The available immunohistochemical stains and their

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1 Immunohistochemistry in Gynecologic Pathology An Example-Based Practical Update Natalia Buza, MD; Pei Hui, MD, PhD Context. Immunohistochemical stains are routinely applied in the pathology diagnostic workup of gynecologic lesions. The rapidly expanding repertoire of available markers helps in refining the diagnostic criteria for each disease entity; however, limitations and pitfalls exist. Objective. To provide a practical, relevant update on the use of immunohistochemistry for commonly encountered differential diagnoses in gynecologic pathology. Data Sources. Literature review and authors diagnostic experience. Conclusions. Immunohistochemistry is a helpful adjunct tool in gynecologic pathology. Although the primary application is in the diagnostic workup, novel prognostic and predictive markers, and immunostains to screen for genetic cancer syndromes, have also been recently introduced in the field. (Arch Pathol Lab Med. 2017;141: ; doi: /arpa RA) The available immunohistochemical stains and their routine application in diagnostic surgical pathology continue to expand with our better understanding of pathogenesis, characterization of new disease entities, and increasing clinical demand for better tumor prognostication and prediction of response to targeted therapies. This article provides a focused review of the most recent developments on the use of immunohistochemistry in selected diagnostic gynecologic pathology topics in an organ-based fashion. While most immunomarkers discussed here fall under the diagnostic category, certain prognostic and predictive markers and markers of screening for inherited cancer syndromes are also included. Accepted for publication December 27, From the Department of Pathology, Yale University School of Medicine, New Haven, Connecticut. The authors have no relevant financial interest in the products or companies described in this article. This manuscript was presented at the First Chinese American Pathologists Association (CAPA) Diagnostic Pathology Course: Best Practices in Immunohistochemistry in Surgical Pathology and Cytopathology; August 22 24, 2015; Flushing, New York. Reprints:PeiHui,MD,PhD,YaleUniversitySchoolofMedicine, 310 Cedar St, LH108, PO Box , New Haven, CT ( pei.hui@yale.edu). LOWER GENITAL TRACT UTERINE CERVIX, VAGINA, AND VULVA Illustrative Example 1 A 38-year-old patient with a history of atypical squamous cells of undetermined significance and positive high-risk human papillomavirus (HPV) test result on Papanicolaou smear underwent a colposcopy examination, which revealed a cervical acetowhite lesion at the 6-o clock position. A biopsy was performed that showed changes morphologically consistent with low-grade squamous intraepithelial lesion (LSIL) with mitotic activity extending up to the second third of the epithelium (Figure 1, A and B). The background cervical tissue had marked chronic inflammation. Should p16 immunostaining be performed to rule out high-grade dysplasia? Both when to order and how to interpret a p16 immunohistochemical study for squamous lesions of the cervix remain a common diagnostic challenge and are discussed in detail below. Human Papillomavirus Associated Squamous Lesions of the Cervix, Vagina, and Vulva. The pathology diagnosis and classification of squamous intraepithelial lesions of the lower anogenital tract continue to be problematic, despite availability of p16 immunostain for at least a decade in the routine practice. 1,2 The Lower Anogenital Squamous Terminology Project and the 2014 World Health Organization classification of female genital tract tumors recommend a new 2-tier system low-grade intraepithelial lesion (LSIL; intraepithelial neoplasia 1 [-IN1], condyloma, koilocytosis) and high-grade intraepithelial lesion (high-grade squamous intraepithelial lesion [HSIL]; -IN2 and -IN3) for reporting precursor lesions of the cervix, vagina, and vulva to reflect the biology and clinical relevance of lesions, which also generated a renewed interest in improving the diagnostic accuracy for these entities. 3,4 While the typical histologic features of squamous intraepithelial lesions are well characterized and illustrated in the literature, several important morphologic mimics and diagnostic pitfalls exist. In addition, the interobserver and intraobserver agreement on histologic grading of cervical and vulvar intraepithelial neoplasia, especially -IN2, based on hematoxylin-eosin stain (H&E) slides alone, has been reported as moderate at best Abortive/transforming infections with high-risk HPV types in high-grade intraepithelial lesions produce increased levels of E7 viral oncoprotein, which binds to and inactivates 1052 Arch Pathol Lab Med Vol 141, August 2017 Immunohistochemistry in Gynecologic Pathology Buza & Hui

2 Figure 1. A and B, Low-grade squamous intraepithelial lesion/cervical intraepithelial neoplasia 1 (LSIL/CIN1) with a mitotic figure (arrow) in the second third of the epithelium, showing only weak patchy staining with p16 immunostain. C and D, High-grade squamous intraepithelial lesion (HSIL/CIN3) with strong, diffuse, blocklike p16 positivity (hematoxylin-eosin, original magnification 34 [A and C]; p16, original magnification 34 [B and D]). Rb, leading to upregulation of p16 (cyclin-dependent kinase inhibitor) by disruption of the negative feedback loop. 11 Thus, immunohistochemical detection of p16 protein overexpression (p16 positivity) defined as continuous strong nuclear or nuclear and cytoplasmic staining in a blocklike pattern involving at least one-third of the epithelial thickness can significantly aid the diagnosis of high-grade dysplasia, particularly when the differential diagnosis includes benign mimics (eg, atrophy, squamous metaplasia, or reactive changes) and in cases with equivocal morphologic features between LSIL and HSIL (-IN2 category of lesions in the 3-tier classification system) 1,4 (Figure 1, A through D). P16 may also be helpful in evaluation of cauterized cone biopsy margins and small tissue fragments in endocervical curettements. While the use of p16 immunostaining in the above settings has been strongly advocated and also has been shown to improve interobserver agreement, 5,7,12,13 several studies have reported positive p16 staining in normal epithelium and lowgrade lesions as well: in a large meta-analysis of 34 studies on p16 expression in cervical tissue specimens, 2% of all benign cases (ranging between 0.4% 30% in individual studies) and 38% of low-grade intraepithelial lesions (range, 23% 53%) showed positivity 14 (Figure 2, A and B). Negative p16 staining, on the other hand, does not rule out HSIL; the proportion of p16-negative cervical intraepithelial neoplasia 3 (CIN3) cases varies between 8% and 28% 14,15 (Figure 3, A and B). The choice of antibody clone may also affect the sensitivity and positive predictive value of p16 immunohistochemistry although the most commonly used clone is E6H4, various other clones have also been used in prior studies. 14,16 It has also been suggested that diffuse p16 expression in low-grade cervical lesions may predict the risk of progression into high-grade dysplasia (CIN2/3) ; however, others 20 have reported similar rates of regression in p16-positive and p16-negative LSIL cases. Overall, when p16 immunostain is used as an adjunct test in the diagnosis of lower genital tract squamous intraepithelial lesions, it should always be interpreted in conjunction with the morphologic features of the H&E-stained lesions; and in cases with discordant morphology and p16 staining pattern, the morphologic interpretation should prevail. Ki-67, a marker of proliferative activity, is only expressed in basal/parabasal cells in normal squamous epithelium, whereas increased Ki-67 labeling extending into the upper Arch Pathol Lab Med Vol 141, August 2017 Immunohistochemistry in Gynecologic Pathology Buza & Hui 1053

