PANEL OF ANTIBODIES OF VALUE IN DISTINCTION BETWEEN TYPE 1 AND TYPE 2 ENDOMETRIAL CANCER
A dual model of endometrial carcinogenesis is now well established. Type 1 carcinoma, the prototype of which is endometrioid adenocarcinoma, is usually low grade, oestrogen-dependent and arises in a background of endometrial hyperplasia in the perimenopausal or early postmenopausal age group. In contrast, type 2 carcinoma, the prototype of which is uterine serous carcinoma (USC), commonly referred to as papillary serous carcinoma, is a high grade neoplasm which is not oestrogen-dependent and which usually arises in elderly postmenopausal women from an atrophic endometrium. Usually the morphological distinction between an endometrioid adenocarcinoma and USC is straightforward but, on occasions, it may be difficult for a number of reasons (it should be remembered that combined endometrioid and serous neoplasms are not uncommon). Endometrioid adenocarcinoma may have a focal or diffuse papillary growth pattern, including the villoglandular variant, and this may result in overdiagnosis of USC. Conversely a glandular variant of USC exists with little or no papillary formation which may result in underdiagnosis of USC. This is an argument for the nomenclature USC rather than uterine papillary serous carcinoma. The nuclear features are different between endometrioid adenocarcinoma and USC but, in problematic cases, immunostaining with anti-p53 (DO7), MIB1 and estrogen receptor (ER) may be of value². Endometrioid adenocarcinoma is usually positive with ER, exhibits a low to moderate proliferation index with MIB1 and is largely negative with D07, although some endometrioid tumours, especially those of high grade, may be positive with DO7³ and negative with ER. Conversely, USC characteristically exhibits diffuse nuclear positivity with DO7 and a high proliferation index with MIB1 but is negative with ER. The combination of DO7, MIB1 and ER may be useful in diagnosing USC, especially the glandular variant. Conversely, diffuse positivity with ER and negative staining with D07 in a papillary endometrial adenocarcinoma is against a USC. As already stated, the glandular variant of USC may be overlooked and misdiagnosed as an endometrioid adenocarcinoma. Generally, in endometrioid adenocarcinomas with good glandular formation, the nuclear features are low grade and, although exceptions occur, the nuclear and architectural grades usually are broadly similar. In a tumour with good glandular differentiation throughout, but with marked cytologic atypia, a diagnosis of the glandular variant of USC should be considered. MIB1, DO7 and ER may be useful in this scenario, as already described.

A recent study has shown that immunohistochemical staining with ß-catenin and E cadherin may be of value in the distinction between grade 3 endometrioid adenocarcinoma and USC⁴. Nuclear expression of ß-catenin is suggestive of an endometrioid carcinoma whereas moderate or strong E cadherin staining is suggestive of USC.

FOCAL USC AND ENDOMETRIAL INTRAEPITHELIAL CARCINOMA (EIC) ARISING IN ENDOMETRIAL POLYPS OR NON-POLYPOID ENDOMETRIUM
Previous reports have drawn attention to the fact that USC and its precursor lesion endometrial intraepithelial carcinoma (EIC) may rarely involve and be largely confined to otherwise benign endometrial polyps^2,5-7. This is an argument for careful pathological sampling of endometrial polyps. EIC may focally involve polypoid or non-polypoid endometrium and be easily overlooked. With EIC and USC arising in otherwise benign endometrial polyps, there is usually an abrupt transition from glands lined by atrophic or weakly proliferative endometrial epithelium to glands lined by cells with nuclear features characteristic of EIC or USC. Pre-existing glands in EIC are lined by cells with markedly pleomorphic nuclei, a high nuclear to cytoplasmic ratio, nuclear hyperchromatism and a high mitotic rate, often with abnormal mitoses. D07 and MIB1 staining may be useful in highlighting the areas of EIC which could otherwise be missed if they are focal^2,8. The cells of EIC and USC are characteristically strongly positive with D07, exhibit a high proliferation index with MIB1 and are ER-negative. This contrasts with the surrounding endometrial epithelial cells which are ER-positive, D07-negative and exhibit a low proliferation index with MIB1. An occasional problem in endometrial polyps is that degenerative changes may occur resulting in focal, quite marked nuclear atypia. This is especially so when there is associated metaplasia, eg oxyphil metaplasia. In such instances, staining with the aforementioned antibodies may be useful in distinguishing degenerative nuclear atypia (D07-negative, low proliferation index with MIB1, ER-positive) from EIC.

When small numbers of markedly abnormal cells are seen in endometrial biopsy or curettage specimens admixed with atrophic endometrium, a diagnosis of EIC or USC should be suspected, especially in an elderly postmenopausal woman. The aforementioned immunostains may facilitate a correct diagnosis. The effects of radiation may also result in profound nuclear changes resembling EIC and the panel of antibodies detailed may be useful in this instance.

