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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.
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.
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