3 Figure 2. A and B, Low-grade squamous intraepithelial lesion may show discordant ( blocklike ) p16 immunostaining pattern (hematoxylin-eosin, original magnification 310 [A]; p16, original magnification, 310 [B]). two-thirds can be observed in dysplastic epithelium, including condylomas and low- and high-grade squamous intraepithelial lesions (Figure 4, A and D). Low-grade squamous intraepithelial lesion and HSIL have similar staining patterns, hence, Ki-67 is not helpful in this setting. In addition, increased proliferative activity may also be observed in reactive and inflammatory conditions, presenting another potential diagnostic pitfall. 24 Nevertheless, Ki- 67 immunohistochemistry may be a helpful adjunct in separating benign findings (ie, atrophy, squamous metaplasia, fibroepithelial polyps) from HPV-associated lesions and avoiding overdiagnosis. 23,24 ProExC, a novel immunohistochemical cocktail composed of topoisomerase II a (TOP2A) and minichromosome maintenance protein 2 (MCM2) antibodies has also been recently suggested to be a useful marker of high-grade dysplasia 25,26 : strong nuclear staining in more than half of the epithelial thickness showed 90% sensitivity and 79% positive predictive value for HSIL. 27 A combined immunohistochemical panel consisting of ProExC and p16 has been shown to further improve the specificity and positive predictive value for CIN2/3, although with decreased sensitivity. 28,29 As with p16 and Ki-67 immunostains, the results of ProExC staining should be closely correlated with the morphologic findings, and further studies are necessary to evaluate the utility of this marker in routine daily practice. Endocervical Glandular Lesions. Similar to squamous lesions of the lower genital tract, p16 can be used as a surrogate marker for the presence of high-risk HPV in endocervical glandular epithelium. 24 The diagnosis of endocervical adenocarcinoma in situ (AIS) can be particularly challenging in small biopsy or curettage specimens, as reactive changes, tuboendometrial metaplasia, and small fragments of proliferative endometrial epithelium are frequent morphologic mimics. Strong diffuse p16 immunostaining is characteristic of AIS, whereas benign endocervical epithelium is p16 negative (Figure 5, A through D). 24,30,31 Cervical microglandular hyperplasia, tuboendo- Figure 3. A and B, High-grade squamous intraepithelial lesion with discordant, weak, and patchy p16 expression (hematoxylin-eosin, original magnification 320 [A]; p16, original magnification 320 [B]) Arch Pathol Lab Med Vol 141, August 2017 Immunohistochemistry in Gynecologic Pathology Buza & Hui

4 Figure 4. A and B, High-grade squamous intraepithelial lesion (CIN2). Lesion shows strong, blocklike p16 immunoreactivity (C) and increased Ki- 67 labeling extending up to two-thirds of the epithelial thickness in this case (D) (hematoxylin-eosin, original magnifications 34 [A] and 320 [B]; original magnification 310 [C and D]). metrial metaplasia, and endometrium/cervical endometriosis may show patchy p16 immunoreactivity 1,24,32 but without diffuse staining in the absence of high-risk HPV. Paired box 2 (PAX2) immunostain on the other hand has shown diffuse positivity in benign cervical tissues including tubal metaplasia, lobular endocervical glandular hyperplasia, and benign mesonephric proliferations, while AIS and most endocervical and endometrial carcinomas are PAX2 negative. 33 Ki-67 proliferation index is generally increased in AIS, compared with that seen in benign conditions, and may provide additional help in the differential diagnosis. Estrogen receptor (ER), vimentin, and CD10 immunostains can also be added to the immunohistochemical panel to rule out endometriosis: in contrast to most AIS cases, ER and vimentin show diffuse positivity in endometrial epithelium, and CD10 helps in highlighting endometrial stroma (Figure 6, A through D). A small proportion of endocervical adenocarcinomas for example, mesonephric adenocarcinoma and gastric-type mucinous adenocarcinomas are not typically associated with high-risk HPV infection, however, and therefore these entities lack diffuse p16 immunoreactivity in most cases Mesonephric adenocarcinomas of the cervix are commonly positive for CD10, paired box 8 (PAX8), vimentin, and calretinin, while they usually lack staining with ER and carcinoembryonic antigen (CEA). 37,38 Diffuse p16 staining has been described in rare cases of mesonephric adenocarcinoma, despite absence of high-risk HPV, likely due to non-hpv related upregulation of protein expression. 38 Cervical adenocarcinoma with gastric differentiation including adenoma malignum as the well-differentiated end of the spectrum represents another rare, non-hpv related subtype of cervical adenocarcinoma showing morphologic similarities to gastric pyloric type and pancreatobiliary adenocarcinomas. 34,35,39 Poorly differentiated gastric-type adenocarcinomas of the cervix are biologically aggressive, often with ovarian and lymph node metastases at presentation, and are associated with a significantly worse clinical outcome than usual endocervical adenocarcinoma. 39 Cervical gastric-type adenocarcinomas are cytokeratin 7 (CK7) and CEA positive (similar to usual endocervical adenocarcinoma), but are typically negative or only focally positive for p16. They commonly show at least focal CK20 and caudal-related homeobox gene 2 (CDX2) positivity and Arch Pathol Lab Med Vol 141, August 2017 Immunohistochemistry in Gynecologic Pathology Buza & Hui 1055

