Immunohistology of Carcinoma of Unknown Primary Site

Cancer of Unknown Primary Site: Clinical Aspects and Economic Considerations

Patients who present with CUPS by definition have no obvious identifiable primary site despite a careful clinical history, physical examination, radiologic imaging, and biochemical or histologic investigations. In some such patients, radiologic imaging shows multiple liver or lung metastases without a clear visceral primary; in other patients, the disease is sufficiently disseminated such that there is no clear dominant lesion. Studies of patients with malignancies have shown that CUPS accounts for 5% to 15% of all patients who present with a malignancy. ^, The impact of recent improvements in radiologic imaging has reduced this percentage to 3% to 7% of patients who present with a CUPS diagnosis. ^{, ,}

The clinical presentation in a CUPS case depends on a number of factors including age, gender, sites of involvement, and line of differentiation (viz., epithelial, mesenchymal, lymphoid, germ cell, melanocytic). It appears that the tumors that present as CUPS are biologically and clinically different from the known primary tumors that metastasize several years after diagnosis. CUPS patients fail to show any symptoms related to the primary tumor and demonstrate an unpredictable pattern of spread (i.e., a difference in frequency of involvement of a particular site than would be expected of a known primary tumor). The majority of CUPS cases present with multiple sites of involvement with a few presenting with only one or two sites of involvement. Based on the sites of involvement, several clinicopathologic entities have been characterized that are helpful in identifying the primary site.

The liver is one of the single largest repositories for metastatic malignancies of all types, especially for carcinomas. The most common malignancies metastatic to the liver are from the gastrointestinal (GI) tract, with colorectal carcinomas leading this group. Lung and breast carcinomas also commonly metastasize to the liver, as do pancreaticobiliary carcinomas. This entire group of adenocarcinomas may appear similar to primary cholangiocarcinoma of the liver and may simulate some hepatocellular carcinomas, particularly poorly differentiated hepatocellular carcinomas. Prostate carcinoma, although unusual, does metastasize to the liver and can also be confused with cholangiocarcinoma. Thus, for hepatic metastases of unknown primary in women, colorectal, breast, and lung carcinomas are of primary consideration, whereas in men, colorectal, lung, and prostate carcinomas top the list. Malignant melanoma metastatic to the liver is not uncommon, with the highest frequency of liver metastases seen with primary eye melanomas. Pisharodi and associates, in a fine needle biopsy study of 200 malignant aspirates of the liver, found that 32% were hepatocellular carcinomas, 49.5% were readily diagnosed as metastatic carcinomas, and 18.5% were problematic. Of this latter group, IHC contributed to definitive diagnosis in half of the cases.

Along with the liver, the lung is a major repository for metastatic carcinomas, especially adenocarcinomas. Identification of the origin of an adenocarcinoma in the lung is a frequent, difficult, and challenging process for the surgical pathologist because adenocarcinomas not only are the most frequent primary lung tumor, but they are also the most common metastatic tumor found in the lung. Distinction among these tumor types can be especially challenging on scant biopsy materials, such as transbronchial biopsy or fine needle aspiration biopsy (FNAB). Clinical information regarding circumscription and the number of lesions is quite useful. Metastatic tumors often present as multiple circumscribed nodules, whereas primary lung tumors often show a dominant infiltrative lesion, sometimes accompanied by a “ground glass” (i.e., in situ) component. It is important to identify those carcinomas that can be treated by chemotherapy, targeted therapy, or hormonal manipulation, especially metastatic breast, lung, or prostate carcinomas.

In the brain, distinguishing metastatic adenocarcinoma or poorly differentiated carcinoma from a glial tumor is straightforward, although determining the source of the metastasis may be problematic, especially when the occult primary is unknown. ^, Lung carcinomas are the most likely primary to be discovered subsequent to central nervous system (CNS) presentation, and other common primaries include breast, kidney, thyroid, and GI tract. Patients with other adenocarcinomas, such as ovarian, prostatic, and pancreaticobiliary, rarely present with brain metastases, because there is almost always evidence of widespread dissemination of these tumors before the development of cerebral metastases. The site of origin of carcinoma remains unknown in up to 5% of patients. The survival of most patients with carcinomatous brain metastases is in the range of 3 to 11 months.

Patients presenting with skeletal metastases often have primary carcinomas in the lung, breast, kidney, or urogenital region, and imaging studies have been particularly useful in elucidating the primary tumor.

For patients who present with pleural effusions, the breast is the most common primary site for women, with the lung being the most common site for a primary tumor in men. Lymphomas are seen in both sexes.

In women who present with malignant abdominal effusions (malignant ascites), common abdominal sites include müllerian primary (often fallopian tubes and ovaries), whereas men with malignant ascites typically have primary tumor sites in the GI tract, predominantly in the colon, rectum, pancreas, or stomach. Patients with peritoneal carcinomatosis of non-gynecologic origin most often have origins in the stomach, colon, or pancreas and have a median survival of 3 months.

For patients who present with primary lymph node metastases, there may be clues to the primary site of the tumor based on tumor morphology (adenocarcinoma, squamous cell carcinoma, or undifferentiated carcinoma) and the anatomic site of lymph node involvement.

In women presenting with adenocarcinoma in the axilla, the primary tumor is most often found in the ipsilateral breast. For the patient who presents with metastatic adenocarcinoma in the neck, the metastatic workup will begin in the lung (males) or breast (females), although GI and prostate adenocarcinomas both show a predilection for the left side of the neck.

Undifferentiated carcinomas of the head and neck are the most common primary source for metastatic tumors in head and neck lymph nodes, and the majority of these are of squamous mucosal derivation. The prognosis for this group of patients rests largely on the nodal status, with patients having stage N3 lesions carrying a poor prognosis. For a squamous cell carcinoma involving the upper and mid-cervical lymph nodes, a thorough examination of the oropharynx, hypopharynx, nasopharynx, larynx, and upper esophagus by direct vision and fiberoptic nasopharyngolaryngoscopy with biopsy of any suspicious areas is more valuable than further pathologic examination. Advanced diagnostic techniques such as computerized tomography (CT) and positron emission tomography (PET) scans are helpful in determining the primary site. Occasionally, systematic random biopsies of mucosal sites such as nasopharynx, base of tongue, pyriform sinus, and tonsils may reveal an occult tumor. Metastatic squamous cell carcinoma involving lower cervical lymph nodes or at any other site except inguinal nodes is highly suspicious for a lung primary. In addition, occasionally, esophageal squamous cell carcinomas may preferentially involve the lower cervical lymph nodes. The vast majority of patients with squamous cell carcinoma involving the inguinal nodes generally have detectable primary tumor in the anogenital area. Therefore all women must undergo a thorough evaluation of the vulva, vagina, and the cervix, and all men should undergo careful inspection of the penis. Both sexes must also have examination of the anorectal region.

