Cytokeratins

Cytokeratins (CKs) belong to the group of intermediate filament proteins that are intermediate between microfilaments and microtubules. They constitute the cytoskeletal structure of virtually all epithelial cells, both benign and malignant. Some nonepithelial cell types and tumors derived from these may also express CKs. The cytokeratin family of proteins, which are coded by different genes, has been numbered (numbers 1–20).³ The expression of the various CKs in cells and tumors depends on their embryonic origin and also the degree of cellular differentiation.³ One broad group of CKs, type I (CK9–20), has an acidic isoelectric point. The other group, type II (CK1–8), has a basic neutral isoelectric point. Antibodies against CKs help confirm the epithelial lineage of a neoplasm. In this regard, monoclonal antibodies, such as AE1/3 and CAM 5.2, are available that recognize multiple members of the CK family. AE1/3 reacts against almost all of the CK family of proteins (AE1 recognizes most of the type 1 CKs whereas AE3 reacts against most of the type II CKs) while CAM 5.2 reacts against CK8 and CK18. Additionally, antibodies are available that react against specific CKs, for example, CK7, CK20, or CK5/6. The following sections detail the use of various anti-CK antibodies in the diagnosis of female genital tract lesions.

Broad Spectrum Cytokeratins

Broad spectrum anti-CK antibodies, such as AE1/3, often prove of value in confirming the epithelial lineage of a neoplasm. For example, in distinguishing a poorly differentiated carcinoma from sarcoma, melanoma, or lymphoma, reactivity with AE1/3, especially if widespread, favors a diagnosis of carcinoma. However, anti-CK antibodies, such as AE1/3, occasionally react with tumors of melanocytic, mesenchymal, and lymphoid origin.⁴ Smooth muscle tumors may also react with anti-CK antibodies.⁵ This may result in diagnostic difficulties, especially if dealing with an epithelioid smooth muscle neoplasm, and underscores the necessity of using panels of antibodies.

Endometrial stromal neoplasms may also be CK positive.⁶ Broad spectrum anti-CK antibodies are reactive with trophoblastic cells and may be useful in distinguishing intermediate trophoblast from decidua, thus confirming the presence of a placental site.

CAM 5.2

CAM 5.2 does not react against normal squamous epithelium but is reactive against most glandular epithelia. It may help in the diagnosis of vulvar Paget disease, as the Paget cells usually react (the residual squamous cells do not), and this may help to exclude mimics such as melanocytic tumors, pagetoid Bowen’s disease, and mycosis fungoides.

CK7 and 20

A combination of antibodies against CK7 and 20 (differential CK staining) has been widely used in ovarian and peritoneal pathology to distinguish between a primary ovarian or peritoneal adenocarcinoma and a metastatic adenocarcinoma, especially of colorectal origin.1,2,7–9 In general, primary ovarian carcinomas of serous, endometrioid, and clear cell type exhibit diffuse CK7 reactivity and are negative with CK20. Primary ovarian mucinous neoplasms exhibit a more variable immunophenotype. In general, they are diffusely reactive with CK7 and nonreactive or focally reactive with CK20. However, there are exceptions with occasional primary ovarian mucinous neoplasms, especially those of intestinal type, being diffusely CK20 positive. In the distinction between a primary ovarian endometrioid adenocarcinoma and a metastatic colorectal adenocarcinoma with an endometrioid appearance, differential CK staining is very useful alone or as part of a larger panel (Table 36.1). Endometrioid adenocarcinoma is usually diffusely CK7 reactive and CK20 negative while colonic carcinoma generally exhibits the opposite immunophenotype. In the case of an ovarian mucinous neoplasm, differential CK staining is not uncommonly difficult to interpret since many primary ovarian mucinous neoplasms may be CK20 reactive and mucinous colorectal adenocarcinoma may be focally CK7 positive. Rectal adenocarcinomas may also be CK7 positive. Additionally mucinous tumors arising in a teratoma often exhibit a large intestinal immunophenotype with diffuse CK20 immunoreactivity. In this regard, other antibodies (discussed in the following sections) are sometimes of value.