5 Figure 5. A and B, Small fragments of endocervical adenocarcinoma in situ (AIS) in an endocervical curettage with nuclear atypia, hyperchromasia, crowding, and frequent apical mitoses (arrows). P16 immunostaining is strongly and diffusely positive (C) and Ki-67 shows increased proliferation index (D) (hematoxylin-eosin, original magnification 320 [A and B]; original magnification 320 [C and D]). express markers of pyloric-type mucin (MUC6 and HIK1083), and may be only focally positive or negative for PAX8 immunostain (Figure 7, A through F). P53 mutations are not uncommon in these tumors, resulting in an abnormal p53 immunostaining pattern. 34,36 Differentiated Vulvar Intraepithelial Neoplasia. A small proportion of vulvar intraepithelial neoplasia (VIN) cases are unrelated to HPV infection and have been associated with lichen sclerosus and an increased risk of progression to invasive squamous cell carcinoma, compared with usual (HPV-positive) VIN. 40,41 Despite the significant clinical implications, making the morphologic diagnosis of differentiated VIN (dvin) is often very challenging, as the histologic features overlap with those of benign hyperplasia, lichen sclerosus, and lichen planus. The epidermis in dvin is usually thickened and hyperkeratotic and/or parakeratotic with elongated and anastomosing rete ridges. 40 The basal cells show atypical, hyperchromatic nuclei often with increased mitotic figures, while the upper layers are composed of enlarged squamous cells with abundant eosinophilic cytoplasm. Frequent p53 mutations have been found in dvin, 42 making p53 immunohistochemistry a promising candidate for ancillary testing. Differentiated VIN typically demonstrates intense nuclear p53 positivity or less commonly, complete absence of p53 staining ( p53 null phenotype) extending from the basal cell layer to the suprabasal cells, involving one-third to full thickness of the epidermis (Figure 8, A and B). 40,42 44 However, p53 overexpression has also been reported in lichen sclerosus and other nonneoplastic conditions, due to nonmutational upregulation of p53. 45,46 P16 immunostain shows negativity in dvin, 44 and the Ki-67 proliferation index is increased in the basal and parabasal cell layers. 47 An immunohistochemical panel composed of p53, Ki-67, and p16 may be helpful in margin evaluation for dvin in a dvin-associated invasive squamous cell carcinoma. In small biopsy specimens, however, conservative application and interpretation of immunohistochemical markers are suggested to avoid overdiagnosis and unnecessary extensive surgery. Vulvar Paget Disease. The morphologic mimics of primary vulvar Paget disease (PD) include pagetoid VIN/ squamous cell carcinoma in situ and in situ melanoma. In addition, secondary vulvar involvement by colorectal and urinary tract malignancies, although occurring less frequently, should also be considered in the differential diagnosis. Primary vulvar PD is typically positive for CK7, 1056 Arch Pathol Lab Med Vol 141, August 2017 Immunohistochemistry in Gynecologic Pathology Buza & Hui

6 Figure 6. A and B, Endometriosis mimicking endocervical adenocarcinoma in situ (AIS) in a cervical biopsy. P16 immunostaining is patchy in the endometrial glandular epithelium (C) and CD10 immunostain highlights endometrial stroma (D) (hematoxylin-eosin, original magnifications 34 [A] and 310 [B]; original magnification 320 [C and D]). CAM 5.2, CEA, and gross cystic disease fluid protein 15 (GCDFP-15) immunostains (Figure 9, A through D), in contrast to pagetoid VIN and melanoma, which do not express these markers Melanocytic markers HMB-45, Mart-1 (Melan-A), and S100 on the other hand show negativity in vulvar PD. 49 P16 immunostaining may be strongly positive in rare cases of vulvar PD, presenting a potential pitfall when VIN is considered in the differential diagnosis. 52 Cytokeratin 20 usually shows negativity or only focal positivity in primary PD, whereas diffuse CK20 and CDX2 expression suggests secondary spread from a colorectal primary. 53,54 Coexpression of CK7/CK20 and uroplakin-iii or uroplakin-ii is characteristic of PD secondary to urothelial carcinoma. 55,56 Immunohistochemical markers of PD for example, CK7 may also be very useful in highlighting a subtle invasive component or evaluating margin involvement. Most recently, human epidermal growth factor receptor 2 (Her2) overexpression (including scores 2þ and 3þ) has been demonstrated in up to 58% of vulvar PD and has also shown correlation with presence of invasion. 57,58 In addition to its potential diagnostic and prognostic application, Her2 immunohistochemistry in vulvar PD may also be helpful in predicting response to Her2-based targeted therapies. 59 UTERINE CORPUS Illustrative Example 2 A 66-year-old patient presented with several years history of postmenopausal bleeding. Her last Papanicolaou smear was more than 10 years before presentation. Endometrial curettage and cervical biopsies were performed and were interpreted as endometrial endometrioid adenocarcinoma with cervical involvement. The patient then underwent a modified radical hysterectomy with bilateral salpingo-oophorectomy and lymph node dissection. Sections from the hysterectomy showed adenocarcinoma involving the endometrium and endocervix, although with morphologic differences between the 2 sites, raising the possibility of 2 synchronous independent endocervical and endometrial primaries (Figure 10, A through D; and Figure 11, A through D). What immunohistochemical panel would be the most helpful in differentiating between an endocervical and an endometrial primary? Arch Pathol Lab Med Vol 141, August 2017 Immunohistochemistry in Gynecologic Pathology Buza & Hui 1057

7 Figure 7. A, Gastric-type endocervical adenocarcinoma with markedly atypical, mucinous glands infiltrating the cervical stroma. Paired box protein 8 (PAX8) immunostain shows focal, weak nuclear positivity (B) and carcinoembryonic antigen (CEA, monoclonal) shows diffuse positivity (C). Focal staining may be observed with caudal type homeobox 2 (CDX2) (D). P16 typically shows negativity (E), while p53 shows strong diffuse immunoreactivity (F) (hematoxylin-eosin, original magnification 310 [A]; original magnification 310 [B through F]). Adenocarcinoma: Endometrial Versus Endocervical Origin. The origin, endometrial versus endocervical, of small tumor fragments in endometrial or endocervical biopsies and curettements may be difficult to identify by their morphologic features alone. In the most frequent scenario the tumor is composed of low- or intermediategrade glandular proliferation of stratified columnar mucinpoor cells, raising the differential diagnosis of usual-type 1058 Arch Pathol Lab Med Vol 141, August 2017 Immunohistochemistry in Gynecologic Pathology Buza & Hui

