The evolving landscape of immunohistochemistry in cervical and uterine carcinoma in gynecologic oncology: current status and future directions

Sumedha Gupta; Sana Ahuja; Dheer Singh Kalwaniya

doi:10.5468/ogs.24120

Obstet Gynecol Sci > Volume 67(5); 2024 > Article

Gupta, Ahuja, and Kalwaniya: The evolving landscape of immunohistochemistry in cervical and uterine carcinoma in gynecologic oncology: current status and future directions

Review Article

Gynecologic Oncology

Obstetrics & Gynecology Science 2024;67(5):449-466.

Published online: September 4, 2024

DOI: https://doi.org/10.5468/ogs.24120

The evolving landscape of immunohistochemistry in cervical and uterine carcinoma in gynecologic oncology: current status and future directions

Sumedha Gupta, MS¹, Sana Ahuja, MD²

, Dheer Singh Kalwaniya, MS³

¹Department of Obstetrics & Gynaecology, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India

²Department of Pathology, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India

³Department of General Surgery, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India

Corresponding author: Sana Ahuja, MD Department of Pathology, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi 110029, India E-mail: sanaahuja11@yahoo.com

Received April 27, 2024 Revised May 31, 2024 Accepted August 28, 2024

Articles published in Obstet Gynecol Sci are open-access, distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Immunohistochemistry (IHC) has become an indispensable tool in routine gynecological pathology, particularly with the advancements in molecular understanding and histological classification of gynecological cancers. This evolution has led to new immunostainings for diagnostic and classification purposes. This review describes the diagnostic utility of IHC in gynecological neoplasms, drawing insights from literature reviews, personal experiences, and research findings. It delves into the application of IHC in resolving morphologically equivocal cases, emphasizing its role in achieving an accurate diagnosis. The selection of appropriate immunomarkers for common scenarios encountered in gynecological pathology aids pathologists in navigating complex cases. Specifically, we focus on cervical and endometrial malignancies, elucidating the molecular rationale behind the use of specific immunohistochemical markers. An updated overview of essential immunohistochemical markers provides knowledge for precise diagnosis and classification of gynecological cancers. This review serves as a valuable resource for clinicians and researchers involved in the management and study of gynecological malignancies, facilitating improved patient care and outcomes.

Keywords: Endometrium; Uterus; Cervix; Carcinoma; Immunohistochemistry

Introduction

Morphologic assessment through Hematoxylin & Eosin (H&E) staining remains the primary method for diagnosing Mullerian tract abnormalities, but biomarker studies are increasingly valuable for further characterization. Biomarkers, measurable substances expressed in a cell or tissue-specific manner, aid in distinguishing normal from diseased states and can offer diagnostic, prognostic, and therapeutic insights.

Immunohistochemistry (IHC) is the foremost technique for biomarker evaluation. It involves creating antibodies to identify specific antigens, which are then detected using a secondary antibody system. Commonly utilized biomarkers in the Mullerian tract include PAX8, WT1, p16, p53, desmin, and mismatch repair (MMR) proteins.

When interpreting IHC results, several considerations are crucial: confirming H&E impressions, directing questions for antibody selection, understanding biomarker expression patterns, using limited panels, recognizing antibody specificity and sensitivity, and interpreting staining artifacts with attention to controls.

IHC combines microscopic morphology with molecular identification, aiding in classifying and understanding malignancies. It plays a pivotal role in guiding cancer therapy, exemplified by its routine use in identifying patients eligible for targeted treatments such as trastuzumab and tamoxifen.

With the continual emergence of molecular studies and novel biomarkers, pathologists’ responsibilities have expanded to ensure sample adequacy, perform IHC analyses, and contribute to biomarker development. Molecular studies have enriched the repertoire of IHC markers, enhancing the classification of gynecological malignancies and improving interobserver reproducibility.

IHC is a vital tool in gynecological pathology, facilitating accurate diagnosis, classification, and therapeutic decision-making. Its effective use requires a comprehensive understanding of biomarkers, meticulous technique, and clinical and morphological data integration.

Cervical cancer subtypes

1. Squamous cell carcinoma (SCC)

Cervical SCC is the predominant form of cervical cancer, comprising 80-90% of cases globally. The vast majority (>90%) are linked to high-risk human papillomavirus (HPV) types, notably HPV 16 and 18. However, a small percentage (5-7%) are HPV-independent, often occurring later in life and presenting at an advanced stage [1].

Histologically, SCCs display infiltrating epithelial nests with varying patterns such as keratinizing, non-keratinizing, basaloid, warty, and papillary. HPV-associated tumors commonly exhibit non-keratinizing and basaloid patterns, whereas HPV-independent SCCs typically manifest as keratinizing [2] (Fig. 1).

Morphology alone may not reliably establish HPV association, warranting p16 IHC and molecular HPV typing. Notably, nearly all HPV-associated SCCs exhibit strong and diffuse p16 overexpression in nuclei and cytoplasm, aiding in diagnosis [3].

2. Endocervical adenocarcinoma

Endocervical adenocarcinoma, typically arising from the transformation zone, exhibits glandular formation with stromal or expansile invasion. It can be HPV-associated (commonly HPV 18, 16, and 45) or HPV-independent. HPV status determination is crucial as HPV-associated cases generally have better outcomes.

Grossly, these tumors may present as ulcers, exophytic masses, or a barrel-shaped cervix due to endophytic growth [4].

Histologic subtypes of HPV-associated adenocarcinoma include the usual type (75% of cases), including villoglandular and micropapillary subtypes, and mucinous types such as intestinal, signet-ring cell, and invasive stratified mucin-producing carcinoma (invasive stratified mucin-producing intraepithelial lesion). Block-type p16 expression by IHC is seen in 95% of HPV-associated cases. In cases where doubt persists, HPV presence via in situ hybridization can confirm an HPV-associated adenocarcinoma, which is especially helpful in metastatic scenarios to ascertain endocervical origin. However, diffuse p16 positivity does not always indicate endocervical origin; it could signify high-grade endometrial primaries, notably serous carcinoma [5-7]. In such instances, a panel of immunostains, including carcinoembryonic antigen (CEA), estrogen receptor (ER), progesterone receptor (PR), and vimentin may provide additional insights, though results are not always definitive [8,9] (Fig. 2).

In differentiating between conventional endocervical adenocarcinoma and uterine serous carcinoma or high-grade endometrial primary, p53 staining can be useful. While p53 overexpression or null phenotype typically indicates ovarian or endometrial origin, caution is needed as some primary serous carcinomas of the cervix may exhibit p53 mutant expression. In such cases, tumor location during the examination of the hysterectomy specimen becomes pivotal in differentiation [10,11].

The Silva system categorizes HPV-associated adenocarcinomas into three patterns based on invasion characteristics, affecting prognosis [12].

HPV-independent adenocarcinomas include gastric, clear cell, mesonephric, and endometrioid types.

Gastric-type adenocarcinomas exhibit histologic and immunophenotypic features of gastric differentiation, often displaying foveolar or pyloric appearances and gastrointestinal-type neuroendocrine cells [13]. They may contain neutral cytoplasmic mucin, detectable through stains like Alcian blue or periodic acid-Schiff, although the preferred stains for detecting gastric-type mucin are MUC6 and HIK1083 [14,15]. Gastric type (10-15% of cases) is often associated with Peutz-Jeghers syndrome, demonstrating negative for p16 block expression and mutated p53 expression and presenting at an advanced stage.