The current clinical approach is an attempt to identify favorable prognostic groups in patients with unknown primary tumors so that they can be managed appropriately. This group of tumors includes leukemia/lymphoma, germ cell tumors, small cell carcinoma of the lung, and carcinomas of the breast, ovary, endometrium, adrenal gland, thyroid, and prostate. ^{, ,} When possible, it is useful to separate regional from distant metastases, because locoregional disease is more amenable to treatment. ^, Other favorable clinical features that have been described include location of tumor in the retroperitoneum or peripheral lymph nodes, tumor limited to one or two metastatic sites, a negative smoking history, and young age.

Kirsten and colleagues studied 286 patients with CUPS and concluded that the factors that predicted survival were lymph node presentation, good performance status, and body weight loss of less than 10%. Using a panel of antibodies to determine differentiation of tumors in 41 patients with CUPS, Van der Gaast and associates concluded that the immunohistochemical panel approach to uncover tumor origin is useful for selecting appropriate treatment of patients, especially those who may benefit from combination chemotherapy. ^, Other immunohistochemical studies of CUPS have elucidated the origin of tumors in as few as 5% to as many as 70% of patients. However, most investigators have arrived at the same conclusion: for individual patient therapy, knowledge of site of origin improves patient survival. ^, Furthermore, with the advent of targeted therapy, the benefit of identifying the site of origin may be manifold. With the incorporation of next-generation sequencing in diagnostic testing, it may become less important to identify the site of origin and more useful to find actionable targets for an individual patient. However, finding the site of origin may still have genetic implications and patient and family need to know the tumor source.

The appropriate workup for identifying a primary tumor depends on the patient’s clinical symptoms, age, history, gender, and the likelihood of finding the primary tumor. Patients with CUPS do poorly as a group, with a median survival of 6 to 11 months, and the importance of establishing the origin of the primary site guides therapeutic interventions of hormonal manipulation, chemotherapy, and radiation. The clinician must also take into account the economics of an extensive clinical workup, as well as the inconvenience and discomfort to which the patient is subjected.

The economic considerations of clinical workup in these patients have not been extensively studied. There are few data available on the cost-effectiveness of IHC in surgical pathology. Schapira and Jerrett analyzed the clinical workups in a group of 199 patients and concluded that the search for a primary neoplasm incurred an average cost of US$17,973, with only 19.6% of patients surviving for more than 1 year. As a matter of fact, IHC is probably undervalued and is likely a cost-effective maneuver in the study of CUPS. Radiologic studies themselves have limited value in the management of these patients, and prognosis is not affected. ^, Even autopsies on some of these patients may not detect the primary tumor site because of small size, extensive dissemination, or regression due to therapy. In 1988, Le-Chevalier and coworkers studied 302 autopsy specimens from patients who presented with CUPS. The primary tumor site was located premortem in 27% of patients, at autopsy in 57% of patients, and remained unidentified in 16% of patients. The most common primary tumor sites in this study included pancreas, lung, kidney, and colon/rectum, a list that includes the two malignancies with the highest incidence in both men and women.

Diagnostic Approach to the Study of Cancer of Unknown Primary Site: Specimen Preparation

The goal of the surgical pathologist is to identify the line of differentiation of the tumor and identify those tumors that are within the “treatable” group of tumors, namely, carcinomas of breast, prostate, ovary, endometrium, and thyroid, as well as germ cell tumors and neuroendocrine carcinomas. Hormonal and antihormonal therapies are useful for patients with breast and prostate carcinomas. Neuroendocrine, thyroid, and germ cell tumors may be responsive to suppression by chemical agents. The therapeutic response of other carcinomas is less certain, but the identity of the carcinoma, if available, is useful to determine more useful therapeutic regimens for these patients prospectively. ^{, , ,} Some studies on patients with CUPS demonstrate that up to one-third respond to taxane-based therapies. ^,

Tissue procurement is the first step in the workup for tumors of unknown primary origin, and it is a common practice to obtain tissue by FNAB or core tissue biopsy. The sensitivity of FNAB for metastatic carcinoma in a series of 266 superficial lymph nodes was 96.5%, with no false-positive results and nine false-negative results. Tissue from both FNAB and core biopsies can be triaged for ancillary studies in the same manner. There is also great value in the immunocytochemical study of malignant effusions, and often these are the first samples available by virtue of therapeutic evacuation. Whatever the method of obtaining tissue, it is ideal to be able to monitor the process so that adequate tissue may be obtained to triage the patient’s problem appropriately, namely, triage of the specimen for immunohistology, flow cytometry, and molecular-cytogenetic studies. If there is more than one core available for histology, it is best to place each core in a separate tissue block to maximize the tissue available for IHC and/or molecular genetic studies.

Monitoring the tissue procurement process can be performed with frozen sections, immediate interpretation of FNAB, or tissue imprints. In addition to tissue procurement, the pathologist must define the problem by taking the patient’s age, gender, known risk factors, duration of symptoms, and clinical and radiologic findings. Based on this information and the morphologic appearance of the tumor, the quest for the study of tumor origin begins.

In surgical pathology and cytopathology, poorly differentiated carcinomas can be broadly classified as large cell undifferentiated, small cell undifferentiated, and spindle cell. The starting point for diagnostic interpretation is the standard hematoxylin and eosin (H&E) or Papanicolaou-stained slide. The importance of the histologic morphology should not be underestimated in arriving at a definitive diagnosis. Morphology is the foundation upon which the interpretation of all immunohistochemical studies rests.

In this chapter, the role of diagnostic IHC in diagnosing CUPS is emphasized, especially as it relates to adenocarcinoma/poorly differentiated carcinoma of unknown primary site and germ cell tumors, as they account for over 90% of cases of CUPS. Specific tables are presented that aid in the differential diagnosis of tumors in specific anatomic sites. The role of molecular studies in combination with IHC for patients with CUPS is also discussed.

Determining Site of Origin: Stepwise Approach

Carcinomas form the predominant category (∼90% to 95% cases) of CUPS and will therefore remain the main focus of this chapter. Because virtually all carcinomas show significant positivity for cytokeratins (CKs), carcinomatous differentiation becomes readily apparent when the tumor is diffusely CK positive (with rare exceptions). The simple and broad-spectrum CKs are the initial antibodies of choice for detecting carcinomatous differentiation. More specific subcategorization of the tumor origin is then possible using a variety of site-specific CKs as well as antibodies directed against various cellular products. It is a combination of these cellular antigens that may yield a cost-effective approach to tumor categorization.

The approach to definitive diagnosis of the patient with CUPS effectively follows four to five sequential steps:

• 1.

  Determine the cell line of differentiation using major lineage markers, including keratins, lymphoid, melanoma, germ cell, and sarcoma markers.

• 2.

  Determine the CK type or types of distribution in the tumor cells because some subsets of CKs are unique to certain tumor types.