Differential CK staining is of limited value in distinguishing between a primary ovarian carcinoma and a metastatic adenocarcinoma from other organs, since many of these tumors exhibit a CK7-positive/CK20-negative or focally positive immunophenotype (Table 36.2). However, dual CK7 and CK20 reactivity raises the possibility of a primary neoplasm in the stomach, pancreas, biliary tree, or urinary bladder.^1,2,7–9 Breast, pulmonary, endometrial, and endocervical adenocarcinomas are most commonly CK7 positive and CK20 negative.

CK7 and CK20 staining also helps to confirm that most cases of pseudomyxoma peritonei in women are of appendiceal (or more rarely colorectal) origin rather than originating from a ruptured ovarian mucinous neoplasm.¹⁰ In cases of pseudomyxoma peritonei with coexistent appendiceal and ovarian mucinous neoplasms, the epithelial elements in all locations, i.e., the appendix, ovary, and peritoneum, are usually diffusely CK20 positive and negative or focally positive with CK7, in keeping with an intestinal origin.

CK7 may be of value in the vulva in confirming a diagnosis of Paget disease and excluding mimics such as malignant melanoma and mycosis fungoides. The cells of primary vulvar Paget disease are usually intensely CK7 positive,11,12 a feature that may assist in assessment of margins. Strong CK20 reactivity should result in consideration of secondary Paget disease, from either the colorectum or urinary tract.^11,12

Ck5/6

CK5/6 is often reactive in mesothelial cells (benign or malignant) and is helpful to distinguish a mesothelial from a serous epithelial proliferation (benign, borderline, or malignant); the latter is usually negative.¹³ In this regard, CK5/6 should be used as part of a panel that may include Ber-EP4 (an epithelial marker reactive in most epithelial lesions and generally negative in mesothelial lesions). Other antibodies generally reactive in mesothelial lesions are calretinin, HBME1, thrombomodulin, D2-40, and CD44H (generally negative in epithelial lesions).^13,14 CK5/6 is more likely to be positive in squamous than glandular neoplasms and this may be useful in diagnosis.

Other Cytokeratins

Although the remaining specific CKs have found little place in diagnostic gynecologic pathology, there are a few exceptions. Staining with the high molecular weight CK34β E12 may assist in highlighting the basal cell layer in ectopic prostatic tissue within the cervix.¹⁵ CK18 is especially likely to be positive in undifferentiated endometrial carcinoma and this marker may be useful in distinguishing undifferentiated endometrial carcinoma from undifferentiated sarcoma.¹⁶

Epithelial Membrane Antigen and Ber-EP4

Epithelial membrane antigen (EMA), a glycoprotein found in human milk fat globule membranes, and Ber-EP4, an epithelial-specific antigen to a membrane-bound glycoprotein, help to confirm that a neoplasm has an epithelial lineage. Trophoblast and trophoblastic neoplasms are also reactive. Both markers are commonly used in panels to distinguish an ovarian adenocarcinoma (reactive) from a sex cord–stromal tumor (negative).1,2,17 Ber-EP4 is useful in distinguishing a serous proliferation of the ovary or peritoneum (reactive) from mesothelial derived lesions (negative). Although EMA reactivity is rare in ovarian sex cord–stromal tumors, focal immunoreactivity has been found in 50% of a small series of juvenile granulosa cell tumors.¹⁸ EMA is generally negative in female adnexal tumor of wolffian origin (FATWO). This is diagnostically useful since FATWO may be confused with an epithelial neoplasm, which is usually EMA reactive. EMA is often positive in undifferentiated endometrial carcinoma and may be useful in distinguishing this from undifferentiated sarcoma.¹⁶

Vimentin

Vimentin is the most widely distributed of the intermediate filament proteins and is expressed in virtually all mesenchymal cells, and most mesenchymal neoplasms in the female genital tract. In most cases the distinction of whether a tumor is mesenchymal or epithelial is apparent from the H&E-stained slide, but, when this is not obvious, vimentin staining may be useful, although vimentin positivity is not uncommon in epithelial neoplasms.

In the cervix, vimentin may be used as an aid to distinguish between tuboendometrial metaplasia and endometriosis (usually vimentin reactive) and adenocarcinoma in situ (AIS) (usually vimentin negative).¹⁹ Vimentin may also be useful in differentiating between an endometrial adenocarcinoma of endometrioid type and an endocervical adenocarcinoma of usual type.²⁰ The former usually exhibits diffuse vimentin reactivity whereas endocervical adenocarcinomas are generally negative.