Mesonephric adenocarcinoma of the cervix presents as well-differentiated neoplasms composed of tubules lined by flat to cuboidal epithelium with eosinophilic, colloid-like secretions. They may display heterogeneous morphology, making adjunct IHC useful for differentiation. Mesonephric carcinoma is negative to patchy positive for p16, reflecting its HPV-independent nature. Additional biomarkers used in the differential diagnosis include CEA (generally negative), calretinin (generally positive), CD10 (variably positive), and ER/PR (negative) [16]. The mesonephric type shows differentiation toward Wolffian structures. GATA binding protein 3 (GATA3), a marker indicative of mesonephric/Wolffian lineage, is strongly recommended due to its high sensitivity (98%) and specificity (98%) in identifying cervical mesonephric lesions compared to endocervical and endometrial carcinomas. However, GATA3 staining intensity can vary widely, from focal weak positivity to strong and diffuse, and it does not differentiate between benign and malignant mesonephric lesions [17,18].

Clear cell carcinoma of the cervix resembles its counterparts in the gynecologic tract, featuring tubulocystic, solid, and papillary growth patterns with characteristic hobnail cells, clear to eosinophilic cytoplasm, and high-grade nuclei. Immunostaining for HNF1beta and Napsin A, while similar to endometrial and ovarian lesions, does not aid in identifying the primary site [19,20]. Clear cell carcinomas (3-4%) may express p16 despite HPV negativity.

3. Adenosquamous carcinoma

The mixed epithelial tumor with squamous and glandular differentiation represents 5-6% of cervical carcinomas, showing similar clinical outcomes to cervical adenocarcinoma.

Histologically, it displays both squamous and glandular components, which are distinguishable on routine histology. The squamous part typically exhibits abundant glycogen-rich cytoplasm, while the glandular portion resembles usual HPV-associated adenocarcinoma.

IHC reveals p16 overexpression in both components. Additional stains like CK7, CEA, and PAX8 highlight the glandular part, while p63 and p40 highlight the squamous component [21] (Fig. 3).

4. Neuroendocrine carcinoma

Neuroendocrine carcinoma (NECC) of the cervix, although rare (<5% of cervical cancers), is the most common site for genital tract neuroendocrine tumors. Clinically aggressive, NECC often exhibits rapid metastasis and poor outcomes [22]. Typically, HPV-associated, with HPV types 18 more prevalent than 16, NECC morphologically resembles pulmonary neuroendocrine carcinomas, predominantly small cell type.

Small cell NECC, a morphological diagnosis, presents with a diffuse growth pattern, sometimes showing insular, perivascular, or trabecular patterns. Cytologically, it features uniform cells with scant cytoplasm, hyperchromatic nuclei, abundant mitosis, and necrosis. IHC may show positivity for chromogranin, CD56, synaptophysin, and insulinoma-associated protein 1, though staining patterns vary. Notably, thyroid transcription factor 1 positivity is common but lacks specificity [23] (Fig. 4).

While most NECCs are p16-positive due to HPV infection, p16 cannot discern the site of origin reliably. Hormone production, including adrenocorticotropic hormone and serotonin, may be observed in some cases. Differentiating small cell from large cell NECC can be challenging, requiring a combination of morphological and immunohistochemical assessment [24].

Biomarker for the lower genital tract

1. Programmed cell death ligand 1 (PD-L1)

Patients with HPV infection, SCC, advanced stage, large tumors, poor differentiation, metastatic disease, and prior chemotherapy are often associated with positive PD-L1 expression.

HPV infection may increase programmed cell death protein 1 (PD-1)/PD-L1 expression in the immune microenvironment, leading to high cytotoxic T lymphocyte infiltration and dysfunction. Blocking the PD-1/PD-L1 pathway could suppress antigen presentation and T-cell activation, altering cytokine regulation and promoting immune suppression in the tumor microenvironment.

Advanced or metastatic cervical cancer has a poor prognosis, with a 5-year overall survival rate of less than 5% using conventional radiotherapy and chemotherapy. High-risk HPV infection and elevated PD-L1 expression are common in SCC, potentially enhancing the effectiveness of immune checkpoint inhibitors (ICIs) due to increased inflammation in the tumor microenvironment.

While PD-L1 expression is not correlated with prognosis using conventional radiotherapy, high PD-L1 expression is linked to poorer outcomes.

PD-L1 expression varies with histopathological features; it is higher in SCC than in adenocarcinoma and poorly differentiated tumors. Young patients with poorly differentiated SCC show high PD-L1 [25].

Food and Drug Administration (FDA)-approved PD-L1 IHC assays (22C3, 28-8, SP263, and SP142) are used in clinical practice, with 22C3 being the most common. High concordance exists among 22C3, 28-8, and SP263, with SP263 showing higher sensitivity [26].

2. PD-1

PD-1 inhibits adaptive and innate immune responses and is expressed on activated T, NK, and B cells, macrophages, dendritic cells, and monocytes, particularly on tumor-specific T cells. PD-1 has dual roles as it helps maintain immune tolerance by reducing ineffective or harmful immune responses but also facilitates cancer cell evasion by interfering with protective immunity [27].

The FDA has approved PD-1 ICIs (nivolumab, pembrolizumab) for several cancers, and they are being explored for cervical cancer. Clinical trials reveal the potential benefits of pembrolizumab, nivolumab, and atezolizumab in cervical cancer, with ongoing studies further investigating their efficacy [4].

Distinction of endocervical and endometrial adenocarcinoma

Distinguishing between adenocarcinomas originating from the endocervix and endometrium is crucial for effective patient management. However, this differentiation can be challenging due to morphological similarities and the possibility of tumor involvement in both sites, as observed in imaging studies, biopsy specimens, and hysterectomy evaluations. Additionally, the primary site may not always be accurately represented by the dominant tumor component in hysterectomy specimens, complicating diagnosis further.

Immunohistochemical analysis is commonly employed to determine tumor origin, but the choice of markers depends on specific subtypes of endometrial and endocervical adenocarcinomas under consideration. It is important to note that there is not a one-size-fits-all panel of markers for distinguishing between all subtypes. The selection of markers varies based on factors such as the subtype of endometrial adenocarcinoma (endometrioid or serous), its grade, and the type of cervical neoplasm (HPV-related or unrelated subtypes like gastric-type or mesonephric adenocarcinoma). Thus, tailored marker panels are essential for accurate diagnosis in each case.

Distinction between high-risk HPV related endocervical adenocarcinoma and low-grade endometrial endometrioid adenocarcinoma

Distinguishing between high-risk HPV-related endocervical adenocarcinoma and common endometrial endometrioid adenocarcinoma poses challenges due to several factors. 1) Shared cellular characteristics: both types can exhibit mucinous and endometrioid-like features. While high-risk HPV-related endocervical adenocarcinomas typically display a hybrid of these features, true endometrioid differentiation is rare. Conversely, most endometrial adenocarcinomas are of the endometrioid type but may show varying degrees of mucinous differentiation. 2) Shared architectural patterns: both tumor types often exhibit predominant glandular architecture, as well as papillary and villoglandular growth patterns. 3) Involvement of both sites: biopsy or hysterectomy specimens may show involvement of both the endometrium and endocervix, complicating diagnosis. And 4) lack of identifiable precursor lesions: precursor lesions may be overgrown by carcinoma or simulated by tumor extension, making it challenging to identify the primary site.

In routine practice, distinguishing between low-grade endometrial endometrioid adenocarcinoma and high-risk HPV-related endocervical adenocarcinoma can be achieved using selected immunohistochemical markers. While traditional markers like CEA and vimentin have been used, the current recommendation emphasizes the utility of p16 and hormone receptors (estrogen and progesterone receptor, ER/PR) due to their reliability in distinguishing between these tumor types. High-risk HPV-related endocervical adenocarcinomas typically display diffuse moderate to strong p16 expression, reflecting HPV-mediated molecular alterations [28]. In contrast, endometrial endometrioid adenocarcinomas often exhibit patchy p16 expression, usually <80% of cells, with variable intensity and scattered negative areas. Additionally, ER/PR expression is typically absent in HPV-related endocervical adenocarcinomas but present in most endometrial endometrioid adenocarcinomas [29] (Table 1).