• 3.

  Determine whether there is coexpression of vimentin.

• 4.

  Determine whether there is expression of supplemental antigens of epithelial or germ cell derivation, that is, carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA), placental alkaline phosphatase (PLAP), or SALL4. This step can be combined either with step 3 or step 5.

• 5.

  Determine whether there is expression of cell-specific products, cell-specific structures, transcription factors or receptors that are unique identifiers of cell types—for example, neuroendocrine granules, peptide hormones, thyroglobulin, PSA, NKX3.1, inhibin, gross cystic disease fluid protein-15 (GCDFP-15), GATA-binding protein 3 (GATA3), villin, uroplakin, TTF-1, transcription factor CDX2 or PAX8.

Step One: Screening Immunohistochemistry

An abbreviated first-line panel to determine the line of differentiation should be composed of epithelial markers (pankeratin AE1/3 and CAM5.2, often used together), lymphoid marker (leukocyte common antigen or LCA), and melanocytic marker (S100 or SOX10). Vimentin has previously been considered a mesenchymal marker, but it may be expressed quite diffusely in many poorly differentiated carcinomas. As a matter of fact, vimentin coexpression in a carcinoma may point toward a specific primary site. Diffuse strong expression of any of the above markers is generally suggestive of a particular line of differentiation.

Line of Differentiation: Lymphoid

The above first-line panel generally leads to a more extensive workup. At this point, if the tumor is strongly positive for LCA and negative for keratins, further workup is directed toward classifying the lymphoma using pan-B-cell (CD20, CD79a, and PAX5), pan-T-cell (CD3), and other markers. ^, If LCA is +/−, and pan-B and pan-T cell markers are negative, and the morphology is still suggestive of lymphoid neoplasm, it is not unreasonable to think about a myeloid neoplasm and perform myeloid markers for myeloid sarcoma. This is one diagnosis that may be missed even after an expensive and extended immunohistologic evaluation. The stains that are helpful in demonstrating myeloid lineage are myeloperoxidase, lysozyme, CD33, and CD117 (also known as KIT). CD43 and CD68 stains are also positive in myeloid sarcomas. ^{, ,}

Line of Differentiation-Melanocytic

A diffuse strong staining with S100, negative for CK in a tumor of unknown origin is good evidence that it may be a melanoma. However, this still needs to be confirmed by additional melanoma markers such as human melanoma black 45 (HMB-45), melan-A, tyrosinase, or SOX10. This is because S100 is not a specific (although very sensitive) marker for melanoma. S100 is also expressed by some carcinomas ^, and sarcomas (liposarcoma, chondrosarcoma, and neural tumors). ^, Although the typical variants of these sarcomas are easy to diagnose on H&E alone, the unusual variants such as dedifferentiated liposarcoma, mesenchymal chondrosarcoma, or a malignant peripheral nerve sheath tumor (MPNST) may pose a challenge to distinguish from melanomas. In some practices, SOX10 is used preferentially as a screening marker. Of note, MPNST typically shows limited S100 or SOX10 staining (in around 40% of cases); diffuse staining for these markers nearly excludes MPNST. Additional melanoma markers should be performed for definitive diagnosis. Similarly, a pathologist should also be careful in distinguishing melanoma from carcinoma based on only a limited number of immunostains. As mentioned earlier, some carcinomas may show strong S100 expression or expression of SOX10; an additional pitfall is that some melanomas may show polyclonal CEA and/or focal CAM5.2 keratin immunoreactivity. Therefore, caution is advised when the diagnosis is heavily based on immunohistochemical expression of markers. In addition, it has recently been recognized that some metastatic epithelioid (or spindle cell) malignant neoplasms represent dedifferentiated (or undifferentiated) melanomas without expression of any conventional markers; IHC using mutation-specific antibodies (e.g., BRAF V600E) or sequencing can be helpful to confirm the diagnosis in such cases ( Fig. 8.1 ). ^,

Line of Differentiation: Mesenchymal

Vimentin does not show sufficient specificity to be useful as a general mesenchymal marker. Most, but not all, sarcomas are negative for epithelial markers. Epithelioid sarcomas show extensive expression of CK and EMA, and synovial sarcoma may have a well-defined biphasic pattern that shows strong staining for epithelial markers in the glandular component, whereas the spindle cell component shows limited expression of epithelial markers. The sarcomas that need to be considered in a CUPS case are the ones that do not demonstrate a particular line of differentiation on morphology alone. These sarcomas may have small round blue cell tumor morphology (Ewing sarcoma, desmoplastic small round cell tumor and rhabdomyosarcoma), spindle and epithelioid cells (synovial sarcoma, clear cell sarcoma, angiosarcoma), and pure epithelioid cells (epithelioid sarcoma). Proximal-type epithelioid sarcoma is particularly challenging to distinguish from metastatic “large cell” carcinoma, since this tumor type typically presents in the inguinal region (a common site of lymph node metastasis) and shows diffuse CK expression. Loss of SMARCB1 (INI1) can be helpful to confirm the diagnosis of epithelioid sarcoma ( Fig. 8.2 ). ^, Although a number of immunohistochemical stains are available to further classify a sarcoma into a defined category, many stains are not specific enough to provide a definitive diagnosis. In recent years, much more specific markers that correlate with molecular genetic alterations have been introduced, in some cases replacing genetic studies. ^, Occasionally a high index of suspicion is required to make the correct diagnosis ( Fig. 8.3 ). However, IHC may be performed to narrow down the differential diagnosis and streamline the molecular tests that need to be ordered. ^, For example, a small round blue cell tumor positive for CD99 in a child or young adult should be evaluated for EWSR1 gene rearrangement ( Fig. 8.4 ). In the past, fresh-frozen tissue was considered the best sample for molecular testing; however, nearly all currently available assays can now be performed on formalin-fixed, paraffin-embedded (FFPE) tissue. ^, Triage for suspected sarcoma cases can be performed as shown in Table 8.1 .

