Houghton, Oisin MB; McCluggage, W. Glenn MD
TP63 is a p53 homologue gene located at 3q27-29. p63 protein is the transcription factor product of the TP63 gene. Alternative splicing and different translation initiation results in 2 N-terminal domain isoforms, TA and ΔN, and 3 C-terminal isoforms, α, β, and γ1; as such, 6 potential forms of p63 exist. Despite its relationship to the tumor suppressor protein p53, p63 is believed to act as an oncogene with regard to the ΔNp63 isoform whereas the TAp63 isoform has a tumor suppressor function.1
p63 is involved in the development and maintenance of stratified epithelia.2 The various forms of p63 have different functional roles and a balance between the TA and ΔN forms is required for proper epidermal development.3 p63 is expressed strongly in myoepithelial cells and in the basal cells of stratified epithelia and the prostate. It plays an important role in the development of the breast, urothelium, prostate gland, and squamous epithelium.1 The ΔNp63 isoform is thought to be the key regulator of myoepithelial, squamous and basal cell differentiation.4 In p63-/- mice, there is abnormal development of squamous epithelium, sweat glands, salivary glands, and mammary glands.2 In addition, the prostate develops without basal cells and sinus vaginal epithelium differentiates as columnar epithelium.4,5
In the following sections, the expression of p63 in the normal tissues of the female genital tract is described followed by its expression in various pathologic conditions and its potential diagnostic applications in several scenarios in the female genital tract. Several p63 antibodies are available, most reacting against all the p63 isoforms but some against either the TA or ΔN isoforms.6 It is stressed that only nuclear immunoreactivity with p63 is of importance and that cytoplasmic staining should be ignored, as its biologic significance is unclear.
p63 EXPRESSION IN THE NORMAL FEMALE GENITAL TRACT
In the female genital tract, p63 is expressed in the basal and parabasal layers of mature cervical, vaginal, and vulval squamous epithelium4 (Fig. 1A). Immature squamous metaplasia in the cervix is also p63 positive (Fig. 1B), as is atrophic cervical squamous epithelium. While glandular cells in the gynecologic tract are generally p63 negative, subcolumnar reserve cells and reserve cells in cases of reserve cell hyperplasia at the transformation zone of the cervix are p63 positive (Fig. 1C). Cervical transitional metaplasia is also p63 positive (as discussed later, p63 is expressed in benign urothelium and in many urothelial neoplasms, including those arising within the ovary). Scattered p63-positive cells are present within the normal endometrium and these have been postulated to be reserve cells or stem cells capable of undergoing various types of metaplasia.7 p63 positivity is also present within oocytes in the ovary and focal immunoreactivity has been observed within fallopian tube epithelium.4 Walthard rests (nests of transitional epithelium commonly seen around the ovary and fallopian tube) exhibit positive nuclear immunoreactivity with p63 (Fig. 2).
EXPRESSION OF p63 IN PATHOLOGIC LESIONS AND DIAGNOSTIC UTILITY IN THE FEMALE GENITAL TRACT
Tubulosquamous Polyp of Vagina
This is a recently described polyp characterized by expansile nests of squamous epithelium within a hypocellular fibrous stroma.8,9 Tubules are present within the squamous epithelium and these characteristically stain positively with prostatic markers, more frequently prostatic acid phosphatase than prostate-specific antigen. Characteristically, the tubules are composed of a double cell layer of epithelium with an inner cuboidal and an outer flattened layer. The squamous cells and the outer layer of the tubules stain positively with p63 (personal observations, W.G.M.). The outer cell layer also stains with 34βE12. Positivity with p63 and 34βE12 is characteristic of the basal cell layer of normal prostatic glands and suggests that the outer cell layer in tubulosquamous polyp is similar to prostatic basal cells. There is a similar staining pattern in ectopic prostatic tissue within the cervix and it is likely that this is related to vaginal tubulosquamous polyp.
