Immunos for female genital tract

International Journal of Gynecological Pathology 25:101–120, Lippincott Williams & Wilkins, Baltimore Ó 2006 International Society of Gynecological Pathologists

Review

Immunohistochemical and Functional Biomarkers of Value in Female Genital Tract Lesions W. Glenn McCluggage

Summary: There has been a marked expansion in the literature pertaining to the use of immunohistochemical markers in female genital tract pathology, especially with regard to diagnosis. This review provides a survey of the antibodies commonly used in the diagnosis of gynecological lesions grouped as to function or type. Prognostic or predictive markers are also discussed where appropriate, although few of these are of value in everyday practice. It is stressed that when immunohistochemistry is used diagnostically, panels of markers provide better information than reliance on a single antibody. Key Words: Female genital tract—ImmunohistochemistryVDiagnosis.

67, a marker of nuclear proliferation, is discussed where appropriate, understanding only a handful are sufficiently informative to be used in routine practice. It is stressed that in practice the same antibodies from different sources are not always directly comparable in their range of reactivity and may differ to some degree.

For some markers e.g., lymphoid markers only brief discussion is provided, this topic being too complex for full consideration in the space available.

Recent years have witnessed a marked expansion in the use of immunohistochemical markers in gynecologic pathology (1Y5). Most of the published data relates to the use of antibodies in the diagnosis of gynecological neoplasms but some markers have prognostic or predictive value. In general when immunohistochemistry is used diagnostically, panels of markers provide better information than reliance of a single antibody. The necessity to perform adequate positive and negative controls cannot be overemphasised. As might have been anticipated historically, when any individual antibody is initially thought to be specific for a given tumor, this later proves not to be the case with many antibodies staining diverse tumor types. As an example, calretinin, an excellent marker for mesothelium, is also remarkably sensitive for ovarian sex cord tumors. The focus of this review is to provide a survey of the antibodies commonly used in the diagnosis of gynecologic lesions grouped as to function or type. This is in contrast to most reviews which discuss the value of antibodies in specific scenarios in female genital tract pathology. The value of markers as prognostic or predictive factors, such as Ki-

BROAD SPECTRUM DIFFERENTIATION MARKERS The following biomarkers are commonly expressed in many cell types. They are useful in a wide variety of settings and in differential diagnoses. In some cases, their combinations may be unique to a particular entity.

Epithelial Markers 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, coded by different genes, have been classified and numbered by Moll (numbers 1 to 20) (6).

From the Department of Pathology, Royal Group of Hospitals Trust, Belfast.

Address for correspondence and reprint requests to Professor W Glenn McCluggage, Department of Pathology, Royal Group of Hospitals Trust, Grosvenor Road, Belfast, BT12 6BL, Northern Ireland.

E-mail:glenn.mccluggage@bll.n-i.nhs.uk

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DOI: 10.1097/01.pgp.0000192269.14666.68

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The expression of the various CKs in cells and tumors depends on their embryonic origin and also the degree of cellular differentiation (7). One broad group of CKs, type 1 (CK9-20), have 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 recognise multiple members of the CK family, e.g., AE1/3 reacts against almost all of the CK family of proteins (AE1 recognises most of the type 1 CKs whereas AE3 reacts against most of the type II CKs). CAM 5.2 reacts against CK8 and CK18. Additionally, antibodies are available that react against specific CKs, e.g., CK7 or CK20. The following sections detail the use of various antiCK antibodies in the diagnosis of female genital tract lesions.

Broad spectrum CKs (e.g., AE1/3). Broad spectrum antiCK 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, i.e., an epithelial tumor. However, the antiCK antibodies, such as AE1/3, occasionally react with tumors of melanocytic (8), mesenchymal and lymphoid origin (9). Even smooth muscle tumors may react with antiCK antibodies (10,11). This may result in diagnostic difficulties, especially if dealing with an epithelioid smooth muscle neoplasm and underscores the necessity to use panels of antibodies.

Endometrial stromal neoplasms may also be CK positive on occasion (12). Broad spectrum antiCK antibodies are reactive with trophoblastic cells and often are 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. Besides its utility in diagnosing an epithelial neoplasm, its differential staining of squamous and glandular epithelia often helps in the diagnosis of vulval Paget’s disease. The Paget cells usually react (the residual squamous cells do not) and this may be of value in diagnosis and helping to exclude mimics such as melanocytic tumors, Pagetoid Bowen’s disease and mycosis fungoides.

Cytokeratin 7 and 20. In recent years 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 (13Y17). In general, primary ovarian carcinomas exhibit diffuse CK7 reactivity and are negative with CK20. Primary ovarian adenocarcinomas of serous, endometrioid and clear cell type usually exhibit this immunophenotype. Primary ovarian mucinous neoplasms are more variable. In general, they are diffusely reactive with CK7 and nonreactive or at most focally reactive with CK20. However, there are many exceptions with occasional primary ovarian mucinous neoplasms, especially those which exhibit intestinal differentiation, 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, the former usually being diffusely CK7 reactive and CK20 negative whereas the latter generally exhibits diffuse CK20 reactivity and is CK7 negative. In the case of an ovarian mucinous neoplasm, differential CK staining is not uncommonly difficult to interpret when distinguishing a primary ovarian tumor from a secondary colorectal neoplasm because many primary ovarian mucinous neoplasms may be CK20 reactive and conversely colorectal adenocarcinomas with a mucinous appearance may be focally CK7 positive. Also mucinous tumors arising in a teratoma often have staining patterns of the gastrointestinal component of the teratoma. In this regard, other antibodies (discussed below) are sometimes of value (Table 1).

Differential CK staining is of limited value in distinguishing between a primary ovarian carcinoma and a metastatic adenocarcinoma from other organs, because many of these tumors exhibit a CK7 positive/ CK20 negative or focally positive immunophenotype.

However, dual CK7 and CK20 reactivity raises the possibility of a primary neoplasm in the stomach, pancreas, biliary tree or urinary bladder (Table 2) TABLE 1. Typical reaction patterns in primary ovarian and metastatic colorectal adenocarcinoma Endometrioid ovarian Antibody adenocarcinoma CK7 CK20

Diffuse + j

CA125 CEA

Diffuse + j

A-Catenin Negative, focal, or diffuse + CDX2 j Villin MUC5AC

j Diffuse +

Mucinous ovarian adenocarcinoma

Colorectal adenocarcinoma

Diffuse or focal + Negative, focal, or diffuse + j Negative, focal, or diffuse + j

j Diffuse +

Negative, focal, or diffuse+ Negative, focal, or diffuse + Diffuse +

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j Diffuse + Focal or diffuse + Diffuse + Diffuse + j

IMMUNOHISTOCHEMISTRY IN FEMALE GENITAL TRACT LESIONS TABLE 2. Typical differential cytokeratin reaction patterns in tumors Mucinous ovarian adenocarcinoma Nonmucinous ovarian adenocarcinoma Colorectal adenocarcinoma Cervical adenocarcinoma Endometrial adenocarcinoma Pancreatic/biliary adenocarcinoma Gastric adenocarcinoma Renal cell carcinoma Bladder adenocarcinoma Breast adenocarcinoma Pulmonary adenocarcinoma Mesothelioma

CK7

CK20

+ + j + + + + j + + + +

or + j + j j + or j + or j j + or j j j j j

(16,18Y20). Breast, pulmonary, endometrial and endocervical adenocarcinomas are most commonly CK7 positive and CK20 negative.

CK7 and CK20 staining has also helped 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 (21,22). 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’s disease and excluding mimics such as malignant melanoma and mycosis fungoides, because the cells of primary vulval Paget’s disease are usually intensely CK7 positive (23,24). In addition to its diagnostic value in Paget’s disease, CK7 may assist in assessing the margins. With H&E stained slides, it is often exceedingly difficult to identify that single isolated tumor cell sitting right at the margin. CK20, whereas usually negative, may sometimes be focally reactive in vulval Paget’s disease (23Y25). Strong CK20 reactivity should result in consideration of secondary Paget’s disease, either from the colorectum or urinary tract.