Table 8.1

Sarcomas That Can Present as Cancer of Unknown Primary Site

Sarcoma Type	Age/Site	Morphology	Special Stains/IHC	Ancillary Techniques for Confirmation of Diagnosis
Ewing sarcoma	Usually <30 years. Chest wall, extremities, retroperitoneum, pelvis. Metastases to lungs and bone.	Small round blue cell tumor.	CD99+, NKX2.2+, FLI1+.	RT-PCR for EWSR1-FLI1, EWSR1-ERG, EWSR1-ETV1, EWSR1-E1AF, EWSR1-FEV, FUS-ERG. EWSR1 translocation can also be shown by FISH with EWSR1 break-apart probe.
BCOR-CCNB3 fusion positive sarcoma	Usually <20, male preponderance, bone and deep soft tissue	Ewing-like with angulated nuclei and spindle cell morphology.	CCNB3+, BCOR+, cyclin D1+, SATB2+, CD99+	RT-PCR for BCOR-CCNB3
CIC-DUX4 fusion positive sarcoma	Median age ∼33 years, soft tissue and bone	Atypia and proliferation greater than Ewing sarcoma.	CD99+ (focal, weak), ETV4+, WT1+	RT-PCR for CIC-DUX4. FISH for CIC using break-apart probes.
RMS-alveolar (A), embryonal (E), and pleomorphic (P)	A-RMS: 10–20 years. Extremities and perineum. E-RMS: 3–10 years. Prostate, paratesticular, orbit, nasal cavity. P-RMS: 50+ years. Abdomen, retroperitoneum, chest wall, testes, extremities.	Small round blue cells with alveolar growth pattern in alveolar RMS; round and spindle cells in embryonal; round, spindle, and pleomorphic cells in pleomorphic RMS.	Muscle specific actin (MSA)+, desmin+, myogenin (most specific)+, myoD1+.	RT-PCR for PAX3-FOXO1 and PAX7-FOXO1 in alveolar RMS only. FISH for FOXO1 using break-apart probes.
Desmoplastic small round cell tumor	Young adults, often adolescent boys. Abdomen and pelvis, peritoneal implants.	Round/oval cells in desmoplastic stroma in classic cases, other cases with variable morphology.	Vimentin+, cytokeratin+, EMA+, desmin+, WT1+ (with C-terminal antibody).	RT-PCR for EWSR1-WT1. FISH for EWSR1 using break-apart probes.
Synovial sarcoma	Young adults. Extremities around large joints. Also other locations including kidney, lung and pleura.	Spindle cell or biphasic glandular and spindle cell pattern. Small round cells in poorly differentiated tumor.	EMA+, keratin+ (biphasic tumors), CD99+. Strong nuclear TLE1+. SS18-SSX+ (fusion-specific antibody).	RT-PCR for SS18-SSX1 and SS18-SSX2. FISH for SS18 using break-apart probes.
Clear cell sarcoma (melanoma of soft parts)	Young adults. Deep soft tissue with nodal and lung metastases. Occurs in proximity to tendon, fascia, aponeuroses.	Mixed epithelioid and spindle cells in nested growth pattern.	S100+, HMB-45+, melan A+, SOX10+.	RT-PCR for EWS-ATF1. FISH for EWSR1 using break-apart probes.
Alveolar soft part sarcoma	Young adults—often females. Deep soft tissue. Lung metastases common.	Large polygonal cells, granular cytoplasm, prominent nucleoli, rare mitoses.	TFE3+.	RT-PCR for ASPSCR1-TFE3. FISH for TFE3 using break-apart probes.
PEComas	40–50 years—usually females. Various visceral organs and soft tissue.	Epithelioid and spindle cells with perivascular arrangement, clear to granular cytoplasm.	HMB-45+, melan A+, but S100−. Muscle markers positive.
Epithelioid sarcoma	Young adults. Deep soft tissue of extremities. Metastases to lung, lymph node, and skin.	Epithelioid tumor cells, granuloma-like growth pattern.	Keratin+, EMA+, CD34+, CK5/6−, p63−, loss of SMARCB1 (INI1).
Vascular tumors	Adults. Soft tissue and various visceral organs.	Angiosarcoma: Epithelioid and spindle cell tumor, vasoformative areas. Epithelioid cells arranged in cords in epithelioid hemangioendothelioma.	ERG+, CD31+, CD34+, patchy keratin+. CAMTA1 in epithelioid hemangioendothelioma.
Leiomyosarcoma	Adults. Abdomen, pelvis, and various other locations.	Spindle cells in fascicles.	SMA+, HHF35+, desmin+, caldesmon+, patchy keratin+.
Malignant peripheral nerve sheath tumor	Adults. NF1 patients (50%). Deep soft tissue in association with major nerve.	Spindle cells with perivascular accentuation, abundant mitoses, necrosis+/−. Rarely epithelioid.	S100+ (weak, patchy, 30%–50%), SOX10+ (30%–50%), loss of H3K27me3, CD99+. Negative for HMB-45, melan A, and vascular markers.	.
Chordoma	Adults, usually males. Sacrococcygeal, thoracolumbar spine.	Physaliferous cells, vacuolated cytoplasm, mucoid stroma.	Brachyury+ (nuclear), S100+, keratin+, CK7−/CK20−, EMA+.
Extraskeletal myxoid chondrosarcoma	Adults. Deep soft tissues of extremities. Metastases may be confused with myoepithelial type carcinomas.	Cords of spindle and epithelioid cells in myxoid stroma.	S100+/−, NSE+, synaptophysin+/−, INSM1+, keratin-, chromogranin−.	RT-PCR for EWSR1-NR4A3 and other NR4A3 fusions. FISH for NR4A3 using break-apart probes.
Angiomatoid fibrous histiocytoma	Extremities of children and young adults.	Nodular distribution of ovoid and spindle cells with blood-filled cystic cavities, and surrounding dense lympho-plasmacytic infiltrate.	Desmin+ (∼50%), EMA+ (<50%), CD99+, keratin−, S100−.	RT-PCR for EWSR1-ATF1, EWSR1-CREB1, or FUS-ATF1. FISH for EWSR1 using break-apart probes.
Endometrial stromal sarcoma	Adult females. Abdominopelvic region. Distant metastases to lungs.	Oval/round to spindle cells. Vague resemblance to proliferative pattern endometrial stroma.	CD10+, ER+, BCL2−, CD34−, SMA and desmin positivity with smooth muscle differentiation.	FISH for JAZF1 using break-apart probes better than RT-PCR for JAZF1-SUZ12 fusion.
Gastro-intestinal stromal tumor	Adults. GI tract. Abdominopelvic region. Metastases often to the liver.	Spindle or epithelioid cells.	DOG1+, CD117+, CD34+, often negative for S100, actin and desmin.	KIT or PDGFRA activating mutations.

EMA , Epithelial membrane antigen; ER , estrogen receptor; FISH, fluorescence in situ hybridization; GI, gastrointestinal; IHC , immunohistochemistry; RMS , rhabdomyosarcoma; RT-PCR, reverse transcriptase polymerase chain reaction; WT1 , Wilms tumor protein.

Line of Differentiation: Epithelial

Carcinoma comprises approximately 90% of cases of CUPS. Within the carcinoma category, the overwhelming majority of tumors are adenocarcinomas (∼70%). The poorly differentiated carcinoma group comprises approximately 15% to 20%, and the remaining tumors represent either squamous cell carcinoma (5%) or neuroendocrine carcinomas (5%). CK stains are an excellent marker of epithelial differentiation and are strongly and diffusely expressed in carcinomas. However, examples of keratin positivity have been described in almost all tumor types including sarcomas, melanomas, and even lymphomas. Despite these disturbing reports, when an epithelioid tumor is overwhelmingly positive for pankeratin stains, a diagnosis of carcinoma must be seriously evaluated. The CKs are further discussed in step two.