8 Figure 8. A, Differentiated vulvar intraepithelial neoplasia (dvin) with marked nuclear atypia and hyperchromasia of the basal cell layer. P53 immunostaining is strongly positive in the lower one-third to two-thirds of the epithelium (B) (hematoxylin-eosin, original magnification 320 [A]; original magnification 320 [B]). Figure 9. A, Vulvar Paget disease. The neoplastic glandular cells in the epidermis stain strongly with cytokeratin 7 (CK7) (B), carcinoembryonic antigen (CEA) (C), and human epidermal growth factor receptor 2 (Her2) (D) (hematoxylin-eosin, original magnification 310 [A]; original magnification 310 [B]; original magnification 320 [C and D]). Arch Pathol Lab Med Vol 141, August 2017 Immunohistochemistry in Gynecologic Pathology Buza & Hui 1059

9 Figure 10. A, Endocervical adenocarcinoma, usual type. P16 immunostaining is strongly and diffusely positive (B), carcinoembryonic antigen (CEA, monoclonal) shows cytoplasmic and membranous staining (C), and estrogen receptor (ER) shows only focal weak positivity (D). Vimentin immunostaining is negative in the tumor cells (not shown) (hematoxylin-eosin, original magnification 310 [A]; original magnification 310 [B through D]). endocervical versus endometrioid endometrial adenocarcinoma. Endometrioid adenocarcinoma arising in the endocervix is less common, accounting for only approximately 5% of all endocervical adenocarcinomas. Endocervical adenocarcinomas of usual- and endometrioid-type are almost always associated with high-risk HPV infection, 60 and therefore they exhibit strong and diffuse nuclear staining with p16 immunostain as a result of inactivation of Rb by HPV E7 protein. 61 In contrast, endometrial endometrioid adenocarcinoma is negative or only focally and weakly positive for p16 in the glandular component, although areas of squamous differentiation may show more diffuse staining. 1,61,62 Additional immunohistochemical stains CEA, vimentin, and hormone receptors (ER and/or progesterone receptor [PR]) are also helpful and should be included in the diagnostic panel (Table 1) (Figure 10, A through D; and Figure 11, A through D). Monoclonal CEA antibody typically shows diffuse membranous and cytoplasmic staining in endocervical adenocarcinomas, whereas most endometrioid adenocarcinomas of endometrial origin express vimentin, ER, and PR However, the immunohistochemical profile may be inconclusive showing nontypical expression patterns with the 4 or 5 markers in a significant number of tumors. Detection of high-risk HPV in the tumor genome (by in situ hybridization or polymerase chain reaction based methods) may provide additional discriminatory power in such cases Nevertheless, the results of immunohistochemistry and other ancillary tests Table 1. Immunohistochemical Profile of Primary Endocervical Versus Endometrial Adenocarcinoma Endocervical Adenocarcinoma (Usual and Endometrioid Types) Endometrial Adenocarcinoma (Endometrioid Type) p16 þ (diffuse) /þ (patchy) CEA þ ER /þ þ PR þ Vimentin þ Abbreviations: CEA, carcinoembryonic antigen; ER, estrogen receptor; PR, progesterone receptor; þ, positive;, negative; /þ, focal or patchy positivity Arch Pathol Lab Med Vol 141, August 2017 Immunohistochemistry in Gynecologic Pathology Buza & Hui

10 Figure 11. Endometrioid endometrial adenocarcinoma (A) can be differentiated from an endocervical primary by weak and patchy p16 expression (B), strong diffuse vimentin expression (C), estrogen receptor (ER) positivity (D), and carcinoembryonic antigen (CEA) negativity (not shown) (hematoxylin-eosin, original magnification 34 [A]; original magnification 34 [B through D]). should always be interpreted in correlation with the clinical, radiologic, and gross pathologic findings. Endometrial Adenocarcinoma. Distinction between the different histologic subtypes of endometrial adenocarcinoma (EAC) has major clinical and prognostic implications; therefore, if precise pathologic classification cannot be achieved on the basis of morphologic features alone, an immunohistochemical workup should be pursued even in small biopsy specimens. Among type II endometrial carcinomas, serous carcinoma has been reported to harbor p53 mutations in up to 90% of cases, 69 correlating with an abnormal p53 protein expression pattern by immunohistochemistry 70 : in most cases p53 mutation results in a strong and diffuse nuclear staining in more than 75% of tumor cells (Figure 12, A through D), whereas approximately 10% of serous EACs show complete absence of p53 immunoreactivity due to nonsense mutations or homozygous gene deletion In addition, p53 mutation appears to be an early event in serous carcinogenesis, as identical p53 mutations have been detected in a series of serous EACs and concurrent serous endometrial intraepithelial carcinomas. 74 Clear cell and endometrioid EACs, on the other hand, most often have a wild-type p53 immunostaining pattern, that is, weak focal staining in tumor cell nuclei (Figure 13, A through D; Table 2) However, p53 overexpression by immunohistochemistry has also been reported in a small proportion of FIGO grade 3 endometrioid EACs 78 and been shown to correlate with adverse clinical outcome in morphologically ambiguous Table 2. Immunohistochemical Profile of Different Histologic Subtypes of Endometrial Carcinoma Serous Clear Cell Endometrioid p53 þþþ or complete /þþþ /þþþ (grade 3) absence ( null ) p16 þþþ /þ /þ (patchy) ER þ/ a þþþ PR /þ þþ HNF-1b /þ þþþ /þ Napsin /þ þþþ /þ AMACR þþ/ Abbreviations: AMACR, a-methylacyl coenzyme A racemase; ER, estrogen receptor; HNF-1b, hepatocyte nuclear factor 1; PR, progesterone receptor; þ, weak positivity; þþ, moderate positivity; þþþ, strong positivity;, negative; /þ: focal or patchy positivity. a Serous endometrial adenocarcinomas not infrequently show ER expression at varying degrees. Arch Pathol Lab Med Vol 141, August 2017 Immunohistochemistry in Gynecologic Pathology Buza & Hui 1061