Positive reactivity with uroplakin III is also suggestive of secondary Paget’s disease from the urinary tract (25).

Cytokeratin 5/6. CK 5/6 is often reactive in mesothelial cells, be they normal, reactive or neoplastic, and therefore is often helpful, as part of a panel, to distinguish mesothelial proliferations from epithelial (26). This is often problematic in ovarian and peritoneal pathology, the main differential diagnosis usually being between a serous epithelial proliferation (benign, borderline or malignant) and a mesothelial proliferation (benign or malignant). Mesothelial proliferations generally react

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with CK5/6 whereas epithelial lesions are often, although not always, negative. In this regard, CK5/6 should be used as part of a panel which may include Ber-EP4 (an epithelial membrane marker reactive in most epithelial lesions and generally negative in mesothelial lesions).

Other antibodies generally reactive in mesothelial lesions are calretinin, HBME1, thrombomodulin and CD44H (generally negative in epithelial lesions) (27).

Other Cytokeratins. Although the remaining specific CKs have found little place in diagnostic gynecological pathology, assessment of the qualitative changes in the cells expressing various CKs, plus other noncytokeratins are proving helpful in assessing the progression from normal cervical squamous epithelium to low grade and high grade CIN to invasive squamous carcinoma. In brief, reactivity with CK18, 19 and 14 in the basal cell compartment increases whereas the expression of CK13 decreases (28). Staining with the high molecular weight CK 34A E12 may assist in highlighting the basal cell layer in ectopic prostatic tissue within the cervix (29).

Epithelial Membrane Antigen (EMA) and BER-EP4 EMA, a glycoprotein found in human milk fat globule membranes and Ber-EP4, an epithelial specific antigen to a membrane bound glycoprotein, are both similar and help to confirm that a neoplasm has an epithelial lineage.

Trophoblast and trophoblastic neoplasms are also reactive. Both are commonly used in panels to distinguish an ovarian adenocarcinoma such as a serous or endometrioid carcinoma (reactive) from a sex cord-stromal tumor (negative) (30,31). Ber-EP4 is useful in distinguishing a serous adenocarcinoma of the ovary or peritoneum and implants in the peritoneum (reactive) from mesothelial derived lesions (negative). Although EMA reactivity is rare in ovarian sex cord-stromal tumors (even though reactivity with anticytokeratin antibodies is not uncommon), focal immunoreactivity inexplicably is common (50% of a small series) in ovarian juvenile granulosa cell tumors (32). EMA is generally negative in the female adnexal tumor of wolffian origin (FATWO) (33). This is diagnostically useful because FATWO may be confused with an epithelial neoplasm, which is usually EMA reactive.

MESENCHYMAL CELL MARKERS Vimentin Vimentin is the most widely distributed of the intermediate filament proteins and is expressed in virtually all mesenchymal cells. Most mesenchymal neoplasms in the Int J Gynecol Pathol, Vol. 25, No. 2, April 2006

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female genital tract are reactive for vimentin. Although on occasion there is a need to determine whether a tumor is mesenchymal or epithelial, in which case vimentin staining may be important, in most cases the distinction is apparent from the H&E stained slide, making vimentin determination of little practical value.

There are a few situations where vimentin staining may be of value. In the cervix, vimentin may be used as an aid to distinguish between tuboendometrial metaplasia and endometriosis (usually vimentin reactive) and AIS (usually vimentin negative) (34). Vimentin may also be useful in differentiating between an endometrial adenocarcinoma of endometrioid type and an endocervical adenocarcinoma (35Y38). The former usually exhibit diffuse vimentin reactivity whereas endocervical adenocarcinomas are generally negative. The situation is less clear with a mucinous adenocarcinoma of the endometrium and an endometrioid adenocarcinoma of the cervix, although it has been suggested that vimentin reactivity is more dependent on the pattern of differentiation (endometrioid vs. mucinous) than the site of origin (endometrial vs. cervical) (36).

Vimentin may be of use in distinguishing between a microglandular variant of endometrioid or mucinous adenocarcinoma of the endometrium (usually vimentin positive) and cervical microglandular hyperplasia (vimentin negative) (39). A positive vimentin stain may assist in confirming that a primary cervical adenocarcinoma is of mesonephric derivation (40).

Smooth Muscle Markers Multiple biomarkers reactive against smooth muscle antigens are currently available. These include > smooth muscle actin (> SMA), desmin and h-caldesmon. These markers are helpful in several diagnostic scenarios, especially in helping to confirm smooth muscle differentiation within a neoplasm, such tumors potentially occurring at any site within the female genital tract. In using these antibodies, it is important to remember that some smooth muscle neoplasms, especially malignant and epithelioid variants, are negative or only focally reactive (41). Of the three most commonly used antibodies, h-caldesmon is the most specific, but is less sensitive than desmin. Desmin is not a specific smooth muscle marker, as it also stains skeletal muscle.

In the uterine corpus, the main value of smooth muscle markers is in establishing a diagnosis of a smooth muscle neoplasm, either benign or malignant. An antibody panel composed of desmin, h-caldesmon and CD10 (discussed later) helps distinguish cellular leiomyomatous neoplasms from endometrial stromal neoplasms (Table 3). In general, leiomyomatous neoplasms are diffusely reactive with

TABLE 3. Typical reaction patterns in endometrial stromal and smooth muscle neoplasm Antibody

Smooth muscle neoplasm

Desmin > SMA

Diffuse + Diffuse +

h-Caldesmon CD10

Diffuse + Negative, focal, or diffuse + Diffuse +

Oxytocin receptor

Endometrial stromal neoplasm j or focal + Negative, focal, or diffuse + j Diffuse + j

desmin and h-caldesmon (41Y43). CD10 is usually negative or focally reactive, although some cellular leiomyomatous neoplasms and leiomyosarcomas may also be diffusely positive (44Y46). Endometrial stromal neoplasms are usually diffusely CD10 reactive, and negative (most cases) or maximally focally positive with desmin and h-caldesmon (44Y46). > SMA is of limited value because many endometrial stromal neoplasms are diffusely positive, an indication that considerable immunophenotypic overlap exists between uterine smooth muscle and endometrial stromal neoplasms. This is perhaps not unexpected because these two cell types develop from a common progenitor within the uterus. Uterine tumor resembling ovarian sex cord-tumor, sex cord-like areas within endometrial stromal neoplasms and uterine perivascular epithelioid cell tumor (PEComa) are also variably positive with smooth muscle markers (47).

In the cervix, > SMA is useful to distinguish normal endocervical glands or nonneoplastic endocervical glandular lesions from the well differentiated glands of adenoma malignum. The presence of many > SMA positive stromal cells suggests a desmoplastic response to tumor (48). This is usually accompanied by loss of estrogen receptor (ER) expression in the stromal cells.

In the vulvovaginal region, many of the wide range of relatively site specific mesenchymal neoplasms such as angiomyofibroblastoma, aggressive angiomyxoma and superficial cervicovaginal myofibroblastoma are positive with smooth muscle markers, especially desmin (49).

Thus, these markers are of no value in confirming that a mesenchymal lesion represents a leiomyomatous neoplasm. However, negative staining with smooth muscle antibodies is of value in diagnosing cellular angiofibroma, which in contrast to most other neoplasms in the differential diagnosis, does not usually react (49Y51). It has been speculated that cellular angiofibroma exhibits fibroblastic differentiation whereas most of the other neoplasms mentioned are myofibroblastic in origin.

Another tumor that commonly shows reactivity with desmin is intraabdominal desmoplastic small round cell tumor (in females this may present as a primary ovarian

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IMMUNOHISTOCHEMISTRY IN FEMALE GENITAL TRACT LESIONS neoplasm), usually with paranuclear dot-like immunoreactivity (52). This is useful in diagnosis, especially in differentiating this neoplasm from the wide range of Bsmall blue cell tumors^ that may involve the ovary and peritoneum. Desmin sometimes assists in the distinction between benign and malignant mesothelial proliferations.