DISTINCTION BETWEEN ENDOMETRIAL AND ENDOCERVICAL ADENOCARCINOMA
With small biopsy specimens the histological distinction between a primary endometrial and endocervical adenocarcinoma may be difficult, although careful morphological examination usually allows a confident distinction. In some cases, tumour is present in both endometrial and endocervical biopsies and preoperative imaging procedures do not help in establishing the site of origin. The preoperative distinction between an endometrial and an endocervical tumour is important since primary surgical treatment may differ. Endometrial adenocarcinoma is usually treated by simple hysterectomy whereas endocervical adenocarcinoma is generally managed by primary radiation therapy or by radical hysterectomy and pelvic lymphadenectomy. Difficulties may also occur when in the resection specimen the tumour involves the lower uterine segment and upper endocervix. Morphological clues as to the site of origin include coexisting atypical endometrial hyperplasia, stromal foam cells or squamous morules, suggesting an endometrial origin or foci of cervical adenocarcinoma in situ (AIS) suggesting an endocervical origin. When there is doubt, immunohistochemical studies may assist. Recent reports have addressed the value of vimentin, monoclonal CEA and ER staining^9-11 and this combination of antibodies may be of value in the distinction between a primary endometrial and endocervical adenocarcinoma. Endometrioid type endometrial adenocarcinomas are characteristically diffusely positive with ER and vimentin but negative with CEA (squamous morules often exhibit strong positivity with CEA). In contrast, primary cervical adenocarcinomas of usual type are characteristically, although not always, CEA-positive and vimentin and ER-negative (occasional cases exhibit focal weak nuclear positivity with ER). However, in some cases, there is immunohistochemical overlap. The question also arises as to the immunophenotype of mucinous adenocarcinoma of the endometrium and endometrioid adenocarcinoma of the cervix ie whether the immunophenotype of these neoplasms is more dependent on the site of origin or the pattern of differentiation. It should be stated that, in this author's opinion, endometrioid adenocarcinomas of the cervix are rare, although in some institutions such a diagnosis is frequently made. A recent study addressed the question of whether the immunophenotype of mucinous carcinoma of the endometrium and endometrioid carcinoma of the cervix is more dependent on the site of origin or the differentiation of the tumour¹¹. It was concluded that ER staining is more dependent on the site of origin (positivity is more common in endometrial than endocervical carcinoma), whereas vimentin positivity is more dependent on the diffentiation of the tumour (being more common in endometrioid than mucinous neoplasms). However, these authors concluded that if a tumour exhibited strong positive staining with vimentin and ER, then it was almost certainly of endometrial origin.

Human papilloma virus (HPV) studies have also been found to be of value in the distinction between an endometrial and an endocervical primary, especially if combined with some of the markers listed above¹². HPV is much more common in endocervical than endometrial adenocarcinomas, although it should be acknowledged that several studies have identified HPV in a proportion of endometrial adenocarcinomas^13,14. A recent study found a combination of immunohistochemical staining with ER and progesterone receptor (PR) and in situ hybridisation for HPV to be extremely useful in distinguishing endometrial from endocervical adenocarcinomas¹².

The cyclin-dependent kinase-4 inhibitor (CDK4-I), also known as p16, is the product of the INK4-A gene and specifically binds to cyclin D-CDK4/6 complexes to control the cell cycle at the G1S interphase. Increased expression of p16 has been shown in high grade cervical squamous intraepithelial lesions and in low grade lesions associated with high risk HPV types^15-17. Positive staining of cervical AIS and adenocarcinoma has also been shown^18,19. A recent study has shown that p16 staining may be of value in distinguishing between an endometrial and an endocervical adenocarcinoma²⁰, albeit with some overlap. In general, endocervical adenocarcinomas exhibit diffuse positivity with p16 involving 100% of cells. Endometrial adenocarcinomas are usually focally positive although small numbers of cases exhibit diffuse positivity. The morphologically benign squamous elements of endometrial adenocarcinomas are often diffusely positive with p16. Whether those endometrial adenocarcinomas which are diffusely positive with p16 are associated with HPV should be addressed by further studies. It can be concluded that p16 may be of some value in the distinction between an endometrial and an endocervical adenocarcinoma, especially if used in conjunction with the other antibodies mentioned. Strong diffuse positivity of 100% of cells with p16 favours an endocervical origin.