11 Figure 12. Serous endometrial intraepithelial carcinoma (A) and serous endometrial carcinoma (C) show strong, diffuse nuclear immunoreactivity with p53 in most cases (B and D) (hematoxylin-eosin, original magnification 310 [A and C]; original magnifications 34 [B] and 310 [D]). EAC cases with overlapping serous and endometrioid morphologic features. 79 Serous EAC is also typically strongly immunoreactive with p16 owing to non-hpv driven upregulation of protein expression, in contrast to the weak and patchy p16 staining of endometrioid and p16 negativity in most clear cell EACs Immunohistochemical stains for hormone receptors ER and PR typically show positivity in low- and intermediate-grade endometrioid EAC, while clear cell EACs generally show negativity. 75,83,84 It should be noted that serous EACs frequently show weak or patchy ER positivity. 81,85 87 Diagnostic markers of clear cell carcinoma hepatocyte nuclear factor 1 (HNF-1b), napsin, and a- methylacyl coenzyme A racemase (AMACR) have been proposed with variable sensitivity and specificity results among studies. 84,88 92 A panel of multiple immunohistochemical markers ER, PR, HNF-1b, and napsin has been found to have the highest specificity and positive predictive value for distinction between clear cell and endometrioid EAC in a recent study. 91 In addition to diagnostic applications, immunohistochemistry is also increasingly performed in endometrial carcinomas for Lynch syndrome screening and for guiding targeted therapies. Mismatch repair (MMR) protein immunostains MLH1, PMS2, MSH2, and MSH6 are helpful ancillary tools in identifying tumors with MMR deficiency and in guiding further genetic counseling and testing. Mismatch repair deficiency is common in sporadic EAC, identified in up to 40% of tumors with endometrioid histology. 69,93 Loss of nuclear staining for both MLH1 and PMS2 is most likely a somatic alteration due to hypermethylation of the MLH1 promoter. 94 Isolated loss of PMS2, MSH2, or MSH6 staining and loss of both MSH2 and MSH6 indicate a high likelihood of germline mutation, requiring additional genetic workup. 95 While MMR deficiency/microsatellite instability occurs nearly exclusively in the endometrioid and undifferentiated subtypes among sporadic EACs, tumors arising in patients with Lynch syndrome have been reported to have nonendometrioid (serous, clear cell, and carcinosarcoma) histologic types in almost equal proportion to endometrioid ones HER2/neu amplification and Her2 protein overexpression is present in approximately one-third of serous EACs. 99 The Her2 expression pattern is often lateral/basolateral with lack of staining in the apical portion of cell membrane and shows marked heterogeneity within the tumor in more than 1062 Arch Pathol Lab Med Vol 141, August 2017 Immunohistochemistry in Gynecologic Pathology Buza & Hui

12 Figure 13. Clear cell (A and B) and endometrioid (C and D) endometrial adenocarcinomas have wild-type (ie, focal, weak) p53 expression patterns (hematoxylin-eosin, original magnification 310 [A and C]; p53, original magnification 310 [B and D]). 50% of cases (Figure 14, A and B). 99,100 The role of targeted therapy against Her2 in serous endometrial cancer has been proposed, and patients are currently being enrolled at multiple institutions in the United States in a randomized phase II study evaluating the efficacy of adding trastuzumab to the chemotherapeutic regimen in advanced or recurrent Her2-positive tumors. Uterine Mesenchymal Tumors. Immunohistochemistry is often necessary to distinguish between endometrial stromal and smooth muscle differentiation in uterine mesenchymal neoplasms. CD10 typically shows diffuse, strong staining in endometrial stromal tumors (endometrial stromal nodule and low-grade endometrial stromal sarcoma), whereas expression of smooth muscle actin (SMA), desmin, muscle-specific actin (MSA), and h-caldesmon is characteristically seen in smooth muscle tumors. However, there is considerable overlap in the immunohistochemical profile between the 2 groups, presenting a potential diagnostic pitfall when a single marker or limited panel is used. Nearly half of cellular leiomyomas show CD10 expression, and SMA and desmin immunostains frequently show positivity in endometrial stromal tumors. 101,102 H- caldesmon appears to be a more specific marker for smooth muscle, as it only stains areas with morphologically apparent smooth muscle differentiation within endometrial stromal tumors. 101,103,104 A combination of immunohistochemical stains CD10, desmin, and h-caldesmon is recommended when the differential diagnosis includes leiomyoma or its variants versus endometrial stromal nodule and low-grade endometrial sarcoma (Table 3). Table 3. Immunohistochemical Profile of Smooth Muscle and Endometrial Stromal Tumors Leiomyoma/ Leiomyoma Variants LESS HESS (High-Grade Component) ER þ þ PR þ þ CD10 /þ þ Desmin þ /þ h-caldesmon þ Cyclin D1 /þ (heterogeneous) þ (diffuse) Abbreviations: ER, estrogen receptor; HESS, high-grade endometrial stromal sarcoma; LESS, low-grade endometrial stromal sarcoma; PR, progesterone receptor; þ, positive;, negative; /þ, focal or patchy positivity. Arch Pathol Lab Med Vol 141, August 2017 Immunohistochemistry in Gynecologic Pathology Buza & Hui 1063

13 Figure 14. Human epidermal growth factor receptor 2 (Her2) immunostain in serous endometrial carcinomas (A) showing a characteristic lateral/ basolateral membranous staining pattern (B) with lack of staining in the apical portion of cell membranes (hematoxylin-eosin, original magnification 320 [A]; Her2, original magnification 320 [B]). Figure 15. A, Fragments of low-grade endometrial stromal sarcoma in an endometrial curettage specimen. The tumor cells are strongly and diffusely positive for CD10 immunostain (B), but also show diffuse cytokeratin AE1/AE3 (CK AE1/AE3) expression (C). Epithelial membrane antigen (EMA) immunostaining (D) is negative; note the internal positive control of detached epithelial fragment in the upper left corner. Desmin immunostaining is negative (not shown) (hematoxylin-eosin, original magnification 34 [A]; original magnification 34 [B through D]) Arch Pathol Lab Med Vol 141, August 2017 Immunohistochemistry in Gynecologic Pathology Buza & Hui