Distinction between high-risk HPV related endocervical adenocarcinoma and high-grade endometrial endometrioid adenocarcinoma

While a panel of immunohistochemical markers can help distinguish high-risk HPV-related endocervical adenocarcinomas from high-grade endometrial endometrioid adenocarcinomas, it is limited in distinguishing between an endometrial and cervical origin for other high-grade carcinomas like endometrial serous adenocarcinoma and undifferentiated carcinoma. Despite differences in morphological features, overlap can occur between these types of tumors [30]. For instance, both endocervical adenocarcinomas and endometrial serous adenocarcinomas may exhibit papillary and glandular architecture with high-grade nuclear features.

Endometrial serous adenocarcinomas commonly display diffuse/strong p16 expression and negative or focal hormone receptor expression, potentially leading to misdiagnosis as a primary cervical adenocarcinoma. In such cases, p53 expression and HPV studies may aid in diagnosis, with most endometrial serous adenocarcinomas showing aberrant p53 staining due to TP53 mutations [31].

However, high-risk HPV-related endocervical adenocarcinomas usually exhibit a heterogeneous p53 expression pattern, distinct from the aberrant patterns seen in serous adenocarcinomas. The coexistence of high-risk HPV and TP53 mutations is rare. Thus, aberrant p53 staining suggests a non-cervical primary. Additionally, p63 staining may assist in distinguishing poorly differentiated primary cervical SCC [32]. While theoretically possible, primary cervical serous adenocarcinomas are extremely uncommon, with many cases likely representing high-risk HPV-related cervical adenocarcinomas with serous-like morphology or misclassified primary endometrial or upper genital tract serous adenocarcinomas involving the cervix (Table 2).

Endometrial carcinoma

1. Endometrioid adenocarcinoma

The most common epithelial tumor of the uterus is well-differentiated endometrioid carcinoma. However, other subtypes like serous carcinoma, clear cell carcinoma, and carcinosarcoma also occur, sometimes resembling each other and high-grade endometrioid carcinoma. Distinguishing them is crucial for prognosis and treatment planning, including chemotherapy and radiation.

Biomarkers play a key role in 1) identifying carcinoma subtypes, 2) excluding metastases from non-Mullerian sources like the cervix or ovary, and 3) distinguishing poorly differentiated carcinomas from undifferentiated ones and carcinosarcomas. Commonly used biomarkers for uterine carcinoma include keratins, PAX8, p53, p16, ER, PR, alpha-methylacyl-CoA racemase (AMACR), Naspin, HNF1b, and Wilms' tumour gene 1 (WT1) [33,34].

PAX8, a member of the “paired box family” of genes, is crucial during the organogenesis of the Mullerian tract, thyroid gland, and kidney. Its absence leads to poorly formed genital tracts in female mice. PAX8 expression is observed in both benign epithelial structures and corresponding malignancies, making it a reliable biomarker to confirm uterine and upper Mullerian tract origin of a carcinoma, especially in metastatic cases [35,36]. It is sensitive to serous, endometrioid, and clear cell carcinomas of the uterus and ovary and occasionally in endocervical adenocarcinomas [37,38].

Endometrioid carcinoma of the uterus typically shows positive staining for PAX8, ER, and PR, with patchy expression of p16. A diffuse p16 expression and either the absence or focal staining of hormone receptors may suggest serous carcinoma or endocervical origin, the latter possibly expressing CEA. In challenging cases, HPV in situ hybridization can aid in diagnosing endocervical adenocarcinoma (Fig. 5).

While most cases of endometrioid carcinoma demonstrate heterogeneous p53 staining (wild-type pattern), a subset may exhibit strong diffuse staining (mutant pattern) or complete absence (null mutant pattern), particularly in highgrade cases. Loss of staining for MMR proteins, suggestive of microsatellite instability, is more common in the endometrioid subtype. Regardless of biomarker expression, the presence of squamous and/or mucinous differentiation serves as a valuable morphologic clue for endometrioid differentiation [39,40].

2. Uterine serous carcinoma (USC)

USCs are typically linked with p53 mutations, evident through strong and diffuse p53 expression (mutant pattern) or complete absence of p53 expression (null mutant pattern). In contrast, wild-type/heterogeneous p53 expression in atypical cells, especially within endometrial polyps, suggests reactive atypia or a non-serous carcinoma. Serous carcinomas often show diffuse p16 expression, which should not be confused with endocervical adenocarcinoma [41].

While ovarian serous carcinomas are usually WT1 positive, the majority of USC are expected to be WT1-negative. This difference can aid in distinguishing synchronous primary tumors from metastatic ones. However, morphological features such as deep uterine wall invasion and lymphovascular invasion should also be considered, as WT1 sensitivity and specificity in these sites are not absolute [42] (Fig. 6).

USC exhibits distinct molecular profiles and aggressive metastatic behavior. Traditional platinum-based chemotherapy shows limited effectiveness against USC, with high rates of resistance and recurrence, necessitating the development of novel targeted therapies. Human epidermal growth factor receptor 2 (HER2) has emerged as a significant oncogene in USC, with HER2 protein overexpression observed in approximately 30-35% of cases.

Trastuzumab, the first HER2-directed monoclonal antibody, was FDA-approved in 1998 for HER2-positive metastatic breast cancer. It exerts tumoricidal effects through antibody-dependent cellular cytotoxicity and inhibition of HER2-mediated signaling, thereby blocking cell proliferation and angiogenesis. Trastuzumab is approved for HER2-positive early-stage and metastatic breast cancer, as well as HER2-positive gastric, and gastroesophageal junction adenocarcinoma. The National Comprehensive Cancer Network guidelines recommend trastuzumab, combined with carboplatin-paclitaxel, for patients with stage III/IV or recurrent USC with HER2 overexpression [43].

3. Uterine clear cell carcinoma

Clear cell carcinoma of the uterus is a rare subtype with unique morphological features like tubulopapillary growth and clear cytoplasm. It often lacks response to chemotherapy and is associated with AT-rich interactive domain 1A (ARID1A) gene abnormalities [44]. While ARID1A staining is not commonly used clinically, recent studies highlight the utility of novel biomarkers like Napsin A, racemase/AMACR/p504S, and HNF1b in diagnosis. Although not entirely sensitive or specific, Napsin A and AMACR strongly support a clear cell carcinoma diagnosis, especially in the absence of ER and PR [45,46] (Fig. 7).

These biomarkers are particularly helpful when distinguishing endometrioid carcinoma with clear cell features from true clear cell carcinoma. Additionally, clear cell carcinomas typically show heterogeneous p53 staining and patchy or negative p16 expression, unlike serous carcinoma. However, morphological features and a panel of biomarkers should be utilized to ensure accurate diagnosis, as some clear cell carcinomas may exhibit mutant p53 expression [47] (Table 3).

4. Endometrial carcinoma with spindle cell component

Poorly differentiated uterine malignant neoplasms with epithelioid and spindle cell morphology pose a diagnostic challenge, including dedifferentiated carcinoma, undifferentiated carcinoma, carcinosarcoma, uterine sarcoma, and rarely, metastatic melanoma. Dedifferentiated carcinoma is often diagnosed based on morphological features, particularly when a well-differentiated component is adjacent to a poorly differentiated one. Biomarkers like cytokeratins and epithelial membrane antigen (EMA) can aid in identifying well-differentiated epithelial components in dedifferentiated carcinoma and carcinosarcoma. In contrast, undifferentiated carcinomas typically lack cytokeratin expression but may show focal EMA reactivity [48].

