Gynecologic tract

Gross findings

LSIL (VIN 1) typically appears as a pale macule, papule, or hyperkeratotic lesion on clinical gross examination. HSIL (VIN 2 and VIN 3) typically appear as conspicuous white or erythematous macules, papules, or nodules on clinical gross examination.

Microscopic findings

Histologic sections of LSIL show epithelial thickening with dysplastic changes, including hypercellularity, nuclear enlargement with increased nuclear-to-cytoplasmic ratio, hyperchromasia, and increased mitosis, limited to the lower third of the epithelium. The lower third of the epithelium shows decreased cytoplasmic maturation, but normal maturation is seen in the middle to upper third. Superficial koilocytes with hyperchromatic, occasionally bi- or multinucleated, nuclei, and cytoplasmic halos are characteristic ( Fig. 2.1 A). Both low- and high-risk HPV types are associated with LSIL (VIN 1).

Histologic sections of HSIL show epithelial thickening with dysplastic changes, including hypercellularity, nuclear enlargement with irregular nuclear membranes, increased nuclear-to-cytoplasmic ratio, hyperchromasia, and increased mitosis (occasionally with atypical mitotic figures), involving the middle to upper two thirds of the epithelium. A lack of maturation is noted, with cells appearing more basal-/parabasal-like ( Fig. 2.1 B). Lesions are characterized as moderate dysplasia (VIN 2) when dysplastic changes involve the lower two thirds of the epithelium, and severe dysplasia (VIN 3) when full-thickness dysplastic changes are noted. Koilocytes may occasionally be present.

Differentiated vulvar intraepithelial neoplasia

Differentiated vulvar intraepithelial neoplasia (DVIN) is HPV-independent and frequently arises in a background of vulvar dermatoses, particularly lichen sclerosus and lichen planus. The simplex type/DVIN is considered high grade. A substantial number of cases of DVIN are associated with TP53 mutations. Three less common types of HPV-independent vulvar squamous intraepithelial neoplasms termed differentiated exophytic vulvar intraepithelial lesion (DEVIL), vulvar acanthosis with altered differentiation (VAAD), and basaloid DVIN have been described. Like DVIN, DEVIL and VAAD are HPV-independent. Mutations in PIK3CA have been described in DEVIL. HRAS mutations were found to be common in VAAD.

DVIN lesions are typically observed in areas of lichen sclerosus or lichen planus and may be associated with pruritus, pain, and burning. They typically appear erythematous or white, may have a rough irregular surface, and may be associated with hyperkeratosis or ulceration. DVIN lesions tend to be more clinically/grossly subtle than those observed in classic VIN. As a result, a high degree of clinical suspicion is warranted in patients with a history of lichen sclerosus or lichen planus.

Histologic sections may likewise be subtle due to the highly differentiated nature of the lesion. Sections of DVIN show epidermal thickening with hyper and/or parakeratosis and elongated rete ridges. The keratinocytes typically appear enlarged with large nuclei, prominent nucleoli, and abundant eosinophilic cytoplasm. The granular layer may be inconspicuous. Key diagnostic clues include premature keratinization (keratinocytes with abundant pink cytoplasm are abnormally located in the basal third of the epidermis), dyskeratosis of individual keratinocytes, and basal keratinocyte atypia (including karyomegaly, nuclear hyperchromasia, macronucleoli, and atypical mitosis). Given the association of DVIN with TP53 mutations, mutant p53 immunohistochemical staining patterns are frequently present and the p53 immunohistochemical stain can be a diagnostic adjunct. Patterns of p53 staining that have been found to correlate with TP53 mutations include the following: strong nuclear staining in consecutive basal tumor cells; diffuse strong nuclear staining in basal and parabasal layers; complete absence of staining in the setting of a positive internal control; diffuse cytoplasmic staining in the setting of a positive internal control.

Both DVIN and HSIL show acanthosis and significant keratinocyte atypia. In contrast to HSIL, which shows a loss of maturation, DVIN shows increased (abnormal) maturation, with superficial-type keratinizing cells being present in the basal third of the epidermis ( Fig. 2.1 C). The keratinocyte atypia in DVIN is typically restricted to the basal layer of the epidermis, while that of HSIL is more diffuse involving the middle third and upper layers. There, however, can be morphologic overlap, and in such cases, taking clinical features into account (i.e., patient’s age, presence/history of lichen sclerosus or other dermatoses, sexual history, HIV status, or any history of abnormal cervical PAP smears) may be informative. Additionally, because DVIN is HPV-independent, and HSIL is HPV-associated, immunohistochemical staining for p16 (and/or direct HPV detection tests) is useful (see Fig. 2.2 ); p53 immunohistochemical stain may also be of use, if correctly interpreted ( Fig. 2.3 ). Features useful in distinguishing usual-type VIN from DVIN are summarized in Table 2.1 .