14 Figure 16. A, Metastatic ovarian mucinous carcinoma from an appendiceal primary. The tumor cells show lower gastrointestinal immuphenotype: homeobox protein 2 (CDX2) positivity (B), cytokeratin 7 (CK7) negativity (C), and cytokeratin 20 (CK20) positivity (D) (hematoxylin-eosin, original magnification 310 [A]; original magnification 310 [B through D]). Endometrial stromal tumors may also express cytokeratins (CK AE1/AE3 cocktail and various individual cytokeratins) focally or diffusely, 105 which may lead to erroneous classification as an epithelial neoplasm (Figure 15, A through D). Additional epithelial markers for example, epithelial membrane antigen (EMA) usually help in resolving the diagnosis. 106 C-kit (CD117) expression has also been recently reported in both low- and high-grade endometrial stromal sarcomas mimicking gastrointestinal stromal tumor; however, anoctamin 1 (ANO1, DOG1) immunostain, another diagnostic marker of gastrointestinal stromal tumor, has shown negativity and no mutations were identified in the KIT gene. 107,108 High-grade endometrial stromal sarcoma is typically negative for ER, PR, and CD10 immunostains and shows diffuse, strong staining with cyclin D1 in more than 70% of tumor cell nuclei. 109 In contrast, cyclin D1 staining is usually negative or only focal, heterogeneous in low-grade endometrial stromal sarcoma. 110 Cyclin D1 expression, however, should always be interpreted in the context of a broader immunopanel, as uterine leiomyosarcomas may also be cyclin D1 positive. 111 OVARY Illustrative Example 3 A 52-year-old patient presented with a 10-cm incidental multicystic left adnexal mass on abdominal ultrasonography and underwent total hysterectomy, bilateral salpingooophorectomy, and appendectomy. Sections from the left ovary showed a mucinous neoplasm mimicking a primary ovarian borderline mucinous tumor (Figure 16, A through D). In addition, examination of the appendix revealed a 3- cm lesion at the appendiceal tip with similar morphologic features. What immunohistochemical stains would be helpful in confirming a metastatic ovarian tumor of appendiceal primary? Primary Ovarian Carcinomas. The differential diagnosis between the histologic subtypes of primary ovarian carcinoma can often be resolved by thorough assessment of H&E microscopic features. However, significant morphologic overlap exists, especially between high-grade ovarian carcinomas (ie, high-grade serous, clear cell, and endometrioid adenocarcinomas), requiring additional immunohistochemical workup. P53 mutations are present in nearly all high-grade serous carcinomas of the ovary, 112 resulting in Arch Pathol Lab Med Vol 141, August 2017 Immunohistochemistry in Gynecologic Pathology Buza & Hui 1065

15 Table 4. Immunohistochemical Profile of Different Histologic Subtypes of Primary Ovarian Carcinomas High-Grade Serous Clear Cell Endometrioid p53 þþþ or complete /þþþ /þþþ absence ( null ) WT-1 þþþ ER þ /þ þþþ PR þ /þ þþþ HNF-1b /þ þþþ /þ Napsin /þ þþþ /þ Abbreviations: ER, estrogen receptor; HNF-1b, hepatocyte nuclear factor 1; PR, progesterone receptor; WT-1, Wilms tumor 1; þ, weak positivity; þþ, moderate positivity; þþþ, strong positivity;, negative; /þ, focal or patchy positivity. abnormal strong diffuse or completely absent staining patterns with p53 immunohistochemistry. 113 In contrast, abnormal p53 expression is infrequent in clear cell and endometrioid ovarian carcinomas (Table 4). 114,115 Nuclear immunoreactivity for Wilms tumor 1 (WT-1) is also characteristic of ovarian serous carcinoma, both low- and high-grade subtypes, whereas clear cell and endometrioid carcinomas are WT-1 negative. 116 Similar to their endometrial counterpart, ovarian clear cell carcinomas stain positively with HNF-1b and napsin A, and are most often negative for ER and PR immunostains. 91,117 Primary Ovarian Versus Metastatic Mucinous Adenocarcinoma. One of the most common applications of immunohistochemistry in ovarian pathology is in the distinction between primary and metastatic mucinous carcinomas. The most commonly used markers and their expression patterns are listed in Table 5 (Figure 16, A through D; Table 5) Paired box gene 8 (PAX8) has been recently identified as an immunomarker of Müllerian epithelial tumors, in addition to renal and thyroid neoplasms. 121 More than 90% of nonmucinous ovarian carcinomas have been found to show PAX8 expression; however, only approximately half of primary mucinous carcinomas are PAX8 positive. 122 The immunohistochemical profile of primary mucinous carcinomas of the ovary shows considerable overlap with that of the metastatic ones. Review of a patient s history and the role of gross pathologic examination and thorough clinical and radiologic correlation cannot be overemphasized in this setting. Ovarian Sex Cord Stromal Tumors. A common differential diagnosis for sex cord stromal tumors is primary ovarian carcinoma, which may have sertoliform morphology Table 5. Immunohistochemical Profile of Primary Ovarian Versus Metastatic Mucinous Carcinomas Ovarian Pancreatobiliary Colorectal (Including Appendiceal) CK7 þ þ /þ (focal) CK20 /þ (focal) þ/ þ CDX2 /þ (focal) þ/ þ PAX8 þ/ ER þ/ DPC4 (SMAD4) Normal Loss (50%) Normal Abbreviations: CK, cytokeratin; ER, estrogen receptor; PAX8, paired box 8; þ, positive;, negative; /þ, focal or patchy positivity; þ/, may be positive or negative. Table 6. Immunohistochemical Profile of Ovarian Sex Cord Stromal Tumors Versus Ovarian Carcinomas Sertoli AGCT Cell/SLCT a-inhibin þ þ Calretinin þ þ /þ CK AE1/AE3 /þ /þ þ (punctate pattern) Ovarian Carcinomas CK7 þ EMA þ FOXL2 þ þ SF1 þ þ Abbreviations: AGCT, adult granulosa cell tumor; CK, cytokeratin; EMA, epithelial membrane antigen; FOXL2, forkhead box protein L2; SF1, steroidogenic factor 1; SLCT, Sertoli-Leydig cell tumor; þ, positive;, negative; /þ, focal or patchy positivity. (endometrioid adenocarcinoma) or may mimic adult granulosa tumor. Cytokeratin 7 and EMA immunostains show negativity in sex cord stromal tumors; however, CK AE1/ AE3 may show cytoplasmic dotlike positivity in adult granulosa cell tumor 123 and has also been reported to immunoreact with Sertoli-Leydig cell tumors (Table 6). 124 In addition to well-known immunostains such as inhibin and calretinin, forkhead box protein L2 (FOXL2) and steroidogenic factor 1 (SF1) immunostains have also been recently reported to be specific and sensitive markers for sex cord and stromal tumors of the ovary. 125 Although mutation of the FOXL2 gene is highly specific for adult granulosa cell tumor of the ovary, 126 FOXL2 immunostaining does not correlate with the FOXL2 mutational status, and thus cannot be used to distinguish between different histologic types of sex cord stromal tumors. 125 GESTATIONAL TROPHOBLASTIC DISEASE Illustrative Example 4 An 18-year-old gravida 2, para 0 patient presented at 8 þ 5/7 weeks gestation with ultrasound features suggestive of a hydatidiform mole. Uterine curettage was performed and microscopic evaluation revealed irregularly shaped villi with hypercellular myxoid villous stroma and trophoblastic proliferation, without significant villous hydrops (see Figure 17, C and D). Would p57 immunostain be helpful in this case, and how should the staining pattern be interpreted? Hydatidiform moles have a characteristic genetic composition: complete hydatidiform moles (CHMs) are entirely paternally derived, 127 most often with a diploid homozygous 46XX genotype, whereas partial moles (PHMs) are typically triploid with 1 set of maternal and 2 sets of paternal chromosomes. 128 Thus, paternally imprinted genes, which are preferentially expressed from the maternal allele, are silent in androgenetic CHMs. P57, a cyclin-dependent kinase inhibitor protein encoded by the CDKN1C gene on chromosome 11, is one such example, making p57 immunohistochemistry suitable for differentiating between CHMs and its mimics that contain maternal genetic material. 129 All gestations with maternal genetic material PHMs, hydropic nonmolar abortions, and trisomies show normal p57 protein expression pattern: strong nuclear staining in cytotrophoblasts, intermediate trophoblasts, intervillous trophoblast islands, and villous stromal cells, while syncytiotrophoblasts are uniformly negative. 130 In CHMs, on the other hand, cytotrophoblasts, villous 1066 Arch Pathol Lab Med Vol 141, August 2017 Immunohistochemistry in Gynecologic Pathology Buza & Hui