Undifferentiated carcinoma is characterized by negativity or focal positivity for various markers, including cytokeratins (AE1/AE3, 8, 18, and 8/18), vimentin, EMA, ER, PR, chromogranin, synaptophysin, E-cadherin, and CTNNB1. Approximately 30% exhibit TP53 mutation, 30% show vimentin expression, and 50% have mutations in MMR genes [49]. CD34 expression has been observed in 29% of cases, and loss of expression in SMARCA4 and SMARCA2, components of the SWItch/sucrose non-fermentable chromatin-remodeling complex, has been noted [50,51].

Malignant mixed Müllerian tumors, also known as carcinosarcomas, are aggressive biphasic tumors containing both carcinomatous and sarcomatous components. Typically, morphological features suffice for diagnosis, but in rare cases, distinguishing them from endometrioid carcinomas with spindle cell differentiation may benefit from assessing markers such as high p53/WT1 expression and low ER/PR expression [52].

Mismatch repair immunohistochemistry (MMR IHC)

Lynch syndrome (LS) is a hereditary cancer syndrome linked to mutations in MMR genes, increasing the risk of endometrial and colorectal cancers. Screening for LS in endometrial malignancies involves MMR protein IHC or MSI testing, with MMR IHC alone often preferred due to cost. MMR IHC assesses the expression of four MMR proteins: human mutL homolog 1 (MLH1), postmeiotic segregation increased 2 (PMS2), MutS homolog 2 (MSH2), and MutS homolog 6 (MSH6). Normal expression (reported as “intact”) indicates all four proteins are expressed [53].

MLH1 loss indicates MLH1/PMS2 loss, while MSH2 loss indicates MSH2/MSH6 loss. Isolated PMS2 or MSH6 loss suggests respective gene mutations. Any detectable nuclear staining is considered “intact”, and clonal loss may indicate somatic rather than germline loss [54]. Loss of MLH1 and PMS2 expression is often due to MLH1 promoter methylation, necessitating MLH1 promoter methylation testing before germline testing [55] (Table 4).

Molecular subgroups of endometrial cancer

1. Four molecular subgroup as defined by cancer genome atlas

The Cancer Genome Atlas (TCGA) project categorized endometrial cancers into four molecular subtypes based on mutational burden and copy number alterations. 1) Ultra-mutated cancers with polymerase epsilon (POLE) exonuclease domain mutations, 2) hypermutated cancers with microsatellite instability (MMR deficiency), 3) copy-number-high cancers with frequent TP53 mutations, and 4) copy-number-low cancers with low mutational burden.

POLEmut cancers, with pathogenic POLE mutations, show high mutational burden, strong immune response, early-stage high-grade tumors in younger women, and favorable prognosis, whereas MMR deficient cancers, comprising 25-30% of cases with loss of MMR proteins due to MLH1 promoter hypermethylation or Lynch syndrome, elicit a strong immune response with intermediate prognosis, copy-number-high cancers, high-grade with frequent TP53 mutations and aggressive growth, include serous, carcinosarcoma, clear cell, and high-grade endometrioid cancers with poor prognosis, and copy-number-low cancers, typically endometrioid-type with low mutational burden and positive hormone receptor staining, and have a variable prognosis [56] (Fig. 8).

2. Molecular integrated risk profile

To simplify TCGA classification for clinical use, cost-effective and reliable methods like the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) have been proposed.

PRoMisE group has classified molecular sub-groups of endometrial cancer using surrogate markers in paraffin-embedded tissues. Besides the TCGA molecular groups, additional prognostic clinico-pathologic and molecular factors include substantial lymphovascular space invasion, L1-cell adhesion molecule overexpression, catenin beta 1 (CTNNB1) mutation, and 1q32.1 amplification, especially relevant within the no specific molecular profile sub-group. L1-cell adhesion molecule, a membrane glycoprotein linked to tumor cell adhesion and migration, correlates with TP53 mutations, non-endometrioid histology, high tumor grade, and lymphovascular space invasion, serving as an independent risk factor for loco-regional and distant spread. CTNNB1 mutations stimulate endometrial tissue growth, raising recurrence risk and lowering recurrence-free survival, while 1q32.1 amplification significantly worsens prognosis in no specific molecular profile cases. The ProMisE validation studies highlighted enhanced prognostic significance when combining molecular sub-groups with clinico-pathological factors [57].

Mesenchymal tumors of the uterus

1. Smooth muscle tumors (SMTs)

Uterine mesenchymal tumors encompass various subtypes, primarily derived from smooth muscle cells, such as benign leiomyomas, SMTs of uncertain potential (STUMP), and leiomyosarcomas. Additionally, there are endometrial stromal neoplasms, perivascular epithelioid cell tumors (PEComas), and inflammatory myofibroblastic tumors (IMTs) [58].

SMTs include leiomyomas, STUMP, and leiomyosarcomas, distinguished by their biological potential. Desmin and h-caldesmon are commonly used biomarkers for SMTs, although their utility in subtype differentiation is limited. Ki-67 and hormone receptors (ER and PR) show significant differences between benign and malignant SMTs, with higher expression in leiomyosarcomas [59,60] (Fig. 9). However, they are less effective in distinguishing STUMP from leiomyoma or leiomyosarcoma. Epithelioid and myxoid SMTs pose diagnostic challenges, often overlapping with other mesenchymal tumors like PEComas, high-grade endometrial stromal sarcomas (ESS), and IMTs [61,62]. Additional biomarkers like human melanoma black-45 (HMB45), MelanA, CD10, and cyclin D1 aid in differential diagnosis, with anaplastic lymphoma kinase (ALK) expression in a small subset of myxoid leiomyosarcomas necessitating fluorescent in situ hybridization (FISH) for confirmation [63].

1) Distinction between leiomyosarcoma and leiomyoma

In differentiating leiomyosarcoma (LMS) from leiomyoma, IHC becomes crucial for difficult cases. Diffuse p53 and p16 expression, along with a high Ki-67 proliferation index, suggest leiomyosarcoma. However, some SMTs with uncertain malignant potential and certain leiomyomas may exhibit overlapping patterns, complicating diagnosis [64].

2. Endometrial stromal tumors (ESTs)

ESTs are rare, comprising less than 10% of uterine mesenchymal tumors. They can be subclassified into benign endometrial stromal nodules (ESNs), low-grade endometrial stromal sarcomas (LGESS), high-grade endometrial stromal sarcomas (HGESS), and undifferentiated uterine sarcomas. LGESS and ESNs share similar cytologic features, but their architectural patterns distinguish their malignant potential. Biomarkers like CD10 are key in distinguishing ESTs from SMTs, with ESNs/LGESSs typically expressing CD10 and lacking SMT biomarkers like desmin and h-caldesmon. Though not specific, ER and PR are sensitive markers for ESTs and may guide treatment options [65] (Fig. 10).

HGESS, or high-grade endometrial stromal sarcoma, stands out morphologically, immunophenotypically, and genetically from low-grade EST. Initially recognized in 2014, HGESS is characterized by the YWHAE-NUTM2 gene fusion resulting from t(10;17)(q22;p31), although it can also feature the ZC3H7B-BCOR translocation t(X;22)(p11;q13). These tumors exhibit subtle yet distinct histological variances [66,67].

YWHAE-NUTM2 HGESS presents as infiltrative high-grade stromal proliferation with atypical round-to-epithelioid cytology and a lower-grade spindle cell component. In contrast, ZC3H7B-BCOR HGESS lacks a low-grade spindle cell component, displaying a more myxoid appearance with increased mitotic activity and necrosis.