• DVIN versus squamous hyperplasia: Both DVIN and benign hyperplastic squamous epithelium show acanthosis (i.e., epithelial thickening) and some degree of hyperkeratosis. However, benign squamous hyperplasia displays normal epidermal maturation (with preserved granular layer); it does not show abnormal keratinization and lacks the significant basal keratinocyte atypia that is characteristic of DVIN.

• DVIN versus hypertrophic lichen planus: Like DVIN, lichen planus is often associated with acanthosis and hyperkeratosis and may even have individual dyskeratotic keratinocytes. However, the lesions may be distinguished based on preserved epidermal maturation with accentuation of the granular layer (wedge-shaped hypergranulosis) observed in lichen planus. In addition, lichen planus is typically associated with a lichenoid inflammatory infiltrate and clefting at the dermoepidermal junction. Lichen planus also lacks the significant basal keratinocyte atypia that is characteristic of DVIN.

• DVIN versus lichen sclerosus: Early lichen sclerosus may show psoriasiform epidermal hyperplasia (i.e., elongated rete ridges) similar to DVIN. Lichen sclerosus, however, typically has associated dermal fibrosis (or homogenization in advanced lesions), lichenoid inflammatory infiltrate, and normal epidermal maturation. Lichen sclerosus also lacks the significant basal atypia that is characteristic of DVIN.

• DVIN versus other HPV-independent VIN: The other types of HPV-independent VIN are only just beginning to be recognized. Watkins et al. coined the term differentiated exophytic vulvar intraepithelial lesion (DEVIL) to describe exophytic vulva lesions with prominent acanthosis or verruciform hyperplasia that lack the histomorphologic features of HPV-associated HSIL and also lack sufficient basal atypia to warrant a diagnosis of DVIN. In their study, DEVILs lacked TP53 mutations but were associated with PIK3CA and ARID2 mutations, supporting their categorization as a distinct entity from DVIN. Vulvar acanthosis with altered differentiation (VAAD) was described by Nascimento et al. as an intraepithelial squamous proliferation that displayed marked acanthosis with variable verruciform architecture, loss of granular cell layer with superficial epithelial cell pallor, and multilayered parakeratosis. Both VAAD and DEVIL are distinguished from DVIN by their lack of significant basal keratinocyte atypia. However, there is significant morphologic overlap between DEVIL and VAAD and establishing whether VAAD and DEVIL are distinct entities from each other requires more extensive studies. Ordi et al. described an unusual type of HPV-independent VIN that displayed architectural distortion with a homogenous population of basaloid keratinocytes, mimicking HSIL. All cases were HPV-negative by PCR and p16-negative by immunohistochemistry. In general, due to the varying morphologies and subtleties of HPV-independent vulvar squamous cancer precursors, it is prudent to be wary of acanthotic, hyperkeratotic (or even basaloid) lesions in postmenopausal women, especially those with a history of lichen sclerosus or lichen planus. Ancillary immunohistochemical stains with p16 and p53 may be of value.

Invasive squamous cell carcinoma of the vulva

When HSIL extends into adnexal structures, distinction from invasive squamous cell carcinoma can be difficult, especially on tangential sections. Obtaining deeper sections may be helpful in such cases. Other clues that may help in distinguishing HSIL from invasive carcinoma include paying attention to the spatial orientation of the nests on low-power microscopic evaluation—the nests in HSIL tend to be rounded/organized—and noticing the preserved basement membrane of HSIL on higher power. Invasive squamous cell carcinoma is also typically associated with a desmoplastic stromal reaction that is not seen in HSIL.

A diagnosis of superficially invasive squamous cell carcinoma of the vulva (AJCC pT1a tumor) is rendered if all of the following conditions are met: (1) the tumor is 2 cm or less in size, (2) the tumor is confined to the vulva or perineum, and (3) the depth of invasion is less than or equal to 1 mm. The depth of invasion is measured from the base of epithelial-stromal junction of the adjacent most superficial dermal papilla to the deepest point of stromal invasion. Superficially invasive tumors have low risk of lymph node metastasis and are thus managed by wide local excision (simple partial vulvectomy) without the need for lymph node dissection. Additional surgery (radical vulvectomy with sentinel lymph node biopsy or inguinofemoral lymph node dissection) may be required if a tumor is discovered to have larger size and/or >1 mm of stromal invasion after initial partial vulvectomy.