Benign mesothelial cells are usually desmin reactive whereas the cells of malignant mesothelioma are generally negative, although there is significant overlap.

Skeletal Muscle Markers A variety of skeletal muscle markers are available, including myoglobin, myogenin, myoD1 and sarcomeric actin. These markers assist in confirming the presence of rhabdomyosarcoma (53). Embryonal rhabdomyosarcomas are rare in general in the female genital tract, being most common in the vagina where the differential diagnosis usually includes the Bsmall blue cell tumors of childhood^. Rhabdomyosarcomas rarely arise in the cervix, uterus or ovary. Skeletal muscle markers may also assist in establishing a diagnosis of a pleomorphic rhabdomyosarcoma in the uterus or ovary and in confirming rhabdomyoblastic differentiation in a uterine or ovarian carcinosarcoma.

Endometrial Stromal Markers CD10. CD10 or the common acute lymphoblastic leukemia (CALLA) 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 important in diagnosing an endometrial stromal neoplasm, because most endometrial stromal nodules and endometrial stromal sarcomas (low grade endometrial stromal sarcomas) exhibit diffuse intense reactivity, although fibrous variants may be negative (44Y46,54). In the distinction between an endometrial stromal and a smooth muscle neoplasm, CD10 should be used as part of a panel, as discussed, because 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 positive in mesonephric lesions within the female genital tract. Cervical mesonephric remnants and mesonephric remnants elsewhere within the female genital tract usually exhibit luminal CD10 reactivity (55Y57). CD10 reactivity in a benign cervical glandular lesion is good evidence of a mesonephric origin (55), although prostatic metaplasia may also be reactive (29). However, CD10 is of limited value in confirming a mesonephric origin for an adenocarcinoma

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because many usual endocervical and endometrial adenocarcinomas are also reactive (25). Female adnexal tumor of wolffian origin (FATWO) may be CD10 reactive as this neoplasm most likely has a mesonephric origin (56).

Other uses of CD10 staining in gynecological pathology include the distinction between a metastatic renal clear cell carcinoma involving the ovary (CD10 reactive) (58) and a primary ovarian clear cell carcinoma (CD10 negative). In addition, most trophoblastic cell populations and trophoblastic neoplasms are reactive (57).

CD10 is also of value in confirming the presence of endometrial stroma and in establishing a diagnosis of endometriosis (59). However, this is of limited value in the cervix because a rim of CD10 reactive stromal cells surrounds normal endocervical glands (55). Other gynecological neoplasms that may be CD10 reactive include leiomyosarcoma, carcinosarcoma, undifferentiated uterine sarcoma, ovarian sex cord-stromal tumors, uterine tumors resembling ovarian sex-cord tumors and mixed tumors of the vagina (60Y62). However, CD10 immunoreactivity in these neoplasms is inconsistent and unlikely to be of diagnostic value. In summary, CD10 may be expressed in a much wider range of gynecological neoplasms than was originally appreciated. When used as an aid to diagnosis, CD10 should always be part of a panel, which will depend on the differential diagnoses under consideration.

Mesothelial Markers 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, an epithelial membrane antigen marker, are the two most useful antibodies to distinguish between a serous epithelial and a mesothelial proliferation (27). Most serous proliferations are Ber-EP4 reactive and calretinin negative, the converse being the rule for mesothelial lesions. Nuclear positivity with calretinin is more specific than cytoplasmic staining for mesothelial cells (27).

Calretinin is also found in most ovarian sex cordstromal tumors (63Y66). In comparison to > inhibin, calretinin is a slightly more sensitive, but a less specific, marker of ovarian sex cord-stromal tumors. Calretinin is more likely to be reactive in an ovarian fibroma than > inhibin. However, ovarian adenocarcinomas are more likely to be reactive with calretinin than > inhibin.

In general, neoplasms which are reactive for > inhibin also show reactivity with calretinin. Other gynecological neoplasms that may show calretinin reactivity include the female adnexal tumor of wolffian origin, uterine tumor Int J Gynecol Pathol, Vol. 25, No. 2, April 2006

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resembling ovarian sex cord tumors, sex cord-like areas within endometrial stromal neoplasms and adenomatoid tumors. Mesonephric lesions, both benign and malignant, within the cervix and elsewhere in the female genital tract may show reactivity (55).

Blood Vessel Markers There are several commercially available vascular markers, including CD34,CD31, Ulex, factor 8, Fli-1, podoplanin and thrombomodulin. Some of these are reactive against other cell populations e.g., thrombomodulin stains mesothelial cells. Only CD34 will be considered.

CD34. CD34, a single chain transmembrane glycoprotein, leukocyte differentiation antigen, is expressed by hematopoietic progenitor cells (decreases with maturation), endothelial cells, and fixed connective tissue cells (e.g., fibroblasts in skin). CD34 is inconsistently expressed in several vulvovaginal mesenchymal lesions, including aggressive angiomyxoma, cellular angiofibroma and superficial myofibroblastoma (49Y51,67).

Solitary fibrous tumors rarely occur at various sites within the female genital tract and are CD34 positive (68).

Endometrial stromal neoplasms are CD34 negative, which may be of use in differential diagnosis in that many mimics, especially those in an extrauterine location, are reactive (54). Metastatic gastrointestinal stromal tumor (GIST) to the ovary or elsewhere in the female genital tract usually expresses CD34 (69), as do the rare primary GISTs arising in the vulvovaginal region or rectovaginal septum (70).

NARROW SPECTRUM DIFFERENTIATION MARKERS These are cell type specific (pathognomonic) markers that are often useful to rule in or exclude a targeted question.

Trophoblastic Markers A major application of broad-spectrum cytokeratins, as discussed above, is simple confirmation of presence or absence of implantation site. Because trophoblast cells are reactive for keratin and decidual stroma is not, keratin reactivity is an easy method to identify trophoblast in uterine products of conception or the implantation site when trying to exclude the presence of an ectopic pregnancy. Cytokeratin tends to be more robust than A-HCG or hPL, and the intensity is uniform throughout gestation.

Cytokeratin is not useful to evaluate trophoblastic disease, though. The value of > inhibin and CD10 as trophoblastic markers has already been discussed.

A Human Chorionic Gonadotropin (AHCG) hCG is a glycoprotein comprising a protein core and a carbohydrate side chain, and composed of two dissimilar subunits- > and A. The > subunits are indistinguishable from the > subunits of luteinizing hormone, folliclestimulating hormone and thyroid-stimulating hormone.

The A subunits differ and confer specificity.

A hCG reacts against syncytiotrophoblast but not cytotrophoblast. All trophoblastic neoplasms express reactivity. Choriocarcinoma shows the strongest and most diffuse reactivity. The placental site trophoblastic tumor (PSTT) and epithelioid trophoblastic tumor are less reactive. Trophoblastic elements in mixed germ cell tumors show reactivity, as does syncytiotrophoblast when it sporadically occurs in neoplasms such as dysgerminoma and endometrial carcinoma. AhCG may be found on occasion in a variety of nontrophoblastic neoplasms, including cervical squamous carcinoma (71).

Placental Alkaline Phosphatase (PLAP) The alkaline phosphatases are a heterogeneous group of glycoproteins which are usually confined to the cell surface. PLAP is a dimer of 65kD subunits and is synthesised during the G1 phase of the cell cycle. PLAP is expressed in syncytiotrophoblasts and in some intermediate trophoblastic populations, reactivity being 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 dysgerminomas are also reactive and this may be valuable in diagnosis.

Human Placental Lactogen (HPL) HPL is a member of the gene family that includes human growth hormone and human prolactin. HPL 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 (72,73). Chorion-type intermediate trophoblastic cells are usually negative or focally reactive.