Immunohistochemical analysis reveals that YWHAE-NUTM2 HGESS expresses CD10, ER, and PR in the low-grade spindle cell component but not in high-grade areas, with strong and diffuse cyclinD1 immunoreactivity in high-grade areas. ZC3H7B-BCOR HGESS, on the other hand, frequently expresses CD10 in high-grade areas, is less likely to express ER and PR, and exhibits cyclinD1 expression in nearly all cases [68]. The differential diagnosis for HGESS includes LGESS, myxoid LMS, and IMT, with immunostaining aiding in distinguishing these tumor types. However, genetic confirmation via FISH or next-generation sequencing may be necessary when needed (Table 5).

3. PEComa

PEComa, found in soft tissues and visceral organs, is rare in the gynecologic tract and can mimic symptoms of other uterine tumors. It features atypical epithelioid proliferation with infiltrative growth in the myometrium. Tumor cells typically exhibit clear to eosinophilic cytoplasm, some being multinucleated. A malignant variant is characterized by increased size (>5 cm), high nuclear grade, necrosis, and heightened mitotic activity [69].

PEComas are identified by melanocytic markers like HMB45, MelanA/MART1, and microphthalmia associated transcription factor, though the expression may be focal. Cathepsin K is a sensitive biomarker. Differential diagnosis includes leiomyoma variants, LMS, ESS, and alveolar soft part sarcoma, warranting an immunostain panel including smooth muscle, endometrial stromal, and transcription factor E3 markers [70]. Uterine PEComas may express desmin, SMA, h-caldesmon, and PNL2, distinguishing them from SMTs and ESTs. PNL2 is valuable, showing similar staining to HMB45 and aiding in differentiation from SMTs [71,72].

4. IMT

Uterine IMT is a rare neoplasm often misdiagnosed before its recent recognition. It presents as a hypocellular spindle cell proliferation, easily confused with SMTs and low-grade ESTs, particularly those with myxoid features. Diagnostic markers include ALK expression by IHC, while p53 and p16 expression can help differentiate it from LMS. FISH can confirm the diagnosis by detecting ALK gene rearrangements [73,74].

Conclusion

Biomarkers play a crucial role in assessing lesions within the gynecologic tract. However, it’s essential to ensure that the interpretation of biomarkers aligns with the morphologic question at hand.

Utilizing biomarkers effectively require careful consideration of various factors, including the presence of positive, internal, and negative controls, as well as sensitivity, specificity, and potential staining artifacts associated with IHC. Pitfalls can arise when using biomarkers, underscoring the importance of a meticulous approach to their interpretation. By maintaining awareness of these factors and adhering to best practices in biomarker analysis, healthcare professionals can optimize the accuracy and reliability of diagnostic assessments in gynecologic pathology.

Notes

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

Waived due to literature review.

Patient consent

Waived due to literature review.

Funding information

Nil.

Fig. 1.

A case of HPV-associated cervical squamous cell carcinoma. (A, B) Hematoxylin and Eosin-stained sections exhibited tumor cells arranged in nests and sheets with enlarged hyperchromatic nuclei and moderate eosinophilic cytoplasm. Focal keratinization was also noted (×100 and ×400). (C, D) On immunohistochemistry, tumor cells exhibited nuclear and block positivity for p63 (C) and p16 (D) respectively (×100). HPV, human papilloma virus.

Fig. 2.

A case of HPV-associated endocervical adenocarcinoma. (A, B) Hematoxylin and Eosin-stained sections exhibited tumor cells arranged as back-to-back glands. The tumor cells were columnar and showed moderate pleomorphism with elongated nuclei and scant cytoplasm (×100 and ×400). (C-F) On immunohistochemistry, tumor cells exhibited block positivity for p16 (C) and were negative for ER (D), PR (E), and vimentin (F) (×100). HPV, human papilloma virus; ER, estrogen receptor; PR, progesterone receptor.

Fig. 3.

A case of cervical adenosquamous carcinoma. (A, B) Hematoxylin and Eosin-stained sections exhibited tumor cells arranged as nests and sheets as well as in glandular patterns. Individual tumor cells exhibited moderate pleomorphism (×100 and ×400). (C-F) On immunohistochemistry, the nests of tumor cells (squamous component) exhibited nuclear and block positivity for p63 (C) and p16 (D) respectively (×100). The glandular component showed positive membranous and cytoplasmic expression for EMA (E) and CEA (F) respectively (×100). EMA, epithelial membrane antigen; CEA, carcinoembryonic antigen.

Fig. 4.

A case of neuroendocrine carcinoma of the cervix. (A) Hematoxylin and Eosin-stained sections exhibited sheets and nests of tumor cells with hyperchromatic nuclei and scant cytoplasm (×100). (B-D) On immunohistochemistry, tumor cells showed positive cytoplasmic expression for synaptophysin (B) and chromogranin (C) with a Ki-67 (D) proliferation index of 40% (×100).

Fig. 5.

A case of endometrioid adenocarcinoma of the endometrium. (A) Hematoxylin and Eosin-stained sections exhibited tumor cells arranged as back-to-back glands and solid nests. The tumor cells exhibited moderate pleomorphism with enlarged nuclei and scant cytoplasm (×100). (B-D) On immunohistochemistry, the tumor cells showed nuclear expression of ER (B) and PR (C) and cytoplasmic expression of vimentin (D) (×100). ER, estrogen receptor; PR, progesterone receptor.

Fig. 6.

A case of serous papillary carcinoma of the endometrium. (A, B) Hematoxylin and Eosin-stained sections exhibited a tumor arranged as papillaroid fragments with a fibrovascular core. Individual tumor cells were moderately pleomorphic with enlarged nuclei and moderate eosinophilic cytoplasm (×100 and ×400). (C, D) On immunohistochemistry tumor cells exhibited positive cytoplasmic and nuclear expression for CK7 (C) and p53 (D) respectively (×100).

Fig. 7.

A case of clear cell carcinoma of the endometrium. (A, B) Hematoxylin and Eosin-stained sections exhibited a tumor arranged as complex glandular and papillary architecture with numerous eosinophilic globules. Individual tumor cells were polygonal with moderate clear to eosinophilic cytoplasm with few hobnail cells (×100 and ×400). (C, D) On immunohistochemistry tumor cells exhibited positive cytoplasmic and nuclear expression for Napsin (C) and HNF-1B (D) respectively (×100).

Fig. 8.

Diagnostic algorithm of subclassification of four molecular subtypes. POLE, polymerase epsilon; MMR, mismatch repair; EC, endometrial cancer; MMRd, mismatch repair deficiency; NSMP, non-specific molecular profiling.

Fig. 9.

A case of leiomyosarcoma of the uterus. (A, B) Hematoxylin and Eosin-stained sections exhibited a tumor arranged as long intersecting fascicles and sheets of spindle cells with eosinophilic cytoplasm and hyperchromatic nuclei with moderate to severe pleomorphism (×100 and ×400). (C-F) On immunohistochemistry, tumor cells showed positive cytoplasmic expression for vimentin (C), desmin (D), and smooth muscle actin (E) with a Ki-67 (F) proliferation index of 30% (×100).

Fig. 10.

A case of endometrial stromal sarcoma of the uterus. (A) Hematoxylin and Eosin-stained sections exhibited a tumor composed of nests and sheets of round cells with scant cytoplasm and enlarged hyperchromatic nuclei with arborizing vessels in between (×100). (B-D) On immunohistochemistry, tumor cells showed positive cytoplasmic and nuclear expression for CD10 (B) and ER (C) respectively with a Ki-67 (D) proliferation index of 40% (×100). ER, estrogen receptor.

Table 1.