Paget disease of the vulva

Paget disease is an intraepithelial adenocarcinoma characterized by apocrine/eccrine large cells with abundant cytoplasm (Paget cells). Occasionally, HSIL can display a nested pattern that morphologically mimics Paget disease. Immunohistochemical stains are helpful in distinguishing HSIL from Paget. In contrast to HSIL, Paget cells stain positive for GCDFP-15 (an apocrine marker), CEA, CAM5.2, HER2/neu, and androgen receptors. Paget cells are also mostly negative for p40 and CK5/6. Given that Paget disease is a glandular neoplasm, mucin special stains also tend to be positive in Paget (but negative in HSIL).

Gross findings

Gross findings vary depending on the size of the carcinoma—varying from erythematous, raised lesions to exophytic or endophytic masses with necrosis.

Microscopic findings

Morphologic types : There are various morphologic types of invasive squamous cell carcinoma, such as non-keratinizing (also called usual-type), keratinizing, basaloid, verrucous, papillary, warty, squamotransitional, and lymphoepithelioma-like; HPV has been identified in all described morphologic types. Except for verrucous carcinoma, a well-differentiated tumor with a bulbous pushing (rather than infiltrative) invasive front, which does not metastasize but has a tendency for local recurrence if incompletely excised, squamous cell carcinoma morphologic type has little effect on prognosis once grade and stage have been taken into account. Nevertheless, papillary squamous cell carcinoma and lymphoepithelial-like carcinoma are variants worth noting. Papillary squamous cell carcinoma is superficially exophytic with papillae/connective tissue stroma covered by HSIL-like epithelium but is often associated with an underlying stromal invasive carcinoma, often usual-type. This variant is worth noting because it can be underdiagnosed as HSIL on superficial biopsies. Lymphoepithelial-like carcinoma is composed of poorly defined islands of undifferentiated-appearing squamous cells (i.e., lack keratinization and intercellular bridges) in a background of a dense lymphocytic infiltrate. This variant is worth noting because the associated dense lymphocytic infiltrate may mask the presence of the underlying malignant epithelial cells, which can be highlighted by pancytokeratin, p40, and p16 immunohistochemical stains.

Occasionally a tumor may be composed of clearly distinguishable glandular and squamous carcinoma components—a mixture of a definite adenocarcinoma and squamous cell carcinoma. Such tumors are termed adenosquamous carcinomas. Adenosquamous carcinomas are typically HPV-associated and stage for stage appear to have similar prognosis and behavior to cervical squamous cell carcinoma. Of note, scattered mucin-producing cells may be seen in poorly differentiated squamous cell carcinoma, but their presence is insufficient to render a diagnosis of adenosquamous carcinoma. A diagnosis of adenosquamous carcinoma is rendered when a tumor contains well-developed recognizable glands in addition to a squamous cell carcinoma component.

Grading: The 2014 WHO mentions criteria for grading cervical squamous cell carcinoma that include extent of squamous differentiation, nuclear pleomorphism, size of nucleoli, mitotic frequency, and necrosis; however, details on how to implement such criteria are lacking.

A three-tiered grading system based on tumor budding activity and tumor nest size (TBNS) has been proposed for cervical squamous cell carcinoma. The TBNS system assigns a score from 1 to 3 based on tumor budding activity per 10 high-power fields (HPF): score 1, no budding; score 2: <15 budding foci; score 3, ≥15 budding foci. A score from 1 to 4 is assigned for the smallest cell nest size within the tumor: score 1, > 15 cells; score 2, 5–15 cells; score 3, 2–4 cells; score 4, single-cell invasion. The grade is then assigned based on the total score: grade 1, score 2–3; grade 2, score 4–5; and grade 3, score 6–7. The TBNS system has been found to be a significant prognostic indicator. The system likely assesses a tumor’s dispersion propensity, that is, level of epithelial to mesenchymal transition and metastatic potential.

Squamous intraepithelial lesions

Squamous intraepithelial lesions of the uterine cervix are non-invasive HPV-associated neoplasms. LSILs as associated with transient HPV infections, episomal infections that end up being cleared. The episomal phase of infection is associated with morphologic features, such as koilocytosis, that define LSIL.

In immunocompromised people, those who smoke, those infected with particularly virulent high-risk HPV strains such as HPV types 16 and 18, and those with other less understood risk factors, high-risk HPV infections may persist, allowing for viral integration into the host genome and progression to HSIL. Untreated HSIL has a 30% to 50% risk of progressing into invasive squamous cell carcinoma over 30 years.