Vimentin may help distinguish between a microglandular variant of endometrioid or mucinous adenocarcinoma of the endometrium (usually vimentin reactive) and cervical microglandular hyperplasia (vimentin negative).²¹

CD10

CD10, or the common acute lymphoblastic leukemia antigen, is a cell-surface neutral endopeptidase expressed by lymphoid precursor cells and B-lymphoid cells of germinal center origin. Antibodies against CD10 are widely used in lymphoma panels.

CD10 is useful in diagnosing an endometrial stromal neoplasm, since most endometrial stromal nodules and endometrial stromal sarcomas (low-grade endometrial stromal sarcomas) exhibit diffuse intense reactivity, although fibrous variants may be negative.24,25 In the distinction between an endometrial stromal and a smooth muscle neoplasm, CD10 should be used as part of a panel (Table 36.3), since conventional uterine smooth muscle tumors may be focally reactive and it is not uncommon for cellular and highly cellular leiomyomas (which are not infrequently mistaken for endometrial stromal neoplasms) and leiomyosarcomas to be diffusely reactive.

CD10 is also characteristically reactive in mesonephric glandular lesions within the female genital tract. Mesonephric remnants throughout the female genital tract usually exhibit luminal CD10 reactivity.31,32 CD10 reactivity in a benign cervical glandular lesion is good evidence of a mesonephric origin,³¹ although so-called ectopic prostatic tissue may also be reactive.¹⁵ However, CD10 is of limited value in confirming a mesonephric origin for an adenocarcinoma since many cervical and endometrial adenocarcinomas are also reactive.³¹ FATWO may be CD10 reactive.³³

Other uses of CD10 staining in gynecologic pathology include the distinction between a metastatic renal clear cell carcinoma involving the ovary (CD10 reactive)³⁴ and a primary ovarian clear cell carcinoma (usually CD10 negative). In addition, most trophoblastic cell populations and trophoblastic neoplasms are reactive.³² A wide range of other gynecologic neoplasms may be CD10 reactive, including leiomyosarcoma, carcinosarcoma, undifferentiated uterine sarcoma, ovarian sex cord–stromal tumors, uterine tumors resembling ovarian sex cord tumor, and mixed tumor of the vagina.³⁵ However, CD10 immunoreactivity in these neoplasms is inconsistent and unlikely to be of diagnostic value. In summary, CD10 is expressed in a much wider range of gynecologic neoplasms than was originally appreciated and, when used as an aid to diagnosis, should always be part of a panel, which will depend on the differential diagnoses under consideration.

Calretinin

Calretinin is a 29 kDa calcium-binding protein, best known for its role in the diagnosis of mesothelioma. In the distinction between a mesothelioma and an adenocarcinoma, calretinin should be used as part of a panel. Calretinin and Ber-EP4 are the two most useful antibodies to distinguish between a serous epithelial and a mesothelial proliferation.¹⁴ Most serous proliferations are Ber-EP4 reactive and calretinin negative; the converse is the rule for mesothelial lesions. Nuclear reactivity with calretinin is more specific than cytoplasmic staining for mesothelial cells.¹⁴

Calretinin is also expressed in most ovarian sex cord–stromal tumors, being more sensitive but less specific than inhibin.36,37 Calretinin is more likely to be reactive in an ovarian fibroma than inhibin.

In general, neoplasms reactive for inhibin also show reactivity with calretinin. Other gynecologic neoplasms that may show calretinin reactivity include FATWO, uterine tumor resembling ovarian sex cord tumor, sex cord-like areas within endometrial stromal neoplasms, and adenomatoid tumor. Mesonephric lesions, both benign and malignant, within the cervix and elsewhere in the female genital tract may show reactivity.³¹

CD34

CD34, a single chain transmembrane glycoprotein, leukocyte differentiation antigen, is expressed by hematopoietic progenitor cells, endothelial cells, and connective tissue cells, such as skin fibroblasts. CD34 is variably expressed in several vulvovaginal mesenchymal lesions, including aggressive angiomyxoma, cellular angiofibroma, and superficial myofibroblastoma of the lower female genital tract.²⁸ Solitary fibrous tumors rarely occur at various sites within the female genital tract and are CD34 reactive. Endometrial stromal neoplasms are usually CD34 negative, which may be of use in differential diagnosis in that many mimics are reactive.³⁸ Metastatic GIST in the ovary is usually CD34 positive,²³ as are rare primary GISTs arising in the rectovaginal septum or elsewhere in the female genital tract.³⁹

β-hCG

hCG is a glycoprotein comprising a protein core and a carbohydrate side chain, and composed of two dissimilar subunits, α and β. The α-subunits are indistinguishable from the α-subunits of luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone. The β-subunits differ and confer specificity.