Biomarkers distinguishing endocervical and low-grade endometrial adenocarcinoma

Marker	Endocervical adenocarcinoma (HPV associated)	Endocervical adenocarcinoma (non-HPV related to gastric type)	Low-grade endometrial adenocarcinoma (endometrioid, mucinous)
ER/PR	Negative	Negative	Positive
p53	Wild-type	Wild-type or mutant-type	Wild-type
p16	Positive (diffuse)	Negative to focally positive	Variable positivity
HR HPV	Positive	Negative	Negative

HPV, human papillomavirus; ER, estrogen receptor; PR, progesterone receptor; HR, high-risk.

Table 2.

Biomarkers distinguishing endocervical and high-grade endometrial adenocarcinoma

Marker	Endocervical adenocarcinoma (HPV associated)	High grade endometrial endometrioid adenocarcinoma	Endometrial serous adenocarcinoma
ER/PR	Negative	Usually positive	Negative
p53	Wild-type	Often wild-type, some mutant-type	Mutant type
p16	Positive (diffuse)	Variable positivity	Positive
HR HPV	Positive	Negative	Negative

HPV, human papillomavirus; ER, estrogen receptor; PR, progesterone receptor; HR, high-risk.

Table 3.

Immunohistochemical markers in the differential diagnosis of endometrial carcinoma

Endometrial tumor	Vimentin	ER	PR	ARID1A	Beta-catenin	AMACR	HNF 1 beta	Napsin A	p53	p16	Mutation
EMC	+	+	+	M	M	+/-	-	-	wt	-/+	PTEN and MMR loss
USC	+	-	-	wt	wt	-	-/+	-	M	+	CCNE1 amplification
CCC	+	-	-	wt	wt	-/+	+	+/-	wt>M	-	MMR loss
UC	-	-	-	wt	M	NA	-	NA	M>wt	-/+	E-cadherin and MMR loss
MMMT	+	-/+	-/+	wt	wt	NA	-	NA	M	+	WT1, desmin, CD10, h-caldesmon, myogenin, S100, and Myo-D1

ER, estrogen receptor; PR, progesterone receptor; ARID1A, AT-rich interaction domain 1A; AMACR, alpha-methylacyl-coA racemase; HNF, hepatocyte nuclear factor; EMC, endometrioid carcinoma; M, mutated; wt, wild type; PTEN, phosphatase and tensin homolog; MMR, mismatch repair; USC, uterine serous carcinoma; CCNE1, cyclin e1; CCC, clear cell carcinoma; UC, undifferentiated carcinoma; NA, not applicable; MMMT, malignant mixed mullerian tumor; WT1, wilms’ tumor gene.

Table 4.

Correlation between mutation, immunohistochemistry (IHC) expression of mismatch repair gene (MMR), and microsatellite instability (MSI) status in Lynch syndrome-associated cancer MMR protein expression and mutation

Mutation	MLH1 IHC	MSH2 IHC	MSH6 IHC	PMS2 IHC	MSI
MLH1	-	+	+	-	H
MSH2	+	-	-	+	H
MSH6	+	+	-	+	H/L
PMS2	+	+	+	-	H
EPCAM	+	-	-	+	H

IHC is utilized, with “+” indicating positive nuclear staining and “-” indicating negative nuclear staining, further classified as high (H) or low (L).

MLH, human mutL homolog 1; MSH2, MutS homolog 2; MSH6, MutS homolog 6; PMS2, postmeiotic segregation increased 2; H/L, high/low; EPCAM, epithelial cellular adhesion molecule.

Table 5.

Immunohistochemistry markers in the differential diagnosis of endometrial mesenchymal tumor

Endometrial mesenchymal tumor	Vimentin	ER	PR	p53	p16	CD10	Desmin	h-caldesmon	Remarks
LGSS	+	+	+	wt	-	+	-	-	JAZF1-SUZ12 fusion
HGSS	+	-	-	wt	-	-	-	-	YWHAE-NUTM2A/B fusion, cyclin-D, c-kit, and Ki-67 high
LM	+	+	+	wt	-	-	+	+	Actin
LMS	+	-/+	-/+	M>wt	+	-	+	+	Ki67 high

ER, estrogen receptor; PR, progesterone receptor; LGSS, low-grade endometrial stromal sarcoma; wt, wild type; HGSS, high-grade endometrial stromal sarcoma; LM, leiomyoma; LMS, leiomyosarcoma; M, mutant type.

References

1. Krishnamurti U, Movahedi-Lankarani S, Bell DA. Protocol for the examination of resection specimens from patients with primary carcinoma of the uterine cervix [Internet]. Chicago: College of American Pathologists; c2020 [cited 2024 Jan 11]. Available from: https://documents.cap.org/protocols/cp-gynecologic-uterinecervixresection-20-5000.pdf.

2. Bhatla N, Berek JS, Cuello Fredes M, Denny LA, Grenman S, Karunaratne K, et al. Revised FIGO staging for carcinoma of the cervix uteri. Int J Gynaecol Obstet 2019;145:129-35.

3. Stolnicu S, Barsan I, Hoang L, Patel P, Terinte C, Pesci A, et al. International endocervical adenocarcinoma criteria and classification (IECC): a new pathogenetic classification for invasive adenocarcinomas of the endocervix. Am J Surg Pathol 2018;42:214-26.

4. Diaz De Vivar A, Roma AA, Park KJ, Alvarado-Cabrero I, Rasty G, Chanona-Vilchis JG, et al. Invasive endocervical adenocarcinoma: proposal for a new pattern-based classification system with significant clinical implications: a multi-institutional study. Int J Gynecol Pathol 2013;32:592-601.

5. Yemelyanova A, Vang R, Seidman JD, Gravitt PE, Ronnett BM. Endocervical adenocarcinomas with prominent endometrial or endomyometrial involvement simulating primary endometrial carcinomas: utility of HPV DNA detection and immunohistochemical expression of p16 and hormone receptors to confirm the cervical origin of the corpus tumor. Am J Surg Pathol 2009;33:914-24.

6. Chiesa-Vottero AG, Malpica A, Deavers MT, Broaddus R, Nuovo GJ, Silva EG. Immunohistochemical overexpression of p16 and p53 in uterine serous carcinoma and ovarian high-grade serous carcinoma. Int J Gynecol Pathol 2007;26:328-33.

7. McCluggage WG, Hurrell DP, Kennedy K. Metastatic carcinomas in the cervix mimicking primary cervical adenocarcinoma and adenocarcinoma in situ: report of a series of cases. Am J Surg Pathol 2010;34:735-41.

8. Glenn McCluggage W. Immunohistochemistry as a diagnostic aid in cervical pathology. Pathology 2007;39:97-111.

9. McCluggage WG, Sumathi VP, McBride HA, Patterson A. A panel of immunohistochemical stains, including carcinoembryonic antigen, vimentin, and estrogen receptor, aids the distinction between primary endometrial and endocervical adenocarcinomas. Int J Gynecol Pathol 2002;21:11-5.

10. Staebler A, Sherman ME, Zaino RJ, Ronnett BM. Hormone receptor immunohistochemistry and human papillomavirus in situ hybridization are useful for distinguishing endocervical and endometrial adenocarcinomas. Am J Surg Pathol 2002;26:998-1006.

11. Nofech-Mozes S, Khalifa MA, Ismiil N, Saad RS, Hanna WM, Covens A, et al. Immunophenotyping of serous carcinoma of the female genital tract. Mod Pathol 2008;21:1147-55.

12. Roma AA, Mistretta TA, Diaz De Vivar A, Park KJ, Alvarado-Cabrero I, Rasty G, et al. New pattern-based personalized risk stratification system for endocervical adenocarcinoma with important clinical implications and surgical outcome. Gynecol Oncol 2016;141:36-42.