Step Two: The Cytokeratins—An Overview

The soft epithelial keratin intermediate filaments comprise approximately 20 different keratin polypeptides. The polypeptides, numbered 1 through 20, comprise the type II (basic) keratins and the type I (acidic) keratins ( Table 8.2 ). This family of intermediate filaments is crucial in diagnostic IHC for the identification of carcinomatous differentiation and for identification of specific carcinoma subtypes.

Table 8.2

The Most Common Soft Keratins and Their Distribution

From Quinlan RA, Schiller DL, Hatzfeld M, et al. Patterns of expression and organization of cytokeratin intermediate filaments. Ann N Y Acad Sci . 1985;455:282–306.

Type II (Basic) Keratin	Molecular Weight (kDa)	Typical Distribution in Normal Tissue	Type I (Acidic) Keratin	Molecular Weight (kDa)
CK1	67	Epidermis of palms and soles	CK9 CK10	64 56.5
CK2	65	Epithelia, all locations	CK11	56
CK3	63	Cornea	CK12	55
CK4	59	Nonkeratinizing squamous epithelia	CK13	51
CK5	58	Basal cells of squamous and glandular epithelia, myoepithelial, mesothelium	CK14 CK15	50 50
CK6	56	Squamous epithelia, especially hyperproliferative	CK16	48
CK7	54	Simple epithelia	CK17	46
CK8	52	Basal cells of glandular epithelia, myoepithelial	CK18	45
		Simple epithelia, most glandular and squamous epithelia (basal)	CK19	40
		Simple epithelia of intestines and stomach, Merkel cells	CK20	46

Keratin filaments are formed by tetrameric heteropolymers of two different keratins, two from type I and two from type II, to maintain cellular electrical neutrality. The vast majority of keratins are paired together as acidic and basic types, with rare exception. The classification and numbering system of the keratins is based on the catalog of Moll and associates.

There are 12 keratins with more acidic isoelectric points that form type I (acidic) keratins and 8 keratins with more basic isoelectric points, the type II (basic-neutral) keratins. The keratins are products of two gene families: most genes for type II keratins are localized on chromosome 12, and the genes for type I keratins are localized on chromosome 17. Within each group, the CKs are numbered consecutively from highest to lowest molecular weight in each group. In addition, most low-molecular-weight (LMW) keratins are typically found in all epithelia except squamous epithelium, whereas high-molecular-weight (HMW) keratins are typical of squamous epithelium.

The original methods for identification of the different keratin types in tissues relied on tedious biochemical methods, chiefly performed by Franke and Moll and their associates. ^, More recently, the problem of keratin subtyping has been expedited by the development of numerous monoclonal keratin-specific antibodies. ^, This development was crucial for the ease of keratin subtyping that is now indispensable to the surgical pathologist.

In the last few years, our knowledge on keratins has tremendously increased, with discovery of many new keratin genes (now numbered at 54 keratin genes); however, most newly discovered are expressed in hair follicles. There is even a new consensus nomenclature for mammalian keratins. There are now 28 type I keratin genes (17 epithelial and 11 hair keratins) and 26 type II keratin genes (20 epithelial and 6 hair keratins). The keratins that are functionally useful in determining site of origin have however remained limited (namely keratins 5, 7, 8, 14, 17, 18, 19, and 20).

The detection of keratin, and therefore carcinomatous differentiation, is possible in tumors with extensive necrosis. Judkins and colleagues studied a small number of tumors with necrotic areas, including carcinomas, melanomas, and sarcomas, with a panel of antibodies and found that 78% of carcinomas stained with at least one antikeratin antibody in necrotic areas with 100% specificity.

Distribution of Keratin Antigens in Tissues

Simple Epithelial Keratins

Simple epithelial keratins are the first keratins to appear in embryonic development, as they are expressed in virtually all simple (nonstratified), ductal, and pseudostratified epithelial tissues. ^{, ,} Because these keratins are widespread, they may be useful for the identification of epithelial differentiation. Almost all mesotheliomas and carcinomas, ^{, ,} except squamous cell carcinomas, contain the simple keratins 8 and 18, and a few visceral organs such as liver contain only keratins 8 and 18.

Although identified by many keratin antibodies that recognize a cocktail of keratin peptides (e.g., pankeratin antibodies AE1 and AE3), CAM5.2 and 35BH11 recognize keratins 8 and 18 almost exclusively ( Fig. 8.5 ). This group of antibodies is perhaps the most commonly used to demonstrate the simple keratins in surgical pathology. Because simple keratins are widely distributed in most carcinomas, these antibodies are particularly useful in the initial approach to investigation for carcinomatous differentiation ( Table 8.3 ; see also Table 8.2 ).

Table 8.3

Keratin Antigens and Antibodies

CK Antigen	Antibody	Notes
CK8	35βH11	Carcinomas of simple epithelium
CK8	CAM5.2	Carcinoma of simple epithelium
Pankeratin	AE1/AE3	Carcinomas of simple and complex epithelium
CK1/10	34B4	Squamous cell carcinoma
CK7	OV-TL 12/30	Nongastrointestinally derived carcinomas
CK20	K20	Most gastrointestinal carcinomas; mucinous ovarian, biliary, transitional, and Merkel cell carcinoma
CK19	RCK 108	Most carcinomas; many carcinomas with squamous component; myoepithelial cells
CK1/5/10/14	34βE12	Basal cells of prostate; most duct-derived carcinomas
CK18/19	PKK1	Most carcinomas
CK10/11/13/14/15/16/19	AE1	Most squamous lesions and many carcinomas
CK8/14/15/16/18/19	MAK-6	Most carcinomas

Cytokeratin 19

The lowest molecular weight of the keratin group, CK19 is a simple keratin that has a distribution similar to keratins 8 and 18 and is also present in the basal layer of the squamous epithelium of mucosal surfaces and may be seen in epidermal basal cells. CK19 is a good screening marker for epithelial neoplasms because of its wide distribution in simple epithelia and in many squamous tissues. The monoclonal antibody AE1 (Boehringer-Mannheim, Indianapolis, Indiana) reacts with CK19 as does the AE1/AE3 cocktail (Boehringer-Mannheim). Also reacting in formalin-fixed tissues is a monoclonal antibody to CK19-RCK108 (DAKO, Carpinteria, California). In contrast, CK19 is mostly negative or rarely is seen focally in hepatocellular carcinoma.

