select group of sarcomas are summarized in Table 2, with emphasis on reactivity with key markers.70,71 

Small round cell tumors are a heterogeneous group of tumors that contain morphologically undifferentiated small cells with high nuclear to cytoplasmic ratio and inconspicuous nucleoli. Considerations in the differential diagnosis include small cell carcinoma, Merkel cell carcinoma, Ewing sarcoma/primitive neuroectodermal tumor, poorly differentiated synovial sarcoma, hematopoietic malignancies, rhabdomyosarcoma, desmoplastic small round cell tumor, and neuroblastoma. Metastases from these tumors are often difficult to classify without the use of a panel of markers (Figure 5, A through F). Typical immunoprofiles of these tumors are summarized in Table 3.23,72 

Characterization of type and origin of metastatic tumors requires judicious use of lineage and organ-specific tissue markers. No marker is entirely specific, and for optimal patient care, this tool should be used in the context of the clinical and radiographic findings and careful routine morphologic evaluation.

The author has no relevant financial interest in the products or companies described in this article.