13. Talia KL, McCluggage WG. The developing spectrum of gastric-type cervical glandular lesions. Pathology 2018;50:122-33.

14. Zhao S, Hayasaka T, Osakabe M, Kato N, Nakahara K, Kurachi H, et al. Mucin expression in nonneoplastic and neoplastic glandular epithelia of the uterine cervix. Int J Gynecol Pathol 2003;22:393-7.

15. Lee S, Sahasrabuddhe VV, Mendoza-Cervantes D, Zhao R, Duggan MA. Tissue-based immunohistochemical biomarker expression in malignant glandular lesions of the uterine cervix: a systematic review. Int J Gynecol Pathol 2018;37:128-40.

16. Carleton C, Hoang L, Sah S, Kiyokawa T, Karamurzin YS, Talia KL, et al. A detailed immunohistochemical analysis of a large series of cervical and vaginal gastric-type adenocarcinomas. Am J Surg Pathol 2016;40:636-44.

17. Howitt BE, Emori MM, Drapkin R, Gaspar C, Barletta JA, Nucci MR, et al. GATA3 is a sensitive and specific marker of benign and malignant mesonephric lesions in the lower female genital tract. Am J Surg Pathol 2015;39:1411-9.

18. Roma AA, Goyal A, Yang B. Differential expression patterns of GATA3 in uterine mesonephric and nonmesonephric lesions. Int J Gynecol Pathol 2015;34:480-6.

19. Ju B, Wang J, Yang B, Sun L, Guo Y, Hao Q, et al. Morphologic and immunohistochemical study of clear cell carcinoma of the uterine endometrium and cervix in comparison to ovarian clear cell carcinoma. Int J Gynecol Pathol 2018;37:388-96.

20. Spaans VM, Scheunhage DA, Barzaghi B, de Kroon CD, Fleuren GJ, Bosse T, et al. Independent validation of the prognostic significance of invasion patterns in endocervical adenocarcinoma: pattern a predicts excellent survival. Gynecol Oncol 2018;151:196-201.

21. Rindi G, Klimstra DS, Abedi-Ardekani B, Asa SL, Bosman FT, Brambilla E, et al. A common classification framework for neuroendocrine neoplasms: an International Agency for Research on Cancer (IARC) and World Health Organization (WHO) expert consensus proposal. Mod Pathol 2018;31:1770-86.

22. Howitt BE, Kelly P, McCluggage WG. Pathology of neuroendocrine tumours of the female genital tract. Curr Oncol Rep 2017;19:59.

23. Ganesan R, Hirschowitz L, Dawson P, Askew S, Pearmain P, Jones PW, et al. Neuroendocrine carcinoma of the cervix: review of a series of cases and correlation with outcome. Int J Surg Pathol 2016;24:490-6.

24. Perunovic B, Sunassee A. Small cell neuroendocrine carcinoma [Internet]. Bingham Farms: PathologyOutlines; c2024 [cited 2024 Jan 24]. Available from: https://www.pathologyoutlines.com/topic/cervixsmallcell.html.

25. Huang W, Liu J, Xu K, Chen H, Bian C. PD-1/PD-L1 inhibitors for advanced or metastatic cervical cancer: from bench to bed. Front Oncol 2022;12:849352.

26. Lantuejoul S, Sound-Tsao M, Cooper WA, Girard N, Hirsch FR, Roden AC, et al. PD-L1 testing for lung cancer in 2019: perspective from the IASLC pathology committee. J Thorac Oncol 2020;15:499-519.

27. Han Y, Liu D, Li L. PD-1/PD-L1 pathway: current researches in cancer. Am J Cancer Res 2020;10:727-42.

28. Ansari-Lari MA, Staebler A, Zaino RJ, Shah KV, Ronnett BM. Distinction of endocervical and endometrial adenocarcinomas: immunohistochemical p16 expression correlated with human papillomavirus (HPV) DNA detection. Am J Surg Pathol 2004;28:160-7.

29. Kong CS, Beck AH, Longacre TA. A panel of 3 markers including p16, ProExC, or HPV ISH is optimal for distinguishing between primary endometrial and endocervical adenocarcinomas. Am J Surg Pathol 2010;34:915-26.

30. Saad RS, Mashhour M, Noftech-Mozes S, Ismiil N, Dubé V, Ghorab Z, et al. P16INK4a expression in undifferentiated carcinoma of the uterus does not exclude its endometrial origin. Int J Gynecol Pathol 2012;31:57-65.

31. McCluggage WG, Soslow RA, Gilks CB. Patterns of p53 immunoreactivity in endometrial carcinomas: ‘all or nothing’ staining is of importance. Histopathology 2011;59:786-8.

32. Houghton O, McCluggage WG. The expression and diagnostic utility of p63 in the female genital tract. Adv Anat Pathol 2009;16:316-21.

33. Bartosch C, Manuel Lopes J, Oliva E. Endometrial carcinomas: a review emphasizing overlapping and distinctive morphological and immunohistochemical features. Adv Anat Pathol 2011;18:415-37.

34. Gilks CB, Oliva E, Soslow RA. Poor interobserver reproducibility in the diagnosis of high-grade endometrial carcinoma. Am J Surg Pathol 2013;37:874-81.

35. Laury AR, Perets R, Piao H, Krane JF, Barletta JA, French C, et al. A comprehensive analysis of PAX8 expression in human epithelial tumors. Am J Surg Pathol 2011;35:816-26.

36. Ordóñez NG. Value of PAX 8 immunostaining in tumor diagnosis: a review and update. Adv Anat Pathol 2012;19:140-51.

37. Nonaka D, Chiriboga L, Soslow RA. Expression of pax8 as a useful marker in distinguishing ovarian carcinomas from mammary carcinomas. Am J Surg Pathol 2008;32:1566-71.

38. Shukla A, Thomas D, Roh MH. PAX8 and PAX2 expression in endocervical adenocarcinoma in situ and high-grade squamous dysplasia. Int J Gynecol Pathol 2013;32:116-21.

39. Köbel M, Ronnett BM, Singh N, Soslow RA, Gilks CB, McCluggage WG. Interpretation of P53 immunohistochemistry in endometrial carcinomas: toward increased reproducibility. Int J Gynecol Pathol 2019;38 Suppl 1:S123-31.

40. Baniak N, Fadare O, Köbel M, DeCoteau J, Parkash V, Hecht JL, et al. Targeted molecular and immunohistochemical analyses of endometrial clear cell carcinoma show that POLE mutations and DNA mismatch repair protein deficiencies are uncommon. Am J Surg Pathol 2019;43:531-37.

41. Chen W, Husain A, Nelson GS, Rambau PF, Liu S, Lee CH, et al. Immunohistochemical profiling of endometrial serous carcinoma. Int J Gynecol Pathol 2017;36:128-39.

42. Kommoss F, Faruqi A, Gilks CB, Lamshang Leen S, Singh N, Wilkinson N, et al. Uterine serous carcinomas frequently metastasize to the fallopian tube and can mimic serous tubal intraepithelial carcinoma. Am J Surg Pathol 2017;41:161-70.

43. McNamara B, Mutlu L, Greenman M, Harold J, Santin A. HER2 oncogene as molecular target in uterine serous carcinoma and uterine carcinosarcoma. Cancers (Basel) 2023;15:4085.

44. Glasspool RM, McNeish IA. Clear cell carcinoma of ovary and uterus. Curr Oncol Rep 2013;15:566-72.

45. Fadare O, Liang SX. Diagnostic utility of hepatocyte nuclear factor 1-beta immunoreactivity in endometrial carcinomas: lack of specificity for endometrial clear cell carcinoma. Appl Immunohistochem Mol Morphol 2012;20:580-7.