β-hCG reacts against syncytiotrophoblast but not cytotrophoblast. Choriocarcinoma shows the strongest and most diffuse reactivity. Placental site trophoblastic tumor (PSTT) and epithelioid trophoblastic tumor are less reactive. Trophoblastic elements in mixed germ cell tumors show reactivity, as do isolated syncytiotrophoblast cells in neoplasms such as dysgerminoma and endometrial carcinoma. β-hCG may be reactive on occasions in a variety of nontrophoblastic neoplasms, such as cervical squamous carcinoma.⁴⁰

Placental Alkaline Phosphatase

Placental alkaline phosphatase (PLAP) is a dimer of 65 kDa subunits and is synthesized during the G₁ phase of the cell cycle. PLAP is expressed in syncytiotrophoblast and in some intermediate trophoblastic populations. Reactivity is stronger in lesions derived from chorion-type intermediate trophoblast, such as placental site nodule, than in lesions of implantation site intermediate trophoblast which are usually only focally positive. Ovarian dysgerminoma is also reactive.

Human Placental Lactogen

Human placental lactogen (hPL), a member of the gene family that includes human growth hormone and human prolactin, is expressed in intermediate trophoblast, and is useful in panels to diagnose trophoblastic neoplasms. In general, expression is stronger and more diffuse in placental site trophoblastic tumor than in choriocarcinoma.

Mel-CAM (CD146)

Mel-CAM is expressed in implantation site intermediate trophoblastic cells.⁴¹ Chorion-type intermediate trophoblastic cells are usually negative or focally reactive. Placental site trophoblastic tumor and exaggerated placental site, lesions of implantation site intermediate trophoblast, express Mel-CAM, whereas placental site nodule and epithelioid trophoblastic tumor, lesions of chorion-type intermediate trophoblast, are usually negative. In distinguishing placental site trophoblastic tumor from exaggerated placental site, double immunohistochemical staining with Mel-CAM and MIB1 is of value.⁴¹ In exaggerated placental site, the MIB1 index in intermediate trophoblastic cells is close to zero whereas it is significantly elevated (14 ± 6.9%) in placental site trophoblastic tumor.⁴¹

HLA-G

HLA-G is expressed in all known trophoblastic tumors, including choriocarcinoma, placental site trophoblastic tumor, and epithelioid trophoblastic tumor, as well as in benign trophoblastic lesions, such as placental site nodule and exaggerated placental site.⁴² HLA-G is generally negative in nontrophoblastic uterine neoplasms.⁶⁴ HLA-G reactivity has been demonstrated in some ovarian carcinomas.⁴³

HMB45

HMB45 is probably the most specific marker of malignant melanoma, and it is melanosome associated. HMB45 reactivity is useful to confirm the diagnosis of malignant melanoma at any site within the female genital tract, most commonly in the vulva or vagina. Metastatic melanoma in the ovary can assume an unusual array of morphologic appearances and easily fool the pathologist if there is no history of melanoma. HMB45 may assist in this regard. However, occasional ovarian steroid cell tumors, which may mimic melanoma, are HMB45 reactive.⁴⁴ Another neoplasm characteristically reactive with HMB45 is PEComa.²⁶ This is an uncommon neoplasm, which in the female genital tract most often involves the myometrium.⁴¹ Uterine epithelioid leiomyosarcomas with a clear cell appearance may also express HMB45.⁴⁵

Melan-A (MART-1)

Melan-A, also known as MART-1, is another melanocytic marker of value in the diagnosis of malignant melanoma. Ovarian sex cord–stromal tumors are also commonly reactive.⁴⁶

S-100

S-100 is useful in the diagnosis of malignant melanoma, either primary or metastatic, at various sites within the female genital tract. Other neoplasms in the female genital tract that may be S-100 positive include ovarian sex cord–stromal tumors and cartilaginous areas within carcinosarcomas.