Placental site trophoblastic tumor and exaggerated placental site, being lesions of implantation site intermediate trophoblast, express mel-CAM, whereas placental site nodules and epithelioid trophoblastic tumor, which are lesions of chorion-type intermediate trophoblast, are usually mel-CAM negative. In distinguishing placental site trophoblastic tumor from exaggerated placental site, double immunohistochemical staining with mel-CAM

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IMMUNOHISTOCHEMISTRY IN FEMALE GENITAL TRACT LESIONS and MIB1 has proven of value (72). In exaggerated placental site, the MIB1 index in intermediate trophoblastic cells is close to zero whereas it is significantly elevated (14 T 6.9%) in placental site trophoblastic tumor (72).

HLA-G HLA-G, which is present in all implantation-type and chorion-types of intermediate trophoblast, 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 (74). HLA-G is generally negative in nontrophoblastic uterine neoplasms (74).

HLA-G reactivity has been demonstrated in ovarian carcinomas (75).

Melanocytic Markers There are several available melanocytic markers, including tyrosinase and microphthalmia transcription factor, which will not be discussed in this review.

HMB45 HMB45 is probably the most specific marker of malignant melanoma, being melanosome associated.

HMB45 reactivity is useful to confirm the diagnosis of malignant melanoma at any site within the female genital tract, most commonly found in the vulva or vagina.

Metastatic melanoma in the ovary can assume an unusual array of morphological appearances and may easily fool the pathologist if there is no history of melanoma.

HMB45 may assist in this regard (76). However, occasional ovarian steroid cells tumors, which may mimic melanoma, are HMB45 positive (77). This is true also for melan-A (MART-1), discussed below. Another neoplasm which is characteristically positive with HMB45 is perivascular epithelioid cell tumor (PEComa) (47). This is an extremely rare neoplasm, which in the female genital tract most commonly involves the uterine myometrium (47). Clusters of epithelioid cells with clear cytoplasm coexpress both HMB45 and smooth muscle markers. In addition, uterine epithelioid leiomyosarcomas with a clear cell appearance may also express HMB45 (78,79).

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 positive (80,81).

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S100 S100 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 S100 positive include ovarian sex cord-stromal tumors and cartilaginous areas within carcinosarcomas (82).

Neuroendocrine Markers There are various commercially available neuroendocrine markers, including chromogranin, CD56, synaptophysin and PGP9.5. These vary in their specificity and sensitivity. For example, chromogranin is a highly specific but relatively poorly sensitive neuroendocrine marker whereas CD56 is sensitive but lacks specificity, although in the context of a small cell neoplasm this marker is relatively specific for neuroendocrine differentiation. Neuroendocrine markers may be used to confirm neuroendocrine differentiation within a neoplasm and establish a diagnosis of a small cell or large cell neuroendocrine carcinoma. Reactivity with neuroendocrine markers is not necessary to establish a diagnosis of a small cell neuroendocrine carcinoma because many of these are sparsely granulated and negative with neuroendocrine markers. In contrast, reactivity with neuroendocrine markers is probably necessary for a diagnosis of large cell neuroendocrine carcinoma, although this diagnosis can be strongly suspected on morphology. Rare paragangliomas and typical and atypical carcinoids occur within the female genital tract and these are reactive (83).

Lymphoid Antibodies Markers against lymphoid cells are of value in diagnosing rare hematopoietic malignancies, either lymphoma or leukemia, within the female genital tract.

Several markers may also assist in the diagnosis of a low grade endometritis, usually resting on the morphological identification of plasma cells that may be difficult to visualize with H&E when few in number. Both B and T lymphoid markers may be of use. In the normal endometrium, most lymphoid cells are of T cell or natural killer (NK) cell lineage. B lymphocytes account for under 1% of endometrial leukocytes, being mainly located in lymphoid aggregates. In endometritis, the number of T lymphocytes and NK cells does not differ from controls. However, the use of B lymphoid markers, such as CD20 and CD79a, reveals substantially increased numbers of B cells in unusual locations such as beneath the surface epithelium and intraepithelially (84). In situ hybridisation for kappa and lambda light chains may also be of value in diagnosing endometritis (85), as may Int J Gynecol Pathol, Vol. 25, No. 2, April 2006

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antibodies against plasma cells, such as syndecan and VS38 (86 87).

MARKERS OF ALTERED FUNCTION IN DISEASE STATES The aberrant expression of the following usually indicates a disease state and therefore are helpful in distinguishing normal from disease states. The pattern of aberrant expression may be pathognomonic for a particular disease state, and thus useful in resolving a differential diagnosis.

Tumor Markers CA19.9 CA19.9, an antigen of sialyl Lewisa-containing glycoprotein, is usually reactive in pancreatic, biliary or colorectal adenocarcinoma metastatic to the ovary. Most primary ovarian adenocarcinomas are negative (15), although mucinous neoplasms may be focally reactive.

Carcinoembryonic Antigen (CEA) CEA consists of a heterogeneous family of related oncofetal glycoproteins secreted into the glycocalyceal surface of gastrointestinal cells. The monoclonal antibody to CEA was raised against tumor cells derived from a hepatic metastasis of colorectal carcinoma.

Monoclonal CEA is often helpful to distinguish nonmucinous ovarian adenocarcinomas (usually negative) from colorectal adenocarcinoma (usually reactive), when used as part of a panel (Table 1) (13,15). Primary ovarian mucinous adenocarcinomas are also often reactive. Adenocarcinomas from other organs, such as pancreas and stomach, are variably positive.

As discussed previously, CEA often forms part of a panel to help distinguish endometrioid endometrial adenocarcinoma from endocervical adenocarcinoma (35Y38). 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 carcinomas 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 (88).

CEA is usually reactive in primary vulval Paget’s disease and, like CK7 and CAM 5.2, useful to exclude mimics or assess margins.

CA125 (OC125) CA125 is a mucin-like glycoprotein, which is an antibody to an ovarian carcinoma antigen. Serum CA125 levels are commonly elevated in patients with ovarian cancer, especially of the 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 (2,3). In general, primary ovarian (or peritoneal) adenocarcinomas of serous, endometrioid and clear cell types exhibit diffuse CA125 positivity. 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 including CK7, CK20 and CEA, as well as other markers of colorectal adenocarcinoma which are discussed later. CA125 reactivity is not specific for an ovarian adenocarcinoma, as primary adenocarcinomas of many other organs, including breast, lung, cervix and uterine corpus exhibit reactivity in a proportion of cases.

Mesotheliomas are commonly reactive as are benign mesothelial cells (89).

Inhibin Inhibin is a dimeric 32 kDa peptide hormone composed of an > and A subunit that the ovarian granulosa and theca cells produce. Individual antibodies are available against each subunit. Most ovarian sex cord-stromal tumors show focal to diffuse cytoplasmic reactivity with > inhibin, although fibromas, poorly differentiated Sertoli-Leydig and sarcomatoid granulosa cell tumors are sometimes negative (31,81,90Y99). Small numbers of other ovarian sex cord-stromal tumors are also negative.

Because ovarian sex cord-stromal neoplasms, such as granulosa and Sertoli cell tumors, 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 in confirmation of a metastatic neoplasm, which may occur years or decades later (100). In distinguishing between a sex cord-stromal tumor and an endometrioid carcinoma, the former are almost always negative with epithelial membrane antigen (EMA) (30,31,92) (Table 4). Ovarian sex cord-stromal tumors are unreactive to CK7, but may be focally reactive with a broad spectrum antiCK antibody. Most carcinomas are negative with >-inhibin, although a rare tumor is focally

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IMMUNOHISTOCHEMISTRY IN FEMALE GENITAL TRACT LESIONS reactive. Of import, 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 that close attention must be paid to the morphology of the particular cluster of cells that are immunohistochemically reactive.