46. Fadare O, Zhao C, Khabele D, Parkash V, Quick CM, Gwin K, et al. Comparative analysis of Napsin A, alphamethylacyl-coenzyme A racemase (AMACR, P504S), and hepatocyte nuclear factor 1 beta as diagnostic markers of ovarian clear cell carcinoma: an immunohistochemical study of 279 ovarian tumours. Pathology 2015;47:105-11.

47. Hariri N, Qarmali M, Fadare O. Endometrial serous carcinoma with clear-cell change: frequency and immunohistochemical analysis. Int J Surg Pathol 2018;26:126-34.

48. Li Z, Zhao C. Clinicopathologic and immunohistochemical characterization of dedifferentiated endometrioid adenocarcinoma. Appl Immunohistochem Mol Morphol 2016;24:562-8.

49. Tafe LJ, Garg K, Chew I, Tornos C, Soslow RA. Endometrial and ovarian carcinomas with undifferentiated components: clinically aggressive and frequently underrecognized neoplasms. Mod Pathol 2010;23:781-9.

50. Shah VI, Ramalingam P, McCluggage WG. CD34 expression in undifferentiated endometrial carcinoma. Histopathology 2016;69:894-7.

51. Ramalingam P, Croce S, McCluggage WG. Loss of expression of SMARCA4 (BRG1), SMARCA2 (BRM) and SMARCB1 (INI1) in undifferentiated carcinoma of the endometrium is not uncommon and is not always associated with rhabdoid morphology. Histopathology 2017;70:359-66.

52. Tipps AM, Plaxe SC, Weidner N. Endometrioid carcinoma with a low-grade spindle cell component: a tumor resembling an adnexal tumor of probable Wolffian origin. Ann Diagn Pathol 2011;15:376-81.

53. Stewart AP. Genetic testing strategies in newly diagnosed endometrial cancer patients aimed at reducing morbidity or mortality from lynch syndrome in the index case or her relatives. PLoS Curr 2013;5:ecurrents.eogt.b59a6e84f27c536e50db4e46aa26309c.

54. Watkins JC, Nucci MR, Ritterhouse LL, Howitt BE, Sholl LM. Unusual mismatch repair immunohistochemical patterns in endometrial carcinoma. Am J Surg Pathol 2016;40:909-16.

55. Pai RK, Plesec TP, Abdul-Karim FW, Yang B, Marquard J, Shadrach B, et al. Abrupt loss of MLH1 and PMS2 expression in endometrial carcinoma: molecular and morphologic analysis of 6 cases. Am J Surg Pathol 2015;39:993-9.

56. van den Heerik ASVM, Horeweg N, de Boer SM, Bosse T, Creutzberg CL. Adjuvant therapy for endometrial cancer in the era of molecular classification: radiotherapy, chemoradiation and novel targets for therapy. Int J Gynecol Cancer 2021;31:594-604.

57. Ogunmuyiwa J, Williams V. Emerging advances in endometrial cancer: integration of molecular classification into staging for enhanced prognostic accuracy and implications for racial disparities. Cancers (Basel) 2024;16:1172.

58. Oliva E. Practical issues in uterine pathology from banal to bewildering: the remarkable spectrum of smooth muscle neoplasia. Mod Pathol 2016;29 Suppl 1:S104-20.

59. Zhang Q, Kanis MJ, Ubago J, Liu D, Scholtens DM, Strohl AE, et al. The selected biomarker analysis in 5 types of uterine smooth muscle tumors. Hum Pathol 2018;76:17-27.

60. Hakverdi S, Güngören A, Yaldiz M, Hakverdi AU, Toprak S. Immunohistochemical analysis of p16 expression in uterine smooth muscle tumors. Eur J Gynaecol Oncol 2011;32:513-5.

61. Hoang LN, Aneja A, Conlon N, Delair DF, Middha S, Benayed R, et al. Novel high-grade endometrial stromal sarcoma: a morphologic mimicker of myxoid leiomyosarcoma. Am J Surg Pathol 2017;41:12-24.

62. Parra-Herran C, Schoolmeester JK, Yuan L, Dal Cin P, Fletcher CD, Quade BJ, et al. Myxoid leiomyosarcoma of the uterus: a clinicopathologic analysis of 30 cases and review of the literature with reappraisal of its distinction from other uterine myxoid mesenchymal neoplasms. Am J Surg Pathol 2016;40:285-301.

63. Parra-Herran C, Quick CM, Howitt BE, Dal Cin P, Quade BJ, Nucci MR. Inflammatory myofibroblastic tumor of the uterus: clinical and pathologic review of 10 cases including a subset with aggressive clinical course. Am J Surg Pathol 2015;39:157-68.

64. O’Neill CJ, McBride HA, Connolly LE, McCluggage WG. Uterine leiomyosarcomas are characterized by high p16, p53 and MIB1 expression in comparison with usual leiomyomas, leiomyoma variants and smooth muscle tumours of uncertain malignant potential. Histopathology 2007;50:851-8.

65. Hwang H, Matsuo K, Duncan K, Pakzamir E, Pham HQ, Correa A, et al. Immunohistochemical panel to differentiate endometrial stromal sarcoma, uterine leiomyosarcoma and leiomyoma: something old and something new. J Clin Pathol 2015;68:710-7.

66. Micci F, Gorunova L, Agostini A, Johannessen LE, Brunetti M, Davidson B, et al. Cytogenetic and molecular profile of endometrial stromal sarcoma. Genes Chromosomes Cancer 2016;55:834-46.

67. Isphording A, Ali RH, Irving J, Goytain A, Nelnyk N, Hoang LN, et al. YWHAE-FAM22 endometrial stromal sarcoma: diagnosis by reverse transcription-polymerase chain reaction in formalin-fixed, paraffin-embedded tumor. Hum Pathol 2013;44:837-43.

68. Lee CH, Mariño-Enriquez A, Ou W, Zhu M, Ali RH, Chiang S, et al. The clinicopathologic features of YWHAEFAM22 endometrial stromal sarcomas: a histologically high-grade and clinically aggressive tumor. Am J Surg Pathol 2012;36:641-53.

69. Conlon N, Soslow RA, Murali R. Perivascular epithelioid tumours (PEComas) of the gynaecological tract. J Clin Pathol 2015;68:418-26.

70. Folpe AL, Mentzel T, Lehr HA, Fisher C, Balzer BL, Weiss SW. Perivascular epithelioid cell neoplasms of soft tissue and gynecologic origin: a clinicopathologic study of 26 cases and review of the literature. Am J Surg Pathol 2005;29:1558-75.

71. Rao Q, Cheng L, Xia QY, Liu B, Li L, Shi QL, et al. Cathepsin K expression in a wide spectrum of perivascular epithelioid cell neoplasms (PEComas): a clinicopathological study emphasizing extrarenal PEComas. Histopathology 2013;62:642-50.

72. Gulavita P, Fletcher CDM, Hirsch MS. PNL2: an adjunctive biomarker for renal angiomyolipomas and perivascular epithelioid cell tumours. Histopathology 2018;72:441-8.

73. Bennett JA, Nardi V, Rouzbahman M, Morales-Oyarvide V, Nielsen GP, Oliva E. Inflammatory myofibroblastic tumor of the uterus: a clinicopathological, immunohistochemical, and molecular analysis of 13 cases highlighting their broad morphologic spectrum. Mod Pathol 2017;30:1489-503.

74. Haimes JD, Stewart CJR, Kudlow BA, Culver BP, Meng B, Koay E, et al. Uterine inflammatory myofibroblastic tumors frequently harbor ALK fusions with IGFBP5 and THBS1. Am J Surg Pathol 2017;41:773-80.