Cervical Microglandular Hyperplasia
Microglandular hyperplasia is a common morphologic finding in the cervix, especially during the reproductive years; the diagnosis is usually straightforward. p63 stains the reserve cells of microglandular hyperplasia with a characteristic pattern, the reserve cells typically having a subcolumnar location10 (Fig. 3). In early microglandular hyperplasia, staining is confined to scattered cells whereas in later lesions, there is positivity of subcolumnar reserve cells and metaplastic squamous cells.10 Although recognition of this pattern of p63 staining may be helpful in the distinction between cervical microglandular hyperplasia and microglandular areas on the surface of a uterine endometrioid adenocarcinoma,11 the diagnostic utility is limited as some endometrioid adenocarcinomas are focally p63 positive, predominantly, but not exclusively, in metaplastic foci.7
Preinvasive, Premalignant Cervical Lesions
There has been only limited investigation of p63 staining in preinvasive, premalignant cervical lesions. In one study, p63 staining in cervical intraepithelial neoplasia I (CIN I) was typically localized to the basal and parabasal cells whereas in CIN II and CIN III, p63-positive nuclei extended into the middle and upper layers.12 In contrast, p63 was usually negative in adenocarcinoma in situ. In another study, p63 was uniformly expressed in all dysplastic cells of CIN III13 (Fig. 4). p63 has also been shown to be positive in scattered cells, mainly with a basal location, in stratified mucin producing intraepithelial lesion in the cervix. This is a form of reserve cell dysplasia with morphologic features of both CIN and adenocarcinoma in situ.14
Ectopic Prostatic Tissue in Cervix
The ectopic prostatic tissue has rarely been described in the cervix.15,16 It is controversial whether this represents a metaplasia of endocervical glands or a developmental abnormality. Similar to the possibly related vaginal tubulosquamous polyp, p63 stains the outer (basal) cell layer of the glandular elements, and also the squamous component, which is a common feature of this lesion (Fig. 5) (personal observations, W.G.M.). This is similar to the pattern of immunoreactivity in normal eutopic prostate.17
Morphologic Subtypes of Cervical Carcinoma
p63 staining may be useful in establishing a cervical carcinoma to be squamous in type. Small cell neuroendocrine carcinoma (SCNEC) and small cell squamous carcinoma may be difficult to distinguish on morphology alone. The distinction between a large cell neuroendocrine carcinoma (LCNEC) and a poorly differentiated squamous carcinoma may also be problematic.18 The difficulties may be compounded in small biopsy specimens. The distinction between a SCNEC or LCNEC and a squamous carcinoma is important as management may differ markedly. For example, surgical resection may not be undertaken, even with an early stage neuroendocrine carcinoma. Furthermore, the choice of adjuvant therapy may differ between a neuroendocrine carcinoma and a squamous carcinoma. Prophylactic cranial irradiation may be given in the case of a neuroendocrine carcinoma. Although neuroendocrine markers (such as chromogranin, synaptophysin, PGP9.5, and CD56) may assist, these can be negative in SCNEC, especially chromogranin, which is the most specific neuroendocrine marker available. Moreover, CD56 is relatively nonspecific, being positive in some squamous carcinomas. In this scenario, p63 may be useful in that diffuse positive nuclear staining is present in most squamous carcinomas whereas SCNEC and LCNEC are usually negative or occasionally focally positive19,20 (Fig. 6). However, occasional poorly differentiated cervical squamous carcinomas are p63 negative (personal observations, W.G.M.).
p63 is also of use in the distinction between a poorly differentiated squamous carcinoma and a poorly differentiated adenocarcinoma since, as discussed, squamous carcinoma is typically diffusely positive whereas adenocarcinoma is negative or focally positive.19,20 In adenosquamous carcinoma of the cervix, the squamous component is usually p63 positive and the glandular elements negative. It can be concluded that in cervical neoplasms, p63 is a powerful marker for squamous differentiation and, when diffusely expressed, excludes glandular or neuroendocrine differentiation.19 Other morphologic types of cervical carcinoma shown to be p63 positive include adenoid basal carcinoma, lymphoepithelioma-like carcinoma and papillary transitional carcinoma.19–21
Normal and Pathologic Lesions of the Endometrium
p63 is expressed in basal and subcolumnar cells in the fetal endometrium in a pattern similar to that of reserve cells in the cervix.7 These p63-positive cells become inconspicuous in the reproductive years in cyclical endometrium with positive staining confined to individual scattered basal and suprabasal cells.7 In endometrial polyps and postmenopausal endometria, focal clusters of p63-positive cells may be identified in inactive glands or in surface reparative type epithelium.7 Metaplastic epithelium, either alone or in association with hyperplasias or carcinomas, exhibit the most intense staining with p63, primarily in basal or subcolumnar cells. It has been suggested that p63-positive cells in the normal endometrium represent cells with a basal or reserve cell phenotype, similar to reserve cells at the transformation zone of the cervix, and may be a form of stem cell with the potential for multidirectional differentiation.7
Squamous differentiation in the endometrium and ovary is often seen in the context of endometrioid proliferative lesions, such as endometrial hyperplasias, endometrioid adenocarcinomas of the uterus, and endometrioid neoplasms of the ovary. The squamous elements may be of 2 types, namely typical squamous elements in which features such as keratinization and prominent intercelluar bridges are seen, or the so-called squamous morules in which typical squamous features are absent. A recent study has shown that while typical squamous elements in endometrioid proliferative lesions are usually p63 positive, morules are typically negative, although sometimes there is positive nuclear staining of cells at the periphery of the morules.22