DISTINCTION BETWEEN USC AND OVARIAN SEROUS CARCINOMA
USC and ovarian serous carcinoma exhibit identical morphological features. In some cases, there may be widespread involvement of the uterus, the ovaries and other organs and determining the site of primary tumour may be difficult. A small USC without obvious myometrial involvement may occasionally disseminate widely to involve the ovaries, omentum or peritoneum and with ovarian primaries there may be extensive uterine involvement. In these scenarios it may be difficult or impossible to distinguish between a USC and an ovarian serous carcinoma. This is important since adjuvant therapies for a uterine and ovarian primary may differ. It is also possible that in some of these cases there are coexistent independent primaries within the ovary and uterus. Molecular studies may assist and, in some cases of USC with minimal or no myometrial infiltration but with ovarian involvement, such studies have confirmed that the ovarian tumour represents metastatic disease rather than an independent primary²¹. A recent study has examined the value of the WT1 (Wilm's tumour gene) antibody (which is positive in most ovarian serous carcinomas²²) in distinguishing between a USC and an ovarian serous carcinoma²³. In that study all USC were negative with WT1 whereas 97% of ovarian serous carcinomas were positive. It was concluded that WT1 may be a useful immunohistochemical marker to distinguish between a USC and an ovarian serous carcinoma. Clearly this requires confirmation by further studies.

CD10 IN ENDOMETRIAL ADENOCARCINOMAS
In recent years, the value of CD10 as a marker of endometrial stromal neoplasms has been extensively studied^24-26. CD10, although not specific for endometrial stromal neoplasms (some smooth muscle tumours may be focally positive), may be useful as part of a panel, which should include α-smooth muscle actin, desmin and h-caldesmon, in distinguishing between an endometrial stromal and a smooth muscle neoplasm. Recently, CD10 positivity was shown in a presumed mesonephric adenocarcinoma of the uterus and this antibody was proposed as a useful immunohistochemical marker of cervical mesonephric lesions²⁷. CD10 characteristically exhibits a luminal staining pattern in mesonephric lesions. However, a recent study performed by us has shown that most endometrioid carcinomas of the uterus are also positive with CD10 as are a third of usual endocervical adenocarcinomas (manuscript submitted for publication). The conclusion is that CD10 is neither a specific nor a useful immunohistochemical marker of mesonephric adenocarcinoma.

Since CD10 stains normal and neoplastic endometrial stroma, several studies have addressed the value of CD10 in distinguishing between involvement of adenomyosis by endometrial adenocarcinoma and true myoinvasion^28-30. This distinction may be difficult morphologically and has important implications regarding staging and treatment. In general these studies have shown than even around the myoinvasive component of endometrial adenocarcinoma there is often a small rim of CD10 positive mesenchymal cells. I have also consistently noted this phenomenon of staining of periglandular mesenchymal cells with CD10 in the cervix (manuscript submitted for publication). It can be concluded that CD10 is of no value in distinguishing true myoinvasive endometrial adenocarcinoma from involvement of adenomyosis.

MISCELLANEOUS USES OF IMMUNOHISTOCHEMISTRY IN ENDOMETRIAL CARCINOMA
Some endometrial adenocarcinomas, either of endometrioid or mucinous type (more commonly mucinous) may have a microglandular growth pattern^31-33. There may be an associated neutrophilic infiltrate and the morphological appearances can bear a close resemblance to cervical microglandular hyperplasia (MGH). A microglandular growth pattern may also be seen, not uncommonly, on the surface of many otherwise typical endometrial adenocarcinomas³⁴ and these foci may be preferentially sampled on endometrial biopsy. Although cervical MGH usually occurs in the premenopausal years, it may also occur in the postmenopausal age group. In general, careful morphological examination allows a distinction between microglandular adenocarcinoma and cervical MGH, but immunohistochemistry may be of value in problematic cases. The proliferation marker MIB1 may be of value since, in most cases, cervical MGH has a low proliferation index (less than 10%)¹⁸. The MIB1 index would be expected to be greater in adenocarcinoma. However, since adenocarcinomas with a microglandular growth pattern are usually low grade neoplasms, it is possible that these may also exhibit a low proliferation index. A recent study investigated the value of a large panel of antibodies in distinguishing between microglandular adenocarcinoma and cervical MGH³⁵. Vimentin immunoreactivity was a strong pointer towards an adenocarcinoma since most were positive whereas cervical MGH was invariably negative.

Occasionally the differential diagnosis in an endometrial biopsy or curettage specimen lies between a metaplastic process and a carcinoma. Clear cell metaplasia may closely resemble endometrial clear cell carcinoma. Although marked nuclear atypia and a high mitotic rate are indicative of a malignant lesion, occasional clear cell carcinomas may be deceptively bland and may exhibit a low mitotic count. Obviously the presence of a mass lesion on hysteroscopy is a pointer towards a carcinoma. ER and D07 staining may be of value since most clear cell carcinomas are negative with ER and positive with D07, although this is not invariably so³⁶. Conversely, clear cell metaplasia would be expected to be positive with ER and negative with D07.

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