16 Figure 17. A and B, Hydropic nonmolar abortions show nuclear p57 staining in villous cytotrophoblast and stromal cells. C, Early complete hydatidiform mole with hypercellular, myxoid stroma, and circumferential trophoblastic proliferation. P57 immunostaining is absent in villous cytotrophoblast and stromal cells (D). Note the internal positive control in decidua in the upper portion of image (hematoxylin-eosin, original magnification 310 [A and C]; p57, original magnification 310 [B and D]). intermediate trophoblasts, and villous stromal cells lack p57 immunoreactivity, while p57 staining is retained in intervillous intermediate trophoblasts, villous endothelial cells, and maternal decidua, serving as internal positive controls 131 (Figure 17, A through D). However, p57 expression is not helpful in separating PHMs from other nonmolar hydropic gestations. Furthermore, there are several potential pitfalls of p57 immunohistochemistry that one must be aware of. The expression pattern is different between trophoblastic cell types (see above); absence of staining in cytotrophoblasts, villous intermediate trophoblasts, and villous stromal cells is characteristic of CHMs. Rare exceptions to this staining pattern exist: CHMs with retention of maternal chromosome 11 result in normal p57 expression and rare PHMs lack p57 staining owing to loss of maternal chromosome Mosaic androgenetic/biparental mosaic/chimeric gestations and twin (CHM and nonmolar) gestations can further complicate the immunohistochemical interpretation. 135,136 More precise adjunct diagnostic testing for hydatidiform moles molecular genotyping has become recently available in large academic centers, and various diagnostic algorithms have been proposed that integrate genotyping and p57 immunohistochemistry Immunohistochemical markers are often used in the diagnostic workup of gestational trophoblastic lesions. All 3 types of trophoblasts express low-molecular-weight cytokeratins (eg, CK18, CK8, CK AE1/AE3, CAM 5.2), 140,141 which are widely available and may be used to help in confirming intrauterine pregnancy/implantation site or identifying placental site nodule (PSN) in endometrial curettements. 142 Additional pan-trophoblastic markers also include hydroxyl-delta-5-steroid dehydrogenase (HSD3B1) and HLA-G, although these are less commonly available in routine pathology laboratories. Most recently, expression of GATA binding protein 3 (GATA3), a zinc finger transcription factor, has been described in a wide spectrum of trophoblastic lesions, including normal and molar gestations (Figure 18, A and B), PSN, and exaggerated placental site reaction (EPS) and trophoblastic neoplasms most choriocarcinomas, epithelioid trophoblastic tumor (ETT), and placental site trophoblastic tumor (PSTT). 146,147 GATA3, however, is a multispecific immunostain, which also frequently shows positivity in breast and urothelial Arch Pathol Lab Med Vol 141, August 2017 Immunohistochemistry in Gynecologic Pathology Buza & Hui 1067

