Carcinoma of Unknown Primary Site

"Carcinomas of unknown primary" present in an enlarged lymph node, as nodules in the liver, or as a malignant ascites or pleural effusion, most commonly. In the vast majority of these cases, immunohistochemical studies can identify the most probable primary site, rule out certain potential primary sites, or at least narrow the field of potential primary sites to a short list.

Harnessing the power of IHC techniques in this setting requires a comprehensive knowledge of the available markers, and based on their relative sensitivities and specificities, the most appropriate markers to run in a given clinicopathologic setting.

There are three classes of antibody markers that can be of assistance in analyzing carcinomas of unknown primary: (a) antibodies to cytokeratins and cytokeratin subsets; (b) antibodies to tumor or cell type specific markers, recognizing that this 'specificity' is generally not absolute; (c) antibodies to subset markers, i.e., proteins expressed in clusters of carcinomas. In given clinical situations, antibodies from more than one of these classes are employed.

• Antibodies to Cytokeratins
• Antibodies to Singular Carcinoma Markers
• Antibodies to 'Subset' Markers

Antibodies to Cytokeratins

Cytokeratins are a complex set of at least 20 proteins, expression of which is the hallmark of epithelium and their corresponding tumors-carcinomas. Experience has taught us that the preferred approach to employing anti-cytokeratin antibodies is as follows:

It is useful to employ a 'pan keratin' anti-cytokeratin antibody, if necessary, to confirm the diagnosis of carcinoma in histologic settings where this diagnosis is not obvious. We generally employ the OSCAR anti-cytokeratin monoclonal antibody, developed by PhenoPath Laboratories' scientists, which performs similarly to the AE1/AE3 monoclonal antibody cocktail, which together identify many (but not all) of the 20 members of the cytokeratin protein family. Anti-'pan cytokeratin' antibodies can confirm that a tumor is a carcinoma, but cannot provide additional evidence of possible primary site.

Cytokeratins 7 and 20 are two individual cytokeratin proteins that have a partially overlapping but unique distribution among normal epithelium and corresponding carcinomas. When studied 'coordinately,' cytokeratins 7 and 20 expression patterns can help guide the identification of the most likely primary site. Thus, hepatocellular carcinomas generally display a CK7-negative, CK20-negative immunophenotype, and carcinomas of lung metastatic to liver generally display a CK7-positive, CK20-negative immunophenotype, while carcinomas of the colorectum metastatic to liver almost always display a CK7-negative, CK20-positive immunophenotype.

It is important not to base interpretation of the most likely primary site solely on the CK7 and CK20 results, as the table below summarizes the 'modal' CK7/CK20 immunophenotypes, i.e., those most commonly encountered in a given tumor. For example, while the CK7+/CK20- immunophenotype is seen in most breast carcinomas, 10% of these tumors can display a CK7+/CK20+ immunophenotype.

Antibodies to Singular Carcinoma Markers

These are a set of antibodies to proteins expressed in selected carcinomas. While many of these markers, when first discovered, were initially referred to as 'site-specific' markers (as indicated in names such as 'thyroid transcription factor-1', or TTF-1), with time, the absolute specificity of most of these markers has been called into question. (For example, TTF-1 is expressed in both thyroid and lung carcinomas, as well as neuroendocrine carcinomas of various organs.) However, when used in the context of other markers and with the knowledge of their generally limited cross reactivity, this class of markers can prove highly informative as to primary site. There are two major categories of these singular markers: (a) cytoplasmic or membranous markers, usually a 'terminal differentiation' marker of a given organ or cell type, and (b) nuclear transcription factors, which are nuclear 'switch proteins' that control downstream expression of cytoplasmic and membranous markers. Some important cytoplasmic or membranous 'singular' carcinoma markers include: villin, an excellent marker of GI adenocarcinomas, including gastric and pancreatic carcinomas; mammaglobin, and GCDFP-15, which are sensitive and specific markers of metastatic breast cancer; and the HepPar1 antigen, a novel marker of hepatocellular carcinoma. Important nuclear transcription factors useful as singular carcinoma markers include: (a) thyroid transcription factor-1 (TTF-1), a very sensitive and specific marker of lung and thyroid carcinomas; (b) CDX-2, a marker of colorectal adenocarcinomas but also a subset of adenocarcinomas of the stomach and pancreatobiliary tree; (c) Wilms tumor gene product (WT-1), in the appropriate clinical setting, an excellent marker of serous papillary ovarian carcinoma.

Villin A GI-associated protein which displays a characteristic 'brush border' pattern, is demonstrated in a case of metastatic pancreatic carcinoma.	HepPar1 This cytoplasmic antigen is an excellent marker of hepatocellular carcinoma, and is more sensitive than 'traditional' markers such as alpha fetoprotein.

Antibodies to 'Subset' Markers

The most frequently employed markers that can help identify carcinoma subsets include the following:

Antibodies to ER and PR: Estrogen and progesterone are expressed in a subset of breast cancer cases, and can thus be helpful in distinguishing metastatic breast cancer from tumors in which ER and PR expression is rarely, if ever, seen, e.g., primary lung carcinomas. However, ER and/or PR have been found to be expressed in a number of 'unexpected' settings, e.g., papillary carcinomas of the thyroid and even carcinoid tumors, and cannot be used as 'singular' markers of breast cancer.

p63: This is a nuclear transcription factor, expression of which is largely confined to carcinomas showing squamous, transitional, or myoepithelial differentiation. Thus, metastatic squamous cell carcinomas of all organs, metastatic bladder carcinomas, and many salivary gland carcinomas show strong p63 expression.

Synaptophysin and chromogranin A: These are cytoplasmic markers of neuroendocrine differentiation, expressed across the entire spectrum of neuroendocrine carcinomas, from well differentiated tumors (e.g., carcinoid tumor, in which chromogranin A is highly expressed) to poorly differentiated neuroendocrine carcinomas (e.g., small cell lung carcinoma, in which synaptophysin expression is more common).

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