HSIL may occasionally extend into endocervical glands; extensive glandular involvement by HSIL may be confused with invasive squamous cell carcinoma. Helpful distinguishing features include smooth rounded contours with intact basement membrane in HSIL versus irregular angulated contours in invasive carcinoma. Stromal desmoplasia, detached small tumor nests, and single isolated cells within the stroma are features of invasive carcinoma ( Fig. 2.4 D). A diagnosis of superficially invasive squamous cell carcinoma is rendered if all of the following conditions are met: (1) the tumor has been completely excised, (2) the tumor is not grossly visible, (3) the depth of invasion is less than or equal to 3 mm, and (4) the horizontal spread is less than or equal to 7 mm. The depth of invasion is measured from the base of epithelium involved by HSIL to the deepest point of stromal invasion by carcinoma. After complete cone excision or loop electrosurgical excision procedure (LEEP), tumors diagnosed as superficially invasive squamous cell carcinomas that lack lymphovascular invasion may be managed by observation to spare fertility, if surgical margin clearance is 3 mm or greater. Some histologic features useful in distinguishing invasive squamous cell carcinoma from HSIL are presented in Table 2.2 .

• LSIL versus HSIL: LSIL shows cell maturation and often has associated koilocytic change, whereas cell maturation is often lost in HSIL (i.e., the basal epithelium is not easily distinguished from the superficial epithelium) ( Fig. 2.4 ). However, in flat or tangentially sectioned epithelium or in cases of CIN 2 where some surface maturation occurs, it may be difficult to distinguish LSIL from HSIL. A diagnosis of HSIL should be rendered in the presence of extreme basal/parabasal nuclear atypia, atypical mitosis, and mitosis higher than the basal third of the epithelium. A combination of p16 and Ki67 immunohistochemical stains may be useful in challenging cases. HSIL shows strong and diffuse p16 positivity and Ki67 activity higher than the basal epithelium, while LSIL is p16 negative or if positive, has staining limited to the basal third of the epithelium. In addition, Ki67 is limited to the epithelial base in LSIL. Of note, the immunohistochemical stain results need to be interpreted in conjunction with morphologic findings. Immunohistochemistry is not recommended in cases that are clearly LSIL or HSIL based on morphology. A diagnosis of LSIL should not be changed to HSIL based solely on immunohistochemical staining results.

• Benign, reactive, atrophic, and metaplastic squamous epithelium versus HPV-related squamous intraepithelial lesions: The cytoplasmic clearing in mature glycogenated squamous epithelium and perinuclear clearing in squamous epithelium reacting to infections like trichomoniasis may mimic LSIL. LSIL, however, is characterized by nuclear enlargement, hyperchromasia, and membrane irregularities that distinguish it from benign and reactive processes. Squamous metaplasia and atrophy may show a lack of maturation that mimics HSIL. However, the cellular uniformity, smooth nuclear contours, and lack of mitosis and presence of small nucleoli distinguish squamous metaplasia from HSIL. Atrophy similarly has cellular uniformity, smooth nuclear contours, and lack of mitosis, whereas HSIL often shows enlarged irregular hyperchromatic nuclei that lack appreciable nucleoli in addition to the presence of mitotic figures higher than the basal third of the epithelium. A combination of p16 and Ki67 immunohistochemical stains is useful in challenging cases. HSIL shows strong and diffuse p16 positivity and Ki67 activity higher than the basal epithelium, while squamous metaplasia and atrophy are p16 negative (focal reactivity is considered negative) and have Ki67 limited to the epithelial base.

Gross findings

HPVA endocervical adenocarcinomas grossly appear as ulcers and/or exophytic or endophytic masses in the endocervix.

Microscopic findings

HPVA endocervical adenocarcinomas are invasive tumors morphologically defined by easily detected apical mitosis and apoptosis (i.e., mitosis and apoptosis can be identified at scanning to low-power [4–10×] magnification) and are often diffusely p16 positive by immunohistochemistry ( Fig. 2.5 ). They encompass different morphologic types: usual type, villoglandular, mucinous (intestinal type), mucinous (signet ring cell type), and invasive stratified mucin producing.