With the advent of fine needle aspiration cytology (FNA), > inhibin staining has assumed greater importance in the evaluation of ovarian cysts (101,102). Reactivity of the cells in an aspirate with > inhibin and negative staining with EMA helps confirm the presence of granulosa cells, indicating a follicular rather than an epithelial lined cyst. > inhibin may also help to demonstrate luteinized stromal cells in cases of ovarian stromal hyperthecosis, or in association with ovarian neoplasms of non sex cord-stromal type that have resulted in androgenic or estrogenic manifestations (102).

Other gynecological neoplasms that are variably reactive with > inhibin include female adnexal tumor of wolffian origin (FATWO), cervical mesonephric adenocarcinoma, uterine tumor resembling ovarian sex cord tumor and sex cord-like areas within endometrial stromal neoplasms (33,103Y106). > inhibin also stains some trophoblastic cell populations, syncytiotrophoblast and some intermediate trophoblastic cells showing reactivity whereas cytotrophoblast is negative (107,108). Choriocarcinoma and other trophoblastic neoplasms, especially placental site trophoblastic tumor and epithelioid trophoblastic tumor, may be > inhibin positive. A inhibin is less useful diagnostically than > inhibin because many ovarian and extraovarian carcinomas are reactive (109).

OCT4 OCT4 is an octamer binding transcription factor expressed in both mouse and human embryonic stem and germ cells. Nuclear reactivity is expressed in ovarian dysgerminoma and embryonal carcinoma and in the germ cell component of gonadoblastoma (110). Most ovarian epithelial and sex cord-stromal tumors are unreactive, although occasional clear cell carcinomas have expressed reactivity (110). As clear cell carcinoma and dysgerminoma often superficially resemble each other and in some areas may appear almost identical, the information about OCT4 reactivity must be assessed with great caution.

Testicular seminomas and embryonal carcinomas are also OCT4 reactive.

HIK1083 HIK1083, a monoclonal antibody against gastric gland mucous cell mucin, is characteristically positive in cervical minimal deviation adenocarcinoma of mucinous type (ad-

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enoma malignum) (111,112). This may be diagnostically useful as normal endocervical glands are consistently negative. Focal reactivity may be present in ordinary endocervical adenocarcinomas and less well differentiated areas in adenoma malignum may be negative.

The benign endocervical glandular lesion, lobular endocervical glandular hyperplasia, which can mimic adenoma malignum, may also be positive with HIK1083 (111). It has been suggested that lobular endocervical glandular hyperplasia may, in some cases, be a precursor of adenoma malignum because rare cases with transitional features have been described.

CDX-2 Cdx-2 is a gene that encodes for a transcription factor involved in the development and differentiation of the small and large intestines. Colorectal adenocarcinomas usually exhibit diffuse nuclear reactivity with antibodies against Cdx-2 (113Y116) and this may be useful, as part of a panel (Table 1), in distinguishing between a primary ovarian adenocarcinoma and a metastatic colorectal adenocarcinoma. Complicating interpretation is that whereas primary ovarian endometrioid adenocarcinomas rarely exhibit Cdx-2 reactivity, some primary ovarian mucinous tumors do, the percentage varying widely among studies (113Y116). This reinforces the caution that markers of intestinal differentiation may on occasion be expressed in primary ovarian mucinous neoplasms and that a panel of antibodies should always be used to distinguish between a primary ovarian and metastatic colorectal carcinoma. In summary, Cdx-2 is a relatively sensitive marker of colorectal cancer, but is of limited specificity. > Fetoprotein (> FP) > FP is a glycoprotein composed of 590 amino acid residues that is present in yolk sac tumors and some cases of hepatocellular carcinoma. In the female genital tract, > FP is useful in establishing a diagnosis of yolk sac tumor both of ovarian or extraovarian origin. Primary hepatoid carcinomas of the ovary, metastatic hepatocellular carcinoma and metastatic hepatoid carcinomas from other organs may also be reactive. Some Sertoli-Leydig cells tumors in the ovary express > FP and are associated with an elevated > FP serum level (117).

Hep-PAR1 Hep-PAR 1 is expressed in most hepatocellular carcinomas, ovarian hepatoid yolk sac tumors, primary ovarian hepatoid carcinomas and hepatoid carcinomas metastatic to the ovary from organs in addition to the liver (118). Hep-PAR1 is of no value to distinguish any Int J Gynecol Pathol, Vol. 25, No. 2, April 2006

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of these tumors from each other. Occasional cervical carcinomas, either of glandular or squamous type, may also express Hep-PAR 1 (119).

MUC Antibodies Mucins are high molecular weight glycoproteins. Several mucin genes have been identified or cloned (MUCIMUC12) and monoclonal antibodies to these are available (120). Expression of the mucin gene MUC5AC helps distinguish colonic adenocarcinoma metastatic to the ovary (nonreactive) from a primary ovarian adenocarcinoma (reactive) (14,121). Appendiceal and pancreatic adenocarcinomas typically express MUC5AC (14). Colorectal adenocarcinomas express MUC2. MUC 2 expression in vulval Paget’s disease favors an underlying colorectal adenocarcinoma (122). MUC 2 reactivity is also useful to confirm that pseudomyxoma peritonei is of appendiceal origin (123,124). MUC5AC is expressed in endocervical glands.

CD99 The CD99 antigen, or MIC2 gene product, is a cell surface glycoprotein that is involved in cell adhesion processes. CD99 is important in antibody panels used to diagnose small round blue cell tumors.

In the female genital tract, CD99 helps establish the diagnosis of peripheral primitive neuroectodermal tumor (pPNET) which has rarely been described in the ovary, uterus, cervix and vulva (125). A positive reactivity should be membranous. Cytoplasmic reactivity is less specific.

Ovarian sex cord-stromal tumors also commonly exhibit membranous CD99 positivity, as may uterine tumors resembling ovarian sex cord tumors and sex cord-like areas within endometrial stromal neoplasms (103,104,126).

Tumor Suppressor Genes WT1 The WT1 gene is a tumor suppresser gene located on the short arm of chromosome 11 at p13. Although first reported as a candidate for the main gene implicated in Wilms` tumor development, WT1 immunohistochemical expression is found in other organs but is restricted to the normal tissues of ovary, kidney, testis, spleen and mesothelium. WT1 is expressed in a number of malignancies, including malignant mesothelioma and intraabdominal desmoplastic small round cell tumor (IADSRCT).

Antibodies are available against both the C-terminal and the N-terminal of WT1 and nuclear staining is regarded as positive. IADSRCT is reactive with antibodies against the C-terminal (152). This may be useful in diagnosis and in the distinction of IADSRCT from the other Bsmall blue cell tumors,[ such as rhabdomyosar-

coma and neuroblastoma, which rarely may involve the ovary and peritoneum (127).

Primary ovarian, peritoneal and tubal serous carcinomas are usually WT1 positive (antibody against Nterminal) (128Y133). In a poorly differentiated ovarian carcinoma, nuclear WT1 reactivity favors a serous neoplasm because most endometrioid, clear cell and mucinous carcinomas are negative. Transitional cell carcinomas of the ovary are also commonly WT1 reactive (134). With a disseminated serous carcinoma involving more than one site, diffuse reactivity with WT1 favors an ovarian, peritoneal or tubal primary. Most uterine serous carcinomas are negative or focally reactive, although the literature is somewhat contradictory (128Y133). When dealing with such cases, correlation among clinical, pathological, radiological and serological parameters is critical.

When disseminated adenocarcinoma involves the abdomen and peritoneal cavity in a female, diffuse nuclear WT1 reactivity strongly favors a serous carcinoma arising from the ovary, peritoneum or fallopian tube.

Most pancreatic, biliary, gastric, breast and colorectal carcinomas are WT1 negative. In this regard WT1 should be used as one element of a panel of markers.

