Location of primary tumor is often a challenging question, which may take a good deal of resources to find the right answer especially in cases with limited and sometimes not fully representative biopsy material.

The following approach may help in difficult situations if tumors show uncharacteristic morphology (I) or tumors with morphology of adenocarcinoma (II).

1. Study the morphology of the tumor carefully.
2. Clinical information including sex, age, previous medical history and location of the biopsy will help you to plan your strategy.
3. Check if your morphologic impression fits the clinical data.
4. Decide if you need immunohistochemistry to give your final answer.
5. Always remember to secure sufficient number of unstained sections for further investigations at the beginning of process especially for small biopsies.
6. In case when the tumor shows uncharacteristic (anonymous) morphology which you cannot associate with the primary location, the most rational choice seems to start with wide spectrum antibodies. Use a wide spectrum PanCytokeratin cocktail (it should detect as many as possible classes of cytokeratins), S100, CD45 and optionally Vimentin at the first step. Replacing S100 with SOX10 may have rational arguments, but one has to be aware of differences in the spectrum of reactivity of both antibodies. The combined results of staining usually help narrowing the number of diagnostic choices (see below).
7. The suggested wide spectrum primary panel may be completed with some other antibodies (i.e. SALL-4, Synaptophysin/INSM1, EMA, Melan A, CD30 etc.) if you have clinical or morphological indication or wait for the results of the first round of staining and then apply the extended panel of antibodies as the next step.
8. In case the tumor shows more characteristic morphology for the tumor type or primary origin decide what immunostaining may be of relevance for your final diagnosis and what markers can be useful to support therapeutic decisions.
9. Majority of tumors of unknown origin are metastatic adenocarcinomas. In such cases staining for Cytokeratin 7 and Cytokeratin 20 may be considered as the first step in your investigation (see below).

I. Tumor with “uncharacteristic” morphology

When reading the results of wide spectrum panel it is worth to remember, that:

• PanCytokeratin staining is not always equal with epithelial origin of the tumor, since many other tumor types are also positive.
• S100 protein stains not only melanocytes, neural, chondroid and lipomatous tissues but also some other tumor types including carcinomas, many myoepithelial tumors and lymphomas.    
• CD45 is a reliable hematolymphoid marker when positive, but it is often negative in majority of anaplastic large cell lymphoma (ALCL), most of plasma cell proliferations as well as in some cases of acute leukemia with lymphoid or myeloid differentiation. Slightly less than half of dendritic cell sarcomas are also negative.
• Vimentin is a considered nonspecific marker, but in combination with other markers may be sometimes useful in limiting the number of cases.
• CD45+ usually indicates hematolymphoid origin and is often associated with Vim+.
• CD45+/S100+ profile is often associated with dendritic cell tumors and T-cell lymphomas.
• CD45+/PanCytokeratin+ profile indicates usually aberrant expression of cytokeratins in hematolymphoid tumor. Only exceptional cases of carcinoma show aberrant expression of CD45.
• Combined expression of PanCytokeratin and Vimentin (PanCK+/Vim+) characterizes quite heterogeneous group of tumors including carcinomas (most commonly thyroid, endometrium, kidney, poorly differentiated tumors and sarcomatoid types), mesothelioma, myoepithelial tumors, chordoma and some sarcomas (i.e. epithelioid sarcoma, synovial sarcoma, leiomyosarcoma).
• PanCK+/Vim- profile is seen in most of carcinomas, particularly pancreas, prostate, breast, small cell carcinoma and NUT midline carcinoma. Around halv of the cases of endometrioid carcinoma of the ovary is also negative for Vimentin.
• PanCK+/S100+ profile is common in carcinomas of the ovary, thyroid, kidney, poorly differentiated breast carcinoma, myoepithelial tumors, …
• PanCK-/Vim+ profile speaks in favor of non-epithelial tumors and some types of epithelial tumors including adrenal cortical carcinoma, epithelial-myoepithelial and sarcomatoid/spindle cell carcinoma of ENT origin, solid pseudo papillary tumor of pancreas, ….
• PanCK-/S100+ profile is often seen in melanocytic, neural, chondroid and lipomatous tumors.
• With PanCK-/S100+/Vim+ profile the possibility of malignant melanoma must be always excluded.

II. Tumors with mophology of adenocarcinoma

Tumors showing more or less characteristic morphologic pattern of adenocarcinoma used to show variable expression of Cytokeratin 7 and Cytokeratin 20. Staining for these cytokeratins helps to subgroup primary carcinomas into one of four following expression patterns: CK7-/CK20-, CK7+/CK20-, CK7+/CK20+ and CK7-/CK20+. One should keep in mind that some mesenchymal tumors with epithelioid morphology (i.e. synovial sarcoma, epithelioid sarcoma) in addition to deceptive morphology may also express CK7 or/and CK20.

Identification of primary location will benefit additionally with staining for selected “organ specific” markers (e.g. PSA, Thyroglobulin) or lineage markers including selected transcription factors (e.g. CDX2, PAX8 or TTF1).