Usual type is the most common type of endocervical adenocarcinoma, accounting for approximately 75% of all endocervical adenocarcinomas. It is relatively mucin-poor; intracytoplasmic mucin is present in less than 50% of the cells. Usual-type endocervical adenocarcinomas can be further classified into prognostic groups based on the pattern of stromal invasion. Pattern A tumors carry an excellent prognosis; all studied tumors have been stage I at diagnosis and have not been associated with lymph node metastasis or recurrence. Pattern A tumors are characterized by well-formed glands with preserved lobular architecture and a lack of stromal reaction (i.e., stromal desmoplasia is absent). Single detached cells, detached tumor clusters, solid growth, and lymphovascular invasion must be absent for tumors to be categorized as pattern A. Pattern C tumors display diffuse destructive stromal invasion with desmoplasia, solid growth, and lymphovascular invasion. Extravasated mucin with admixed tumor cells, band-like lymphocytic infiltrate, papillary and/or micropapillary architecture, and confluent glands may be present. Pattern C tumors often present at stage II or higher and are associated with lymph node metastasis (22.5% of the studies cases), recurrences (19.7% of cases), and death from disease. Pattern B tumors are associated with limited (<5 mm; one 4× field) destructive stromal invasion in a background of pattern A. Lymphovascular invasion may be present, but solid growth must be absent. Most patients with pattern B tumors were stage I at diagnosis; less than 5% had lymph node metastasis. Assigning a pattern to usual-type endocervical adenocarcinomas should only be done in a completely excised specimen. Data suggest that regardless of size or depth of stromal invasion, pattern A tumors can be managed with localized complete surgical excision (e.g., cone or LEEP with negative margins) without the need for more radical surgery. Pattern B tumors can be managed with localized complete resection and sentinel node excision to determine the need for adjuvant therapy, while radical surgery may be necessary in pattern C tumors.

Other histologic types of HPVA endocervical adenocarcinoma include: Villoglandular carcinomas which display distinct exophytic long slender papillae. Villoglandular adenocarcinomas typically occur in a younger age group (median age, 34 years) than usual-type adenocarcinomas (median age, 42 years). Mucinous carcinomas comprise cells with intracytoplasmic mucin making up >50% of the tumor population. The mucinous cells may be goblet cells (intestinal type) or signet ring cells (signet ring cell type). Invasive stratified mucin-producing intraepithelial lesion (iSMILE) is characterized by invasive nests of stratified columnar cells with peripheral palisading and variable amounts of intracytoplasmic mucin. At least one study suggests that iSMILE may be more aggressive than usual-type endocervical adenocarcinoma.

Precursor glandular lesions for HPVA endocervical adenocarcinomas include adenocarcinoma in situ (AIS), HPVA, and SMILE. HSIL is occasionally identified as a precursor in HPVA endocervical adenocarcinomas.

HPV-independent endocervical adenocarcinomas (NHPVA adenocarcinomas)

NHPVA endocervical adenocarcinomas are invasive adenocarcinomas with various etiologies unrelated to HPV infection. They encompass different morphologic types, the most common being gastric-type adenocarcinomas, including minimal deviation adenocarcinoma/adenoma malignum. STK11 mutations, gains in chromosome 3p, and loss of 1p have been described in gastric-type adenocarcinomas. Lobular endocervical glandular hyperplasia is a potential precursor lesion for gastric-type adenocarcinomas. Other NHPVA morphologic types of endocervical adenocarcinomas are rare and include: endometrioid (thought to arise from endometriosis), clear cell, serous, and mesonephric.

NHPVA endocervical adenocarcinomas tend to present as larger tumors (median tumor size, 38 mm) and in older women (median age, 55 years) compared with HPVA endocervical adenocarcinomas (median tumor size, 21 mm; median patient age, 42 years). The gastric-type endocervical adenocarcinomas have a significantly worse prognosis than HPVA adenocarcinomas, being more frequently associated with distant metastasis.

In contrast to usual type adenocarcinoma and other HPV-associated subtypes, the NHPVA adenocarcinoma subtypes do NOT have easily identifiable apical mitosis or apoptosis. Mitotic and apoptotic figures may be present but require high-power diligent evaluation to identify. If readily identifiable apical mitotic or apoptotic figures are found even focally, p16 immunohistochemical stain and testing for high-risk HPV types is recommended to avoid misclassifying HPVA adenocarcinomas as one of the NHPVA subtypes. The NHPVA subtypes include:

• 1.

  Gastric type: contains cells with abundant clear, foamy, or pale eosinophilic cytoplasm with distinct cytoplasmic borders and basally located nuclei similar to gastric pyloric glands ( Fig. 2.6 ). Adenoma malignum (minimal deviation adenocarcinoma) is part of the spectrum of gastric-type adenocarcinoma. Gastric-type adenocarcinomas carry a worse prognosis than usual-type adenocarcinoma, being more frequently associated with deep stromal invasion, higher pathologic stage at presentation, and lower disease-free survival (30% 5-year disease-free survival in gastric type versus 74% in usual type).