Ovarian small cell carcinoma of hypercalcemic type is usually reactive with an antibody against the N-terminal of WT1 (127,135). As this small cell tumor often morphologically resembles juvenile granulosa cell tumor (JGCT), a panel of antibodies to WT1, > inhibin and epithelial membrane antigen (EMA) may be of value (Table 5).

Other neoplasms involving the female genital tract that may be WT1 reactive include malignant mesothelioma, adenomatoid tumor (not surprising because these are of mesothelial derivation), endometrial stromal neoplasms, leiomyomatous tumors and ovarian sex cord-stromal tumors (46,136).

DPC4 Dpc 4 (DPC4 (for Deleted in Pancreatic Cancer, locus 4) is a tumor suppressor gene that is inactivated by

TABLE 4. Antibodies of value in distinguishing between ovarian endometrioid adenocarcinoma and sex cordYstromal tumor Antibody

Endometrioid adenocarcinoma

Sex cordYstromal tumor

CK7 EMA > Inhibin Calretinin Broad spectrum CKs

+ + j j or + Diffuse +

j j + + j or focal +

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IMMUNOHISTOCHEMISTRY IN FEMALE GENITAL TRACT LESIONS TABLE 5. Antibodies of value in distinguishing between ovarian small cell carcinoma of hypercalcemic type and juvenile granulosa cell tumor

Antibody

Ovarian small cell carcinoma of hypercalcemic type

Juvenile granulosa cell tumor

WT1 (N-terminal) > Inhibin EMA

Diffuse + (intense) j Focal +

j or focal + (weak) Diffuse or focal + j or focal +

allelic loss in approximately 50% of pancreatic cancers. In such cases there is absence of immunohistochemical staining (14). In contrast, ovarian, colorectal and appendiceal carcinomas are usually Dpc4 reactive (69) because there is no allelic loss. Because pancreatic adenocarcinoma metastatic to ovary may closely mimic a primary ovarian mucinous adenocarcinoma histologically (or even a benign or borderline mucinous cystadenoma), evaluation of Dpc4 reactivity is helpful diagnostically. p53 p53 is a tumor suppressor gene, located on the short arm of chromosome 17, which encodes a 35 kDa nuclear protein involved in regulating cell growth. Mutations of the p53 gene are amongst the most commonly detected genetic abnormalities in human neoplasia. Mutations result in a conformational change of the protein, which becomes stabilized, thus allowing for immunohistochemical detection. The most widely used antip53 antibody is D07. Usually, but not always, a diffuse intense nuclear reactivity is found whenever p53 mutation occurs.

However, lower levels of p53 reactivity may occur without mutation, resulting from stabilized wild type p53 by nonmutational events.

Diffuse intense nuclear p53 reactivity is characteristic of uterine serous carcinoma and p53 is of value in distinguishing this carcinoma, especially those glandular variants without papillary formation, from an endometrioid carcinoma which usually exhibits much lower levels (137Y139). Diffuse p53 staining in a papillary endometrial carcinoma is more in keeping with a serous adenocarcinoma than an endometrioid adenocarcinoma with papillary folds. However, there are many exceptions.

An occasional serous adenocarcinoma may be p53 negative and some endometrioid adenocarcinomas show significant nuclear reactivity. p53 is useful in identifying the precursor lesion of serous carcinoma, namely endometrial intraepithelial carcinoma (EIC). This is often a subtle lesion that not uncommonly involves an endometrial polyp. In this situation, p53 immunohistochemistry may be combined with ER and PR, as dis-

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cussed later. In some studies, the p53 labeling index has proven to be an independent prognostic factor in endometrial carcinoma (140) but in others it is dependent on cell type and other pathological parameters. Some believe that the p53 labeling index is an independent prognostic factor only in endometrioid type endometrial adenocarcinomas (140). Regardless, p53 helps distinguish serous adenocarcinoma, EIC and clear cell carcinoma from benign papillary endometrial proliferations and metaplasias, including the Arias-Stella effect (141,142), a problem most likely to be encountered in small endometrial biopsies. Endometrial metaplasias may exhibit a weak heterogeneous pattern of p53 reactivity (141). Diffuse p53 reactivity is much more common in uterine leiomyosarcomas than benign leiomyomatous neoplasms, including symplastic or atypical leiomyoma (143).

In ovarian carcinomas, diffuse p53 reactivity is more common in serous and undifferentiated carcinomas than in other morphological subtypes (144). Recently, a dualistic pathway of ovarian serous carcinogenesis has been proposed (145,146). Diffuse p53 staining is far more common in high grade compared with low grade ovarian serous carcinoma (147). p53 mutation is likely to occur early in the evolution of high grade ovarian serous carcinoma, because diffuse reactivity has been identified in small microscopic high grade serous carcinomas, especially in prophylactic oophorectomy in patients with BRCA1 and BRCA2 mutations. It is generally absent or focally reactive in low grade tumors and borderline serous tumors, and this is one of several pieces of evidence that the borderline tumor pathogenetically is entirely different from the ordinary high grade serous adenocarcinoma. p53 positivity has also been described in atypical epithelium in ovarian cortical inclusion cysts adjacent to serous carcinomas, so called ovarian dysplasia or ovarian intraepithelial neoplasia (148).

In the vulva, p53 helps distinguish undifferentiated VIN(negative) from simplex VIN(reactive). Strong p53 reactivity confined to the basal cell layer favors differentiated VIN (149), although p53 reactivity may also be seen in lichen sclerosis in the absence of differentiated VIN. p53 reactivity in vulval Paget’s disease helps predict cases in which there is associated dermal invasion (150). p63 The p63 gene is a transcription factor which belongs to the p53 family. p63 protein has six isoforms, three each classified into two groups, designated TA and $ N p63 (151). Cytotrophoblast expresses the $ N p63 isoform whereas chorion-type intermediate trophoblast in the fetal membranes, placental site nodules and epithelioid trophoblastic tumors express the TA p63 isoform (151).

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Intermediate trophoblast in the implantation site and placental site trophoblastic tumor do not express p63. p63 helps distinguish cervical small cell neuroendocrine carcinoma from small cell nonkeratinizing squamous carcinoma (152). Most squamous carcinomas diffusely react whereas neuroendocrine carcinomas, including small cell neuroendocrine carcinoma, are negative or focally reactive (152). In the cervix, p63 is preferentially expressed in immature cells of squamous lineage, including basal and reserve cells (153).

PTEN PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a tumor suppresser gene mutated in a high percentage of endometrioid adenocarcinomas of the endometrium (154). PTEN mutation is generally associated with loss of PTEN immunohistochemical staining (155). PTEN is also mutated in some endometrioid adenocarcinomas of the ovary (156). PTEN mutation occurs early in the development of endometrioid type endometrial adenocarcinoma because mutation with associated absence of immunohistochemical reactivity has been found in over half of the cases of the precursor lesion, called endometrial intraepithelial neoplasia (EIN) in one classification scheme and atypical endometrial hyperplasia in another. Of note, EIN is different from endometrial intraepithelial carcinoma (EIC), the precursor lesion to serous adenocarcinoma of the endometrium.

PTEN-null glands also occur not uncommonly in normal cyclical endometrium (155), especially normal secretory endometrium, and therefore a lack of reactivity cannot be used to diagnose EIN or atypical endometrial hyperplasia.

Furthermore, not all cases of EIN exhibit an absence of PTEN reactivity and in other cases loss of expression precedes the development of morphological features of EIN. The most widely used antiPTEN antibody is 6H2.1.

The PTEN null rate in endometrial adenocarcinoma varies by tumor subtype, ranging from a low of 13% of serous cancers to almost 60% of all endometrioid cancers (155).

PTEN inactivation is highest for those tumors proceeded by an EIN lesion, in which case 66% of EIN and 83% of their associated adenocarcinomas are PTEN-null (155).

Proto-oncogenes BCL-2 bcl-2 is a proto-oncogene, located on chromosome 18, which encodes a 25kDa protein mainly localized to the inner mitochondrial membrane. This extends cell survival by blocking apoptosis.