17 Figure 18. A, Early implantation site in secretory endometrium. GATA binding protein 3 (GATA3) immunostain shows strong nuclear staining in all types of trophoblasts (B) (hematoxylin-eosin, original magnification 310 [A]; original magnification 310 [B]). carcinomas; moreover, this marker may be expressed in a variety of other tumors, including a small proportion of endometrial adenocarcinomas Neoplasms of intermediate trophoblast origin ETT and PSTT can be separated by p63 and hpl immunostains: p63 shows positivity in chorionic-type intermediate trophoblast lesions (PSN and ETT) and negativity in proliferations of implantation site trophoblast (EPS and PSTT), while hpl shows diffuse positivity in EPS and PSTT but typically stains only less than 5% of cells in ETT and PSN. 141,152,153 CONCLUSIONS Novel immunomarkers have been continuously introduced into the practice of gynecologic pathology in the past decade. Beyond diagnostic applications, recent years have witnessed an exciting repertoire of new immunostains in pathology laboratories for screening of inherited cancer syndromes, predicting prognosis and guiding treatment options for gynecologic cancers. In the modern era of precision therapy, the number of immunomarkers for the latter category will likely increase. However, the fundamental pathology diagnostic workup of gynecologic tumors still relies on the histologic evaluation of the H&E-stained tissue samples of lesions. Immunohistochemical stains, particularly new ones, must be interpreted with a good understanding of their limitations, and close correlation with conventional morphology must be sought to avoid misdiagnosis. References 1. O Neill CJ, McCluggage WG. p16 expression in the female genital tract and its value in diagnosis. Adv Anat Pathol. 2006;13(1): Keating JT, Cviko A, Riethdorf S, et al. Ki-67, cyclin E, and p16ink4 are complimentary surrogate biomarkers for human papilloma virus-related cervical neoplasia. Am J Surg Pathol. 2001;25(7): Kurman RJ, Carcanglu ML, Herrington CS, Young RH, eds; International Agency for Research on Cancer, World Health Organization. WHO Classification of Tumours of Female Reproductive Organs. 4th ed. Lyon, France: International Agency for Research on Cancer; World Health Organization Classification of Tumours; vol Darragh TM, Colgan TJ, Cox JT, et al. The Lower Anogenital Squamous Terminology Standardization Project for HPV-Associated Lesions: background and consensus recommendations from the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology. J Low Genit Tract Dis. 2012;16(3): Reuschenbach M, Wentzensen N, Dijkstra MG, von Knebel Doeberitz M, Arbyn M. p16ink4a immunohistochemistry in cervical biopsy specimens: a systematic review and meta-analysis of the interobserver agreement. Am J Clin Pathol. 2014;142(6): Stoler MH, Schiffman M; Atypical Squamous Cells of Undetermined Significance-Low-grade Squamous Intraepithelial Lesion Triage Study Group. Interobserver reproducibility of cervical cytologic and histologic interpretations: realistic estimates from the ASCUS-LSIL Triage Study. JAMA. 2001;285(11): Bergeron C, Ordi J, Schmidt D, et al. Conjunctive p16ink4a testing significantly increases accuracy in diagnosing high-grade cervical intraepithelial neoplasia. Am J Clin Pathol. 2010;133(3): Dalla Palma P, Giorgi Rossi P, Collina G, et al. The reproducibility of CIN diagnoses among different pathologists: data from histology reviews from a multicenter randomized study. Am J Clin Pathol. 2009;132(1): McCluggage WG, Bharucha H, Caughley LM, et al. Interobserver variation in the reporting of cervical colposcopic biopsy specimens: comparison of grading systems. J Clin Pathol. 1996;49(10): Preti M, Mezzetti M, Robertson C, Sideri M. Inter-observer variation in histopathological diagnosis and grading of vulvar intraepithelial neoplasia: results of an European collaborative study. BJOG. 2000;107(5): Doorbar J. Molecular biology of human papillomavirus infection and cervical cancer. Clin Sci (Lond). 2006;110(5): Fernandez S, Wong S, Hui P, Buza N. The impact of P16 immunostain on the diagnostic agreement of cervical biopsies using the new WHO terminology. Mod Pathol. 2015;28:284a 284a. 13. Klaes R, Benner A, Friedrich T, et al. p16ink4a immunohistochemistry improves interobserver agreement in the diagnosis of cervical intraepithelial neoplasia. Am J Surg Pathol. 2002;26(11): Tsoumpou I, Arbyn M, Kyrgiou M, et al. p16(ink4a) immunostaining in cytological and histological specimens from the uterine cervix: a systematic review and meta-analysis. Cancer Treat Rev. 2009;35(3): Agoff SN, Lin P, Morihara J, Mao C, Kiviat NB, Koutsky LA. p16(ink4a) expression correlates with degree of cervical neoplasia: a comparison with Ki-67 expression and detection of high-risk HPV types. Mod Pathol. 2003;16(7): Shain AF, Wilbur DC, Stoler MH, Quade BJ, Kong CS. Test characteristics of specific p16 clones in the detection of high-grade squamous intraepithelial lesions (HSIL). Mod Pathol. 2014;27:307a 307a. 17. Negri G, Vittadello F, Romano F, et al. p16ink4a expression and progression risk of low-grade intraepithelial neoplasia of the cervix uteri. Virchows Arch. 2004;445(6): Hariri J, Oster A. The negative predictive value of p16ink4a to assess the outcome of cervical intraepithelial neoplasia 1 in the uterine cervix. Int J Gynecol Pathol. 2007;26(3): Liao GD, Sellors JW, Sun HK, et al. p16ink4a immunohistochemical staining and predictive value for progression of cervical intraepithelial neoplasia grade 1: a prospective study in China. Int J Cancer. 2014;134(7): Quint KD, de Koning MN, Quint WG, Pirog EC. Progression of cervical low grade squamous intraepithelial lesions: in search of prognostic biomarkers. Eur J Obstet Gynecol Reprod Biol. 2013;170(2): Pirog EC. Immunohistochemistry and in situ hybridization for the diagnosis and classification of squamous lesions of the anogenital region. Semin Diagn Pathol. 2015;32(5): Scurry J, Beshay V, Cohen C, Allen D. Ki67 expression in lichen sclerosus of vulva in patients with and without associated squamous cell carcinoma. Histopathology. 1998;32(5): Pirog EC, Chen YT, Isacson C. MIB-1 immunostaining is a beneficial adjunct test for accurate diagnosis of vulvar condyloma acuminatum. Am J Surg Pathol. 2000;24(10): Arch Pathol Lab Med Vol 141, August 2017 Immunohistochemistry in Gynecologic Pathology Buza & Hui