  

• 2.

  Endometrioid: lacks readily identifiable apical mitosis and apoptosis and has classic endometrioid features—glands lined by columnar cells with pseudostratified nuclei that demonstrate no more than moderate atypia. Squamous metaplasia and/or endometriosis may be present. Of note, in the IECC study, a number of cancers that were initially called endometrioid adenocarcinomas were reclassified as usual-type adenocarcinoma. It is important to exclude HPVA usual-type adenocarcinoma by performing p16 immunohistochemical stain and/or HPV testing before classifying an endocervical adenocarcinoma as endometrioid.

• 3.

  Serous: resemble their endometrial and tubo-ovarian counterparts, being composed of cells with high-grade nuclei displaying slit-like glands, papillary, and/or micropapillary architecture. Endocervical serous carcinomas are exceedingly rare and should only be diagnosed after HPV testing has been performed to exclude HPVA serous-like usual-type adenocarcinomas. In addition, direct extension from the endometrium and drop metastasis from the endometrium, tubes, and ovaries need to be excluded before a diagnosis of primary endocervical serous adenocarcinoma is rendered.

• 4.

  Clear cell: composed of clear or eosinophilic hobnail cells displaying solid, papillary, and/or tubulocystic architecture. Endocervical clear cell adenocarcinomas are rare and are associated with in utero diethylstilbestrol (DES) exposure. Sporadic tumors rarely occur.

• 5.

  Mesonephric: arises from mesonephric remnants, typically in the lateral to posterior cervical wall. Mesonephric carcinomas show a variety of architectural growth patterns (ductal, tubular, retiform, cord-like, papillary, solid, spindle, among others) but have characteristic luminal eosinophilic colloid-like material. Mesonephric carcinomas are pancytokeratin positive and are often also positive for calretinin, PAX-8, and TTF1. They may be p16 immunoreactive focally but are HPV negative. Key features distinguishing HPVA endocervical adenocarcinomas from NHPVA carcinomas are summarized in Table 2.3 .

  TABLE 2.3

  Features Distinguishing HPV-associated Endocervical Adenocarcinomas from Non-HPV-associated Adenocarcinomas

  	HPV-associated Endocervical Adenocarcinomas	HPV-independent Endocervical Adenocarcinomas
  Etiology	High-risk HPV	Not HPV-related; various etiologies, e.g., STK11 mutation in gastric-type, in utero DES exposure in clear cell
  Histology	Easily identifiable apical mitosis and apoptosis at scanning to low power (4–10×) magnification	Apical mitosis and apoptosis are NOT easily identified
  IHC/molecular	Diffusely p16 positive; high-risk HPV detected	p16 negative; HPV negative
  Morphologic types	Usual type: mucin-poor; median patient age 42 years. Important to note pattern of invasion (see text) Villoglandular: exophytic slender papillae; younger patients, median 34 years Mucinous: >50% of tumor cells contain intracytoplasmic mucin; may be intestinal or signet ring iSMILE: stratified columnar cells with peripheral palisading and intracytoplasmic mucin	Gastric type: tumor cells resemble gastric pyloric glands, i.e., have basally located nuclei and abundant clear, foamy, or pale eosinophilic cytoplasm with distinct cytoplasmic borders Endometrioid: columnar cells with pseudostratified nuclei; squamous metaplasia and/or endometriosis may be present Serous: slit-like/gaping glands, papillary and/or micropapillary architecture and high-grade nuclei with ≥3:1 variation in nuclei size and prominent nucleoli Clear cell: clear or eosinophilic hobnail cells with solid, papillary and/or tubulocystic architecture Mesonephric: characteristic luminal eosinophilic colloid-like material; displays variety of growth patterns
  Prognosis	Usual type: depends on pattern of invasion and stage; on average, 74% 5-year disease free survival	Poor: more frequently associated with higher stage and distant metastasis; gastric type has 30% 5-year disease-free survival

  HPV, Human papillomavirus; IHC, immunohistochemistry.