In proliferative endometrium, bcl-2 is diffusely expressed in the gland cell cytoplasm. The activity is re-

duced in the glands of both atypical hyperplasia and endometrioid type adenocarcinomas (157). Furthermore, endometrioid adenocarcinomas of the corpus express bcl2 more commonly than nonendometrioid tumors. In the cervix, bcl-2 is normally expressed in the basal cell layer of the squamous epithelium. Normal fallopian tube epithelium is bcl-2 reactive (158), as are foci of ciliated metaplasia involving the ovarian surface epithelium and the epithelium of cortical inclusion cysts (159).

Tuboendometrial metaplasia and endometriosis in the cervix generally exhibit diffuse cytoplasmic positivity and this may be helpful in the diagnosis of these lesions and in their distinction from AIS, which is generally negative (160,161). Endometrial stromal neoplasms are also commonly bcl-2 reactive but this is of limited value because many other tumors included in the differential diagnosis are also reactive (54).

CD117 (C-Kit) CD117, a transmembrane tyrosine kinase receptor, is expressed in metastatic GIST within the ovary or elsewhere in the female genital tract (69) and in rare primary GISTs arising in the vulvovaginal region or rectovaginal septum (70). Some gynecological sarcomas, including leiomyosarcoma (162,163), may express CD117, as occasionally do other tumors, including uterine carcinosarcoma, ovarian serous carcinoma, and germ cell tumors such as dysgerminoma and gonadoblastoma (147,164).

Cell Cycle & Nuclear Proliferation Markers KI67 (MIB-1) These are several markers of cell proliferation, the two best known being MIB1 (which is reactive against the Ki67 antigen) and proliferating cell nuclear antigen (PCNA).

These proliferation markers, especially MIB1, are of value in diagnosis in several areas of gynecological pathology. MIB1 identifies all cells in non GO phases of the cell cycle, i.e., all proliferating cells. PCNA expression is highest during the S phase of the cell cycle but due to a relatively long half-life persists in cells that are no longer cycling and are in GO. Cervical squamous lesions exhibit an increased MIB1 proliferation index from normal through CIN 3 (165Y168). In normal squamous epithelium, MIB1 positivity is largely confined to the basal and parabasal layers. MIB1 is of little practical value in the separation of adjacent lesions in the CIN spectrum but may be useful in the distinction between CIN and benign mimics such as atrophic squamous epithelium, transitional metaplasia and immature squamous metaplasia, the benign mimics

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IMMUNOHISTOCHEMISTRY IN FEMALE GENITAL TRACT LESIONS exhibiting a low MIB1 proliferation index whereas there is usually substantial to nearly full thickness reactivity (990%) in CIN 3. MIB1 may also be used to evaluate cauterized cervical resection margins, i.e., cauterized CIN 3 from cauterized nondysplastic squamous epithelium (167).

Similarly in the endocervix, MIB1 helps to distinguish endocervical AIS and benign mimics such as tuboendometrial metaplasia, endometriosis and microglandular hyperplasia (MGH) (88,160,161,169,170). Benign mimics usually exhibit a low MIB1 proliferation index of G10% whereas in most cases of AIS the proliferation index is in excess of 30%, usually much greater. In general, only scattered nuclei react in benign mimics whereas most nuclei are positive in AIS and a semiquantitative assessment is all that is necessary. Problematically, there may be overlap at the lower end of the high grade AIS spectrum and the upper end of the benign spectrum (especially in cases of tuboendometrial metaplasia and endometriosis) and MIB1 should be used as part of a panel which can include bcl-2 and p16 (Table 6). MIB1 may also assist in evaluating cauterized cervical resection margins, differentiating precancer from normal.

In the vulva, MIB1 has some utility in the evaluation of squamous lesions (171,172). Koilocytotic changes in the vulva may be less well developed than in the cervix.

True HPV infection of the vulva is associated with clusters of MIB1 reactive cells in the middle and upper thirds of the epithelium (172), which helps to definitively categorize the lesion in which an equivocal diagnosis of condyloma might be made. The cells of high grade VIN express MIB1 throughout much of the full epithelial thickness and this is of value in the distinction of high grade VIN from atrophic squamous epithelium.

MIB1, together with hormone receptors and p53, helps to distinguish endometrioid endometrial cancer from primary clear cell or serous carcinoma. The former generally exhibit a much lower MIB1 proliferation index than serous and clear cell tumors where almost every nucleus is reactive. This may also assist in identifying small foci of EIC, the putative precursor lesion to serous carcinoma. Proliferation indices in endometrial cancers are an independent prognostic factor in some, but not all, studies (173Y175).

Proliferation markers have been used when assessing trophoblastic lesions. There is no significant difference in the proliferation index in chorionic villi between hydatidiform mole and hydropic abortion (176) Reactivity with proliferation markers is largely confined to villous cytotrophoblast. Proliferation markers do not predict progression of hydatidiform moles to persistent tropho-

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blastic disease. MIB1 also aids in distinguishing placental site trophoblastic tumor (PSTT) and an exaggerated placental site (72). In the former, the MIB1 index is significantly elevated (14 T 6.9%) whereas it is nearly zero in the latter (72). Care should be taken to exclude reactivity of small lymphocytes in the latter. MIB1 is also of value in distinguishing between PSTT and choriocarcinoma, because the latter exhibits a much higher proliferation index (72). p16. p16, also known as cyclin dependent kinase-4 inhibitor (CD K4-I), is the product of the INK4-A gene and specifically binds to cyclin D-CDK4/6 complexes to control the cell cycle at the G1-S interphase. In the cervix, diffuse p16 staining usually correlates with the presence of high risk HPV (177Y179). Thus, there is diffuse p16 expression (usually a combination of nuclear and cytoplasmic staining) in high grade CIN. A proportion of low grade CIN is also positive, usually those cases associated with high risk HPV infection (177). In cervical squamous lesions, p16 may assist in identifying small focal areas of high grade CIN and in distinguishing atypical immature squamous metaplasia from high grade CIN involving immature metaplastic squamous epithelium (178,180).

Most neoplastic and preneoplastic endocervical glandular lesions exhibit diffuse p16 positivity because these are generally associated with high risk HPV (160,181,182). p16 may be useful in diagnosing endocervical adenocarcinoma in situ (AIS)(also known as high grade cervical glandular intraepithelial neoplasiaHCGIN), which is usually diffusely reactive, and distinguishing this from mimics such as tuboendometrial metaplasia (TEM) and endometriosis which are either negative or focally reactive (160). In this regard, p16, MIB1 and bcl2 can be used as part of a panel (160) (Table 7). As discussed above, p16 helps distinguish endocervical adenocarcinoma, which exhibits diffuse reactivity, from endometrial adenocarcinoma of endometrioid type, which is negative or focally reactive (183,184). The squamous areas in endometrial adenocarcinomas may be strongly reactive. Rare cases of uterine corpus endometrioid adenocarcinoma will exhibit diffuse reactivity but this is exceptional. Some endometrial serous carcinomas also react diffusely with p16 (183). p16 is useful to distinguish metastatic cervical adenocarcinoma in the ovary (p16 reactive) from primary ovarian endometrioid or mucinous adenocarcinoma (p16 negative) (185). In the vulva, p16 positivity is characteristic of bowenoid, warty or undifferentiated VIN (186,187), because this disease in all stages of its Int J Gynecol Pathol, Vol. 25, No. 2, April 2006

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development is associated with HPV infection. Differentiated or simplex VIN is usually p16 negative because there is no association with HPV (186,187). Similarly, HPV associated vulval squamous carcinomas are p16 reactive whereas those not associated with HPV are negative. p57. p57, also known as Kip2, is a cell cycle inhibitor of cell proliferation and tumor suppressor encoded by a strongly paternally imprinted, maternally expressed gene. p57 is only expressed when maternal DNA is present. A particular use is in the distinction of complete hydatidiform mole from partial hydatidiform mole and hydropic abortion. p57 is expressed in the nuclei of cytotrophoblast and villous mesenchyme in the normal placenta, hydropic villi and villi of partial mole because all have a maternal component (188Y192). In contrast, p57 is negative in the complete mole, because these villi are paternally derived and lack maternal DNA. Positive reactivity in decidua and extravillous trophoblast (it is not known why extravillous trophoblast reacts) acts as an internal positive control. p57 cannot determine whether a trophoblastic neoplasm has arisen from a preexisting partial or complete mole or nonmolar pregnancy (193).