CA19.9

CA19.9, an antigen of sialyl Lewis(a) containing glycoprotein, is usually reactive in pancreatic, biliary, and colorectal adenocarcinoma metastatic to the ovary whereas most primary ovarian adenocarcinomas of serous, endometrioid, or clear cell type are negative, although some serous carcinomas are focally positive. Primary ovarian mucinous neoplasms are commonly focally or diffusely reactive.

Carcinoembryonic Antigen

Carcinoembryonic antigen (CEA) consists of a heterogeneous family of related oncofetal glycoproteins secreted into the glycocalyceal surface of gastrointestinal cells.

Monoclonal CEA helps distinguish non-mucinous ovarian adenocarcinomas (usually negative) from colorectal adenocarcinoma (usually reactive), when used as part of a panel.1,2 Primary ovarian mucinous neoplasms are often reactive. Adenocarcinomas from other organs, such as pancreas and stomach, are variably reactive.

CEA is useful as part of a panel to help distinguish endometrioid-type endometrial adenocarcinoma from endocervical adenocarcinoma of usual type.²⁰ Endocervical adenocarcinomas are usually, but not always, diffusely reactive with CEA. Primary endometrioid adenocarcinomas of the corpus are negative or focally reactive, although the associated squamous elements may be diffusely reactive. CEA staining patterns of primary mucinous adenocarcinoma of the endometrium are not well studied, but at least a proportion are reactive. CEA is usually reactive in cervical AIS and negative in benign endocervical glandular lesions.⁵⁰

CEA is usually reactive in primary vulvar Paget disease and, like CK7 and CAM 5.2, helps exclude mimics and assess margins.

CA125 (Oc125)

CA125 is a mucin-like glycoprotein, commonly elevated in patients with ovarian cancer, especially of serous type. Although elevated levels are not specific for an ovarian cancer, serum CA125 measurements may be useful in diagnosis and especially in the follow-up of patients with ovarian cancer.

Immunohistochemical staining with CA125 helps to distinguish between a primary and a metastatic ovarian adenocarcinoma and in the evaluation of a disseminated peritoneal tumor in a female.1,2 In general, primary ovarian (or peritoneal) adenocarcinomas of serous, endometrioid, and clear cell types exhibit diffuse CA125 reactivity. Primary ovarian mucinous carcinomas are usually negative, as are colorectal adenocarcinomas. In distinguishing primary ovarian adenocarcinoma from a metastatic colorectal adenocarcinoma, CA125 should be used in a panel (Table 36.2) that could include CK7, CK20, estrogen receptor (ER), CDX2, and CEA. CA125 reactivity is not specific for an ovarian adenocarcinoma, as primary adenocarcinomas of many other organs, including pancreas, breast, lung, cervix, and uterine corpus exhibit reactivity in a proportion of cases. Mesotheliomas are commonly reactive, as are benign mesothelial cells.⁵¹

Inhibin

Inhibin is a dimeric 32 kDa peptide hormone composed of an α- and a β-subunit and produced by ovarian granulosa and theca cells. Individual antibodies are available against each subunit. Most ovarian sex cord–stromal tumors show focal to diffuse cytoplasmic reactivity with inhibin (antibody against α-subunit), although fibroma, poorly differentiated Sertoli–Leydig, and sarcomatoid granulosa cell tumors are sometimes negative.52,53 Since ovarian sex cord–stromal neoplasms may be morphologically confused with a wide range of neoplasms, especially endometrioid carcinomas, immunohistochemical evaluation with inhibin (and other sex cord–stromal markers such as calretinin) may be extremely useful in primary diagnosis and also when confirming a metastatic neoplasm, which may occur years or decades later. In distinguishing between a sex cord–stromal tumor and an endometrioid carcinoma, the former is almost always negative with EMA¹⁷ (Table 36.4). Ovarian sex cord–stromal tumors are usually negative with CK7 but may be focally positive with broad spectrum anti-CK antibodies. Most carcinomas are negative with inhibin, although occasional tumors are focally reactive. Activated ovarian stromal cells that occur in association with and at the periphery of any ovarian neoplasm may be reactive with sex cord–stromal markers, so close attention must be paid to the cellular morphology of the particular clusters that are immunohistochemically reactive.