HPVA in situ lesions versus NHPVA in situ lesions (lobular endocervical glandular hyperplasia): HPVA in situ lesions include AIS and SMILE. AIS, HPVA: shows cytologic atypia with nuclear enlargement, crowding, pseudostratification and hyperchromasia, cytoplasmic mucin depletion, and easily recognizable apical mitosis and apoptosis; however, normal lobular endocervical architecture is preserved. Intestinal metaplasia may occasionally be present. SMILE: shows cytologic atypia with nuclear hyperchromasia and easily recognizable apical mitosis and apoptosis but is characterized by stratified epithelium with mucin-containing cells (either present as discrete vacuoles or cytoplasmic clearing) being present in all cell layers. Both AIS and SMILE show strong and diffuse p16 immunoreactivity and are always associated with high-risk HPV, most commonly types 16 and/or 18. Strong diffuse p16 immunoreactivity and high Ki67 mitotic index are useful features helpful in distinguishing AIS and SMILE from benign mimics such as tubal metaplasia. The presence of cilia and absence of apical mitosis and apoptosis are morphologic features present in tubal metaplasia that are helpful in distinguishing it from AIS and SMILE. Lobular endocervical glandular hyperplasia: is a lobular proliferation of endocervical glands displaying pyloric-type epithelium. Lobular endocervical glandular hyperplasia often shows small- to moderate-sized glands surrounding a larger gland. The glands are often benign-appearing and limited to the inner half of the cervical stroma. The cells express pyloric-type mucins, MUC6, and HIK1083. Gains of chromosome 3p and loss of 1p have been identified in some cases of lobular endocervical glandular hyperplasia. Lobular endocervical glandular hyperplasia is a precursor to gastric-type adenocarcinomas. Patients with Peutz-Jeghers syndrome ( STK11 mutations) are susceptible to lobular endocervical glandular hyperplasia and gastric-type adenocarcinomas. Lobular endocervical glandular hyperplasia is distinguished from AIS by the lack of easily identifiable apical mitosis and apoptosis. In addition, lobular endocervical glandular hyperplasia is NOT associated with HPV and as such is p16 negative and without detectable high-risk HPV.

AIS versus invasive endocervical adenocarcinoma: Distinguishing AIS from pattern A invasion in usual-type HPVA endocervical adenocarcinoma can be difficult (and may not be of much clinical significance) as both do not elicit a stromal response. However, increased glandular density with small confluent or cribriform glands and the presence of deep glands suggests an invasive over an in situ process. AIS is distinguished from other patterns of invasion in usual-type adenocarcinoma and other subtypes of endocervical adenocarcinoma by the presence of stromal desmoplasia, lymphovascular invasion, solid cell nests and/or individual malignant cells, or small incomplete malignant glands within the stroma.

Usual-type endocervical adenocarcinoma versus endocervical involvement by endometrial adenocarcinoma: Distinguishing endometrial from endocervical adenocarcinoma on biopsy has important implications for treatment—type and extent of surgery (e.g., total versus radical hysterectomy) and/or chemoradiation options. The distinction can be diagnostically challenging if based solely on morphologic findings. Nevertheless, morphologic clues include readily identifiable apical mitotic and apoptotic figures in HPVA endocervical adenocarcinoma. Clinical and gross pathologic evaluations such as location of the mass are also important clues. Immunohistochemical stains and molecular testing for high-risk HPV infection are helpful adjuncts. Usual-type endocervical adenocarcinoma stains strongly and diffusely positive for p16 and is often estrogen receptor (ER) and progesterone receptor (PR) negative (or only focally positive) and vimentin negative, while endometrial adenocarcinomas (particularly International Federation of Gynecology and Obstetrics [FIGO] grades I and II) are p16 negative, ER and PR positive and vimentin positive. As p16 is a cell cycle protein that can be activated in high-grade tumors independently of HPV status, testing for high-risk HPV may be required in high-grade tumors as FIGO grade III endometrial adenocarcinomas can be p16 positive.

Gross findings

Endometrial endometrioid adenocarcinomas typically appear as nodules, exophytic, or infiltrative masses in the uterine corpus or lower uterine segment.

Microscopic findings

Endometrioid adenocarcinoma typically displays glandular growth pattern with glands lined by stratified columnar cells. Foci of squamous differentiation, mucinous differentiation, secretory change, and ciliated cells may be present. Solid growth pattern may be present, and endometrioid adenocarcinomas are graded based on the amount of solid growth present. Tumors with solid growth comprising 5% or less of the total tumor are grade 1, tumors with 6% to 50% solid growth are grade 2, and those with >50% solid growth are grade 3. Mitosis and necrosis may be present in endometrioid adenocarcinomas as well as severe nuclear atypia (i.e., pleomorphic nuclei with prominent nucleoli; grade 3 nuclei). Tumors with severe nuclear atypia involving greater than 50% of the nuclei are upgraded by one grade; for example, an otherwise grade 1 endometrioid tumor, composed of 95% glands, should be upgraded to grade 2 if the nuclei of cells lining the glands display pleomorphism and prominent nucleoli.