Hormone Receptors Estrogen Receptor (ER) and Progesterone Receptor (PR) Many native normal tissues and tumors arising within the female genital tract exhibit positive immunohistochemical reactivity with antibodies against ER and PR. There are several situations in which these markers have diagnostic value.

Most of the vulvovaginal mesenchymal lesions are reactive with ER and PR, including aggressive angiomyxoma, angiomyofibroblastoma, cellular angiofibroma, superficial cervicovaginal myofibroblastoma and smooth muscle neoplasms (49Y51,67,194). Therefore assessing ER and PR reactivity does not assist in distinguishing between these neoplasms, most of which are thought to arise from the zone of hormone receptor positive subepithelial cells that extends from the cervix to the vulva. The fact that aggressive angiomyxoma is often hormone receptor positive is the rationale for treating these neoplasms with gonadotropin releasing hormone agonists, especially recurrent neoplasms and those tumors not amenable to surgical resection (195).

Endometrial cancers of endometrioid type, often referred to as type 1 cancers, are commonly ER and PR reactive, whereas serous and clear cell tumors, the so called type 2 cancers, are not (196). Pathogenetically, endometrioid cancers often arise on a background of

endometrial intraepithelial neoplasia (EIN) and in some as yet undefined mechanism are often related to abnormal estrogen exposure or abnormal estrogen receptivity. All EIN lesions are reactive for both ER and PR. All anovulatory endometria (this encompasses the balance of Bhyperplasias^) are also all essentially positive.

The serous and clear cell tumors occur in an older population of women, have an adjacent endometrium that is atrophic and are unrelated pathogenetically to estrogen. In practice there may be immunohistochemical overlap with some type 1 cancers being ER and PR negative whereas some type 2 cancers are reactive, albeit usually focally so. Despite this, immunohistochemical staining with ER and PR may assist in distinguishing between the type of neoplastic classes. For example, a papillary endometrioid carcinoma may be mistaken for a serous carcinoma whereas a glandular variant of serous carcinoma without papillary formation may be mistaken for an endometrioid adenocarcinoma. Diffuse strong nuclear reactivity with ER and PR favors an endometrioid adenocarcinoma whereas negative staining or focal reactivity suggests a serous carcinoma. In practice it is useful to combine ER and PR with p53, the latter usually being diffusely reactive in serous carcinomas and negative or only focally reactive in endometrioid cancers.

Similarly, a combination of ER, PR and p53 may be of value in distinguishing problematic endometrial metaplasias and benign papillary proliferations from a small uterine serous carcinoma or its presumed precursor lesion endometrial intraepithelial carcinoma (EIC). Endometrial metaplasias usually exhibit a weak heterogeneous pattern of p53 staining whereas EIC generally exhibits diffuse intense reactivity (141). Quantitative ER and PR are also of prognostic value in endometrial cancers, in that hormone receptor positive neoplasms have a better prognosis than those which are negative. However, the prognostic value of ER and PR staining is not independent of other parameters, such as tumor type and grade.

Many other uterine neoplasms may be ER and PR positive, including endometrial stromal sarcoma and smooth muscle neoplasms, both benign and malignant.

ER assessment as part of a panel helps differentiate endometrioid adenocarcinoma of the endometrium and an endocervical adenocarcinoma (Table 7) (35Y38,183).

This may be a difficult distinction to make when tumor is present in both endometrial and cervical biopsies or in a hysterectomy specimen where tumor involves both the corpus and cervix. Endometrial adenocarcinomas of endometrioid type are generally diffusely ER reactive whereas endocervical adenocarcinomas are negative or at most focally reactive (35,37). In a panel with vimentin, monoclonal CEA and p16 (183,184), endometrioid type

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IMMUNOHISTOCHEMISTRY IN FEMALE GENITAL TRACT LESIONS TABLE 6. Antibodies of value in distinction between cervical tuboendometrial metaplasia and endometriosis and adenocarcinoma in situ Antibody MIB1 Bcl-2 p16

Tuboendometrial metaplasia/endometriosis

Cervical AIS

G30% Diffuse + j or focal +

950% j Diffuse +

endometrial adenocarcinomas are usually vimentin reactive, CEA negative or focally reactive and p16 negative or focally reactive. In contrast, endocervical adenocarcinomas are usually vimentin negative and diffusely reactive with CEA and p16. Squamoid elements in endometrioid adenocarcinomas of the uterus may be both CEA and p16 reactive. Techniques, such as in situ hybridization, to demonstrate human papillomavirus (HPV) may also be of value. Endometrioid adenocarcinoma in the corpus is negative, whereas endocervical adenocarcinoma often is reactive (197).

Other situations in which ER and PR may be of diagnostic value are the distinction between a primary ovarian adenocarcinoma and a secondary adenocarcinoma from outside the female genital tract. In general, ER and, or, PR reactivity suggests an ovarian primary, although many primary ovarian adenocarcinomas are negative. However, ER and PR are of no value in the distinction between a primary ovarian adenocarcinoma and a metastasis from the breast or from elsewhere within the female genital tract.

Androgen Receptor In the cervix and vagina, androgen receptor is reactive in mesonephric remnants and ectopic prostatic tissue (29). Normal endocervical glands are usually negative, although the expression of androgen receptor in the wide range of benign endocervical glandular lesions has not been extensively studied. Normal cervical and vaginal stromal fibroblasts are androgen receptor positive (29).

Other neoplasms in the female genital tract which have been found to express androgen receptor in a variable

TABLE 7. Typical reaction patterns of endometrial adenocarcinoma of endometrioid type and endocervical adenocarcinoma Antibody

Endometrioid type endometrial adenocarcinoma

Endocervical adenocarcinoma

ER Vimentin Monoclonal CEA p16

Diffuse + Diffuse + j or focal + j or focal +

j j Diffuse or focal + Diffuse +

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percentage of cases include endometrial adenocarcinoma, endometrial stromal sarcoma, cervical mesonephric adenocarcinoma and female adnexal tumor of wolffian origin (33,198,199).

Oxytocin Receptor Oxytocin is produced by the posterior pituitary and is released into the systemic circulation in response to stimuli such as parturition and suckling. Oxytocin receptor is present in the nonpregnant uterus, in both the endometrium and myometrium (200). An antibody against oxytocin receptor helps to distinguish a uterine smooth muscle tumor (oxytocin receptor reactive) from an endometrial stromal neoplasm (oxytocin receptor negative) (201).

Cell Adhesion Markers A-catenin A-catenin and adenomatous polyposis coli gene mutations are common in colorectal carcinoma (202).

As a result of these mutations, A-catenin is localized to the nucleus of colorectal carcinomas where it may be detected immunohistochemically. A-catenin may be a useful addition to the panel of antibodies used to distinguish between a primary ovarian adenocarcinoma and a metastatic colorectal adenocarcinoma (Table 1) (203).

Most, but not all, colorectal adenocarcinomas exhibit nuclear reactivity whereas the majority of primary ovarian mucinous neoplasms are negative. Primary ovarian endometrioid adenocarcinomas may exhibit nuclear positivity because they can be associated with A-catenin gene mutation (203) and the squamoid elements are often intensely positive (204). Membranous A-catenin staining is the norm in epithelial cells and only nuclear positivity is of diagnostic value.

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