Cytotrophoblast expresses the ΔNp63 isoform whereas chorionic-type intermediate trophoblast in the fetal membranes (chorion laeve) expresses the TA isoform.23 p63 is positive in placental site nodule (Fig. 7) and epithelioid trophoblastic tumor (lesions derived from chorionic-type intermediate trophoblast) whereas placental site reaction and placental site trophoblastic tumor (lesions derived from implantation site intermediate trophoblast) are p63 negative.23 Thus p63, when used in conjunction with other trophoblastic markers which are differentially expressed in different populations of trophoblast and different trophoblastic lesions, is of use in distinguishing the benign placental site nodule from placental site trophoblastic tumor. Obviously, this marker is of no value in distinguishing placental site nodule from epithelioid trophoblastic tumor. The latter exhibits a MIB1 proliferation index of approximately 10%, in contrast to that of placental site nodule which is close to zero.24 In addition, there is considerably higher expression of cyclin E in epithelioid trophoblastic tumor than in placental site nodule.25 Given the differential expression in different trophoblastic populations, p63 is useful in the diagnostic algorithm of trophoblastic lesions.24
Placental site nodule and epithelioid trophoblastic tumor may be confused with squamous carcinoma, especially when these trophoblastic lesions occur in the cervix, as they sometimes do. All these lesions are typically p63 positive. Other trophoblastic markers, such as cytokeratin 18 and HLA-G, may be of value in the distinction, as may p16 as this is positive in most cervical squamous carcinomas but negative in the trophoblastic lesions.23,25
Ovarian Neoplasms: p63 is a Useful Marker of Transitional Neoplasms
Ovarian neoplasms composed of cells with a urothelial morphology include benign, borderline (proliferating) and malignant Brenner tumor and transitional cell carcinoma. p63 is diffusely expressed in most transitional neoplasms in the urological tract26 and it has been shown that benign and borderline Brenner tumors of the ovary are consistently p63 positive (Fig. 8).26,27 However, in one study most malignant Brenner tumors were p63 negative (the accompanying benign and borderline components were usually positive), as were transitional cell carcinomas.26 It was suggested that while most benign and borderline Brenner tumors exhibit transitional differentiation, ovarian carcinomas which morphologically resemble transitional cell carcinoma are poorly differentiated carcinomas which are not truly urothelial in nature.26 This is in keeping with the results of other immunohistochemical studies using markers such as cytokeratin 7 and 20. The aforementioned study also concluded that loss of p63 expression in an ovarian Brenner tumor is associated with malignant progression.26 In that study, all other morphologic subtypes of ovarian epithelial neoplasm were p63 negative, except for expression in the basal layers of the squamous epithelium in teratomatous neoplasms.26 Another study found p63 nuclear immunoreactivity in cells resembling cervical reserve cells in ovarian Mullerian type mucinous borderline tumors and mixed epithelial borderline tumors.28
Practical uses of p63 staining in ovarian neoplasms might include (a) the distinction between a Brenner tumor (p63 positive) and various mimics, such as adult granulosa cell tumor, which are p63 negative and (b) the distinction between a primary ovarian transitional cell carcinoma (p63 negative) and a metastatic transitional carcinoma from the urological tract (p63 positive).29
Miscellaneous Gynecologic Lesions Expressing p63
p63 expression has been shown in the neoplastic cells of vulval Paget disease secondary to an underlying urothelial carcinoma.30 In contrast, all cases of primary vulval Paget disease were p63 negative.30 Squamous metaplasia of the ovarian surface epithelium in association with peritoneal dialysis has also been shown to be p63 positive.31 Not unexpectedly, transitional metaplasia of the fallopian tube epithelium is p63 positive (personal observations, W.G.M.) (Fig. 9).
1. In the cervix, p63 is expressed in the basal and parabasal layers of mature squamous epithelium, in subcolumnar reserve cells at the transformation zone, in immature squamous metaplasia, atrophy, transitional metaplasia, and in the reserve cells of microglandular hyperplasia.
2. p63 is useful in confirming squamous or transitional differentiation in diverse lesions in the female genital tract.
3. p63 stains the basal cells in vaginal tubulosquamous polyp and ectopic prostatic tissue in the cervix, suggesting that these cells are analogous to prostatic basal cells.
4. Positive p63 staining, when diffuse, is of use in confirming a poorly differentiated cervical carcinoma as squamous in type and in distinguishing a squamous carcinoma from an adenocarcinoma or a neuroendocrine carcinoma.
5. In the endometrum, a population of p63-positive cells exist which may function as a stem cell population and as the source of various epithelial metaplasias.
6. p63 stains the intermediate trophoblast of the chorion laeve and the lesions derived from this, placental site nodule and epithelioid trophoblastic tumor. This may be useful in the diagnostic algorithm of trophoblastic lesions.
7. p63 stains most benign and borderline Brenner tumors of the ovary, in keeping with their urothelial differentiation. p63 staining is absent in the malignant component of most malignant Brenner tumors and ovarian transitional cell carcinomas are negative.
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