Cytokeratin 7

CK7 is a 54-kDa type II simple keratin that has a restricted distribution compared with keratins 8 and 18. Its presence in many simple, pseudostratified, and ductal epithelia and mesothelia is similar in distribution to that of keratins 8 and 18. Much of the data in the literature on CK7 are based on the reactivity patterns of antibody OV-TL 12/30 (DAKO) in FFPE tissues. The OV-TL 12/30 antibody parallels the CK7 immunoreactivity with RCK 105, an antibody for use on frozen sections. In addition, predigestion with protease or heat-induced epitope retrieval (HIER) is required for OV-TL 12/30. The lack of, or extreme paucity of, CK7 distribution in tissues such as colonic epithelium, hepatocytes, and prostatic acinar tissue is used to diagnostic advantage. This antibody identifies transitional cell epithelium ( Fig. 8.6A ) but is predominantly negative in most squamous epithelia. The restricted topography of CK7 makes it especially useful in evaluating the origin of adenocarcinomas, as this keratin is present in most breast, lung, ovarian, pancreaticobiliary, and urothelial carcinomas, but it is either absent or present in only rare cells in colorectal, renal, and prostatic carcinomas ( Box 8.1 , Table 8.4 ). ^{, , ,} CK7 stains squamous cell carcinoma and squamous dysplasias of the cervix. Although conventional hepatocellular carcinomas are usually negative for CK7, it is expressed in the fibrolamellar variant. CK7 is also typically expressed in mammary and extramammary (EM) Paget disease. A diagnostic pitfall in the interpretation of CK7 is that CK7 stains subsets of endothelial cells of normal soft tissues, as well as endothelial cells in venules and lymphatics in intestinal mucosa, uterine exocervix, and lymphoid tissue.

Box 8.1

Dominant CK7/CK20 Immunoprofiles in Select Neoplasms

Modified from Wang MP, Zee S, Zarbo RJ, et al. Coordinate expression of cytokeratin 7 and 20 defines unique subsets of carcinomas. Appl Immunohistochem. 1995;3:99–107; and Chu P, Wu E, Weiss L. Cytokeratin 7 and cytokeratin 20 expression in epithelial neoplasms. A survey of 435 cases. Mod Pathol. 2000;13:962–972.

CK7+/CK20+

• Urothelial carcinoma

• Pancreatic carcinoma

• Ovarian mucinous carcinoma

• Gastric adenocarcinoma

CK7+/CK20−

• Adenocarcinoma of lung

• Small cell carcinoma of lung

• Breast carcinoma, ductal and lobular

• Nonmucinous ovarian carcinoma

• Endometrial adenocarcinoma

• Gastric adenocarcinoma

• Pancreatic ductal adenocarcinoma

• Cholangiocarcinoma

• Mesothelioma

• Squamous cell carcinoma of cervix

CK7−/CK20+

• Colorectal adenocarcinoma

• Merkel cell carcinoma

CK7−/CK20−

• Squamous cell carcinoma, lung

• Prostate adenocarcinoma

• Renal cell carcinoma

• Hepatocellular carcinoma

• Adrenocortical carcinoma

• Some thymic carcinoma

Table 8.4

Cytokeratin 7: Percentage of Tumors With Expression

Abstracted from Chu P, Wu E, Weiss L. Cytokeratin 7 and cytokeratin 20 expression in epithelial neoplasms: a survey of 435 cases. Mod Pathol . 2000;13:962–972.

Tumor	Percentage Expression
Lung, adenocarcinoma	100
Lung, small cell carcinoma	43
Ovary, adenocarcinoma	100
Salivary gland, all tumors	100
Uterus, endometrium	100
Thyroid, all tumors	98
Breast, ductal/lobular	96
Liver, cholangiocarcinoma	93
Pancreas, adenocarcinoma	92
Bladder, urothelial	88
Cervix, squamous cell	87
Mesothelioma	65
Neuroendocrine carcinoma	56
Stomach, adenocarcinoma	38
Head and neck, squamous cell	27
Esophagus, squamous cell	21
Kidney, renal cell	11
Germ cell, embryonal carcinoma	7
Colon, adenocarcinoma	5
Adrenal, carcinoma	0
Prostate, carcinoma	0
Thymus, thymoma	0

Diagnostic Utility of Cytokeratin 7

The specific diagnostic utility of CK7 lies in the fact that there are three dominant patterns of immunostaining:

• 1.

  Tumors that are characteristically strongly and diffusely positive include those of the salivary glands, lung, breast, ovary, endometrium, and bladder, as well as mesotheliomas, neuroendocrine tumors, pancreaticobiliary adenocarcinomas, and the fibrolamellar variant of hepatocellular carcinoma. CK7 is also typically expressed in tumor cells of mammary and EM Paget disease.

• 2.

  CK7 variably stains the tumor cells in biliary and gastric tumors.

• 3.

  Carcinomas that are almost invariably negative but may occasionally show rare CK7+ cells include hepatocellular carcinomas, duodenal ampullary carcinomas, colon carcinomas, renal, prostate, and adrenal cortical tumors.

Strong diffuse CK7 immunostaining is a valuable marker in the diagnostic workup of a carcinoma and may be used as a starting point for further immunohistochemical study. Metastatic carcinomas in the lung that are CK7+ must be differentiated from a primary lung carcinoma with a panel of antibodies, and the IHC workup will be dependent on the patient’s age, gender, and presenting findings. It is important to remember that CK7 may be expressed infrequently in certain tumors (see Table 8.4 ). In general, there is high fidelity of CK7 expression between primary and metastatic carcinomas.

Cytokeratin 20

CK20 is a 46-kDa LMW keratin that was discovered by Moll and associates. The tissue distribution of CK20 is limited predominantly to GI epithelium and its tumors, mucinous tumors of the ovary, and Merkel cell carcinomas. ^{, ,} The limited distribution of CK20 in colorectal, pancreatic, and gallbladder carcinomas, Merkel cell carcinomas, and urothelial carcinomas (see Fig. 8.6B ) is useful in the identification of this group of tumors in primary or even metastatic sites. ^{, ,} When combined with the specific tissue distribution of other keratins such as CK7, it is possible to identify colon cancer metastases in the lung, distinguish pulmonary small cell carcinoma from Merkel cell carcinoma, ^, and distinguish urothelial carcinoma from squamous cell carcinomas. It is of importance to recognize that CK20 in this subgroup of tumors is most often distributed strongly and diffusely. Rare CK20+ cells may be seen in some other neoplasms. Up to 10% of primary pulmonary adenocarcinomas overall and up to 25% of invasive mucinous adenocarcinomas (formerly referred to as mucinous bronchioloalveolar carcinomas) may show CK20+ cells. ^, In addition, colloid adenocarcinoma of the lung shows CK20 immunostaining in more than 50% of cases along with nuclear positivity for CDX2, an intestinal marker. A very small percentage of müllerian and breast carcinomas may also show CK20 positivity.