Endometrial hyperplasia

The term “hyperplasia” as applied to the endometrium encompasses two somewhat related processes: “hyperplasia without atypia” and “atypical hyperplasia/endometrioid intraepithelial neoplasia” (AH/EIN). Hyperplasia without atypia results from prolonged, unopposed, or suboptimally opposed estrogen exposure. Accordingly, epidemiologic associations include obesity, diabetes, and polycystic ovarian syndrome, among others. The changes are proliferative and diffusely present within the endometrium. Alternatively, AH/EIN is a clonal alteration that results in an initially localized cytologic atypia, and which is associated with a substantially increased risk of carcinoma concurrent with, and subsequent to its diagnosis in a sampling specimen. AH/EIN is frequently seen in a background of hyperplasia without atypia, which suggests that unopposed estrogen exposure may be involved in the clonal selection process that eventuates in the development of AH/EIN. The clinicopathologic features of hyperplasia without atypia and AH/EIN are compared in Table 2.4 .

Hyperplasia without atypia versus disordered proliferative endometrium : The term “disordered proliferative endometrium” (DPE) is applied to mitotically active, proliferative-type glands that are devoid of cytologic atypia, but which display scattered gland dilatations (microcysts). Associated glands in the background may display tubular or slightly irregular architecture. Tubal metaplasia is frequent. The changes of DPE are essentially those seen in hyperplasia without atypia but in a more localized form. DPE is frequently found in women with anovulatory cycles and is a manifestation of unopposed estrogen stimulation. It also frequently regresses without treatment.

Hyperplasia without atypia versus AH/EIN : General differences between these two diagnostic categories are outlined in Table 2.3 . Although AH/EIN may be seen in a background of hyperplasia without atypia, the distinction is of ample clinical significance given the marked differences between them in their rates of concurrent or subsequent carcinoma, and accordingly, management approaches. The key diagnostic feature that helps in their distinction is the presence of cytologic demarcation in AH/EIN and the lack thereof in hyperplasia without atypia ( Fig. 2.7 ). The cytologic features of the crowded glands in a putative focus of AH/EIN should be compared with those of the background glands. If AH/EIN is present in an endometrial polyp, their features should be compared with those of the background glands within the polyp. The presence of metaplastic alterations may complicate the determination of cytologic demarcation, but every attempt should be made to compare the focus to the background endometrium. Ancillary immunohistochemical studies may also be helpful: The concurrent loss of PTEN, BAF-250a (ARID1A), and PAX2 expression is substantially more likely to be encountered in AH/EIN than in hyperplasia without atypia.

AH/EIN versus FIGO grade 1 endometrioid adenocarcinoma : The distinction between AH/EIN and grade I endometrioid carcinoma may be difficult. In general, the following features are exclusively seen in carcinoma: Extensive glandular or confluent patterns (i.e., back to back glands, generally exceeding 3 contiguous mm); papillary architectures associated with any other architectural complexity; solid pattern; and cribriform pattern. Features that favor carcinoma include significant cytologic atypia beyond what is typically seen in AH/EIN (including macronucleoli) and a desmoplastic stromal response. Features that may be seen in either category include abundant mitoses, necrosis, and neutrophilic aggregates. If the distinction cannot be readily made in a biopsy, a descriptive diagnosis that acknowledges both possibilities should be rendered.

Endometrial serous carcinoma

Endometrial serous carcinomas are the prototypical type II tumor with aggressive behavior. Endometrial serous carcinomas can metastasize to extra-uterine sites even in the absence of demonstrable myometrial invasion. Serous carcinomas typically display papillary, glandular, or solid architecture with diffuse nuclear pleomorphism and frequent mitosis. Therefore, if one considers upgrading an endometrioid adenocarcinoma because of nuclear atypia, a serous carcinoma should first be excluded, as serous carcinomas can form well-defined glands that mimic endometrioid adenocarcinoma. Immunohistochemical stains are crucial in such settings. An initial limited panel composed of p53, p16, PR, and PTEN is helpful. Most endometrial serous carcinomas (80%–90%) display mutant pattern of p53 immunohistochemical staining, that is, may be diffusely positive (≥75% of nuclei) or completely negative (null pattern) while endometrioid carcinomas are typically p53 wild-type. Likewise, serous carcinomas typically show diffuse strong positive staining with p16, while endometrioid adenocarcinomas are typically p16 negative or have patchy weak expression. Endometrioid adenocarcinomas typically are strongly and diffusely PR positive and typically show PTEN loss, while