Cholangiocarcinomas of liver are also often positive; the central (large duct) type is more likely to have a high labeling index for CK20 in addition to CK7. The positive predictive value using the combination of CK7 and CK20 to predict the presence of metastatic carcinomas of colorectal or pancreaticobiliary origins in the liver, based on clinical outcomes, is close to 0.9. It is important to remember that CK20 may be expressed infrequently in certain tumors ( Table 8.5 ). ^, In general, there is high fidelity of CK20 expression between primary and metastatic carcinomas (see Fig. 8.6C ).

Table 8.5

Cytokeratin 20: Percentage of Tumors With Expression

Abstracted from Chu P, Wu E, Weiss L. Cytokeratin 7 and cytokeratin 20 expression in epithelial neoplasms: a survey of 435 cases. Mod Pathol . 2000;13:962–972.

Tumor	Percentage Expression
Colon, adenocarcinoma	100
Skin, Merkel cell	78
Pancreas, adenocarcinoma	62
Stomach, adenocarcinoma	50
Liver, cholangiocarcinoma	43
Bladder, urothelial	29
Lung, adenocarcinoma	10
Liver, hepatocellular	9
Gut, well-differentiated neuroendocrine tumor	6
Lung, adenocarcinoma	10
Head and neck, squamous cell	6
Ovary, adenocarcinoma	4
Adrenal, carcinoma	0
Breast, ductal/lobular	0
Cervix, squamous cell	0
Esophagus, squamous cell	0
Germ cell, embryonal carcinoma	0
Kidney, renal cell carcinoma	0
Mesothelioma	0
Prostate, adenocarcinoma	0
Salivary gland, all tumors	0
Thyroid, all tumors	0
Thymus, thymoma	0
Uterus, endometrium	0
Lung, carcinoid	0
Lung, small cell	0
Lung, squamous cell	0

Although the prominent expression of CKs is the essential element of epithelial differentiation, on occasion expression of other lineage-specific markers may cloud the issue. Such is the case of finding keratins in nonepithelial tissues (see later) and the rare observation of LCA (CD45) in some undifferentiated or neuroendocrine carcinomas and CD30 in embryonal carcinomas. The use of a panel of antibodies and the pattern and intensity of immunostaining is critically important in these confounding situations.

Key Diagnostic Points: Simple Cytokeratins

• CAM5.2 and AE1/AE3: Broad coverage for detection of carcinomatous differentiation. Both should be used together for screening.

• CK7 (+): Adenocarcinomas of breast, lung, ovary, endometrium, and pancreas; mesothelioma, urothelial carcinomas, thymic carcinomas; cervical squamous cell carcinoma; and fibrolamellar variant of hepatocellular carcinomas.

• CK7 (negative/rare positive): Renal, prostate, adrenocortical, squamous (except uterine cervix), small cell carcinomas, and hepatocellular carcinomas.

• CK20 (+): Colorectal, pancreas (60%), gastric (50%), cholangiocarcinoma (40%), mucinous ovarian, Merkel cell, and urothelial carcinomas (30%).

• CK20 (negative/rare positive): Most breast, lung, and salivary gland carcinomas, hepatocellular, renal, prostate, adrenocortical, squamous, and small cell carcinomas.

• See Box 8.1 for CK7/CK20 immunoprofile of various carcinomas.

Keratins of Stratified Epithelia: Complex Keratins

Keratins of HMW are observed in stratified epithelia and generally are not present in the simple visceral-type epithelia. Basal cells of prostate and myoepithelial cell populations of ducts and glandular tissue also contain an abundance of HMW type II keratins and LMW type I keratins. The antibody 34βE12 or keratin 903 (K903) ^, identifies a cocktail of keratins including Moll types I, II, V, X, XI, and XIV/XV. The practical diagnostic use of this pattern of expression is to identify basal and myoepithelial cells in their respective organs. For example, the staining of myoepithelial cells around ductal carcinoma in situ or sclerosing adenosis can confirm a non-invasive lesion. This keratin of stratified type is also typically present in squamous epithelium and, using antibody K903, is a good antibody for detecting squamous differentiation in an otherwise poorly differentiated carcinoma.

These HMW structural keratins are also commonly seen in duct-derived epithelium (breast, pancreas, biliary tract, lung) and in transitional, ovarian, and mesothelial tissues. ^, The degree of immunostaining of these tissues with HMW keratin antibodies is typically strong and diffuse, a feature that is helpful diagnostically, because HMW keratin immunostaining is seen only focally in visceral epithelial tissues such as colon, stomach, kidney, and liver.

Key Diagnostic Points: Antibodies to Complex Keratins

• Confirms the presence of basal cells of prostate ( Fig. 8.7 ).

  

• Confirms the presence of myoepithelial cells in breast.

• Present in basal cell layer of stratified and squamous epithelium.

• Strong and diffuse in tumors of squamous epithelial differentiation.

• Present in a wide variety of duct-derived carcinomas and mesotheliomas, and most neoplasms that demonstrate tonofilaments ultrastructurally.

Cytokeratin 5 and Cytokeratin 5/6

CK5 and CK6 are basic (type II) polypeptides with molecular weight of 58 and 56 kDa, respectively. Most studies have been performed using antibodies to CK5/6 and have been found useful in the differential diagnosis of metastatic carcinoma in the pleura versus epithelioid mesothelioma. Epithelioid mesotheliomas are strongly positive in all cases ( Fig. 8.8 ), but up to 30% of pulmonary adenocarcinomas will show focal variable immunostaining.

Almost all squamous cell carcinomas, half of urothelial carcinomas, and many undifferentiated large cell carcinomas immunostain with CK5/6 ( Table 8.6 ). CK5/6 has excellent sensitivity and specificity for the detection of squamous differentiation in poorly differentiated carcinomas. ^, p63 is also seen with high frequency in squamous and transitional carcinomas, and when p63 is used with the CK5/6 antibody, it affords high sensitivity and specificity for squamous differentiation. ^,

Table 8.6

Cytokeratin 5/6: Percentage of Tumors With Expression

Abstracted from Chu P, Weiss LM. Expression of CK 5/6 in epithelial neoplasms: an immunohistochemical study of 509 cases. Mod Pathol . 2002;6:6–10.

Tumor	Percentage Expression
Skin, squamous cell	100
Skin, basal cell	100
Thymus, thymoma	100
Salivary gland, all tumors	93
Mesothelioma	76
Bladder, urothelial	62
Uterus, endometrium	50
Pancreas, carcinoma	38
Breast, carcinoma	31
Ovary, carcinoma	25
Liver, cholangiocarcinoma	14
Gut, well-differentiated neuroendocrine tumor	10
Lung, adenocarcinoma	5
Liver, hepatocellular carcinoma	4
Adrenal, carcinoma	0
Colon, adenocarcinoma	0
Germ cell, embryonal carcinoma	0
Kidney, renal cell carcinoma	0
Prostate, carcinoma	0
Stomach, adenocarcinoma	0
Thyroid, all tumors	0

Myoepithelial cells of the breast, glandular epithelium, and basal cells of the prostate express CK5/6, and some carcinomas of ovarian origin may display CK5/6.

Hyperplastic mesothelial cells can be seen on occasion in the sinuses of lymph nodes from the chest or cervical chain, and the differential diagnosis in this instance is metastatic carcinoma. The presence of strong, diffuse CK5/6 in the cells of these nests should aid in identifying them as mesothelial in origin, but this should be confirmed by using more specific markers for mesothelium such as calretinin and WT1.

There has been some renewed interest in these antibodies as both CK5 and CK5/6 are also used to identify the basal-like molecular class of breast cancer. We have previously shown that CK5 (clone XM26) is superior to CK5/6 (clone D5/16B4) antibody in identifying the basal-like phenotype of breast carcinoma with high sensitivity and specificity. Whether CK5 is superior to CK5/6 in other distinctions (mesothelioma vs. carcinoma; or in identifying squamous differentiation) needs to be investigated.

Key Diagnostic Points: CK5 and CK5/6

• Good indicator of squamous and transitional cell differentiation.

• Good discriminator of mesothelial differentiation versus adenocarcinoma in lung.

• Positive in myoepithelial cells of breast and basal cells of prostate.

• Sensitive and specific markers of basal-like phenotype of breast carcinoma.

Keratins in Nonepithelial Cells

Keratins have been documented by IHC, ^, immunoblot, and polymerase chain reaction in several types of tumors in which there is no morphologic evidence of epithelial differentiation. This type of keratin immunostaining has been referred to as anomalous, aberrant, spurious, and unexpected.

The keratins most often found in these nonepithelial mesenchymal tissues or melanocytic lesions are keratins 8 and 18 and, less commonly, keratin 19. Antibodies that detect these LMW keratins have demonstrated positive immunostaining in a variety of FFPE mesenchymal tumors, including leiomyosarcomas (21% to 38%), liposarcoma (21%), rhabdomyosarcoma, MPNSTs (5%), some undifferentiated pleomorphic sarcomas (5%), GI stromal tumors, rare solitary fibrous tumors, angiosarcoma (33%), endometrial stromal sarcoma, and Ewing sarcomas. ^, Keratin usually stains scattered cells in this group of tumors in traditional FFPE tissue, whereas carcinomas and sarcomatoid carcinomas are heavily and diffusely stained ( Fig. 8.9 ). In addition, keratin-positive soft tissue and bone tumors with partial epithelial differentiation are variably stained with keratin in the epithelial areas as expected. This group includes synovial sarcomas, epithelioid sarcoma, chordoma, MPNST, and adamantinoma of long bones. Although some of the soft tissue tumors may mimic metastatic carcinoma morphologically, the finding of sporadic cell immunostaining is unlike the strong, diffuse immunostaining seen in carcinomas, especially when using the broad-coverage antibodies. Frozen tissues fixed in acetone or alcohol, including alcohol-fixed cytologic specimens, yields far more keratin-positive cells, and this can be confusing diagnostically, especially with cytologic specimens for which alcohol is a standard fixative for needle aspiration specimens.

Malignant melanoma also demonstrates immunostaining for keratins 8 and 18, but in FFPE tissues, the prevalence is around 1% of cases, with focal tumor cell staining. ^, Frozen sections and alcohol-fixed melanomas show substantially more positive tumor cells than do formalin-fixed specimens, and it is important to recognize this to avoid misdiagnosing melanoma as a carcinoma, especially in alcohol-fixed cytologic preparations. The consensus regarding keratin immunostaining of nonepithelioid sarcomas and melanomas is that although the presence of keratin is real, as measured by molecular techniques and more sensitive immunohistologic methods (frozen sections, alcohol fixation), the observed nonexpression of keratin staining in these tumors in formalin-fixed tissue is desirable because of its diagnostic usefulness.

Truly “spurious” keratin immunoreactivity has been described in human glial tissue and in some human astrocytomas, especially with antibodies AE1 and 34βE12. In addition, the cocktail AE1/AE3 may cross-react with both normal and neoplastic astrocytes. The spurious keratin immunoreactivity is probably due to cross-reaction with glial cells containing glial fibrillary acidic proteins. This is an obvious pitfall for the misdiagnosis of metastatic carcinoma in the brain. The antibody CAM5.2 does not react with astroglial cells; thus it is best used to detect carcinomatous differentiation in the CNS.

Meningiomas, especially the “secretory variant,” may express keratin in up to one-third of cases.

Epithelial differentiation is simulated in lymph nodes with the LMW keratin-positive fibroblastic reticulum cells of the paracortex ( Fig. 8.10 ). These dendritic cells immunostain with CAM5.2, rarely with AE1/AE3, revealing an extensive network of extrafollicular dendritic processes in lymph nodes, tonsils, and spleen. These keratin-positive cells are a pitfall for the diagnosis of metastatic carcinoma, because the conventional wisdom had been that keratin-positive cells in a lymph node equated with metastatic carcinoma. The pitfall is twofold. When searching for keratin-positive micrometastases in patients with breast carcinoma, one must distinguish the dendritic processes from carcinoma cells that cluster in the subcapsular sinus. Also, needle aspirates and touch imprints of lymph nodes may contain keratin-positive cells without containing metastatic carcinoma; one must be aware of the morphologic features of the keratin-positive cells.

Keratin positivity has been described in plasma cells, plasmacytoma, and in anaplastic large cell lymphoma. For anaplastic large cell lymphoma, keratins may be detected in as many as 30% of cases and, along with some EMA-positive anaplastic lymphoma cells, the definitive diagnosis can be confusing. However, adherence to a broad-spectrum antibody for keratin immunoreactivity will show only focal rare staining at most in these lymphomas. Plasmacytomas likewise should be studied with broad-coverage antibodies in a panel that includes antibodies to CD138 and kappa/lambda light chains.

The majority of keratin immunostaining is performed on FFPE tissues. The duration of formalin fixation is a key factor when trying to optimize the technical performance of keratin immunoperoxidase stains. The fixation time is closely related to the time required for enzymatic predigestion. In general, tissue fixed in 10% formalin for more than 2 days requires greater antigen retrieval, with less time required for tissues fixed briefly (hours) in 10% formalin. Most, if not all, keratin antibodies require epitope retrieval (depending on antibody and fixation duration) for optimal keratin antibody performance.

Key Diagnostic Points: Keratin in Nonepithelial Tumors

• Focal presence in many sarcomas (see text).

• Focal rare presence in melanoma mainly with CAM5.2.

• Plasma cells common; other lymphoid neoplasms rare.

• Common in dendritic cells of lymph nodes mainly with CAM5.2.

• Antibody AE1/AE3 may give spurious positive keratin result in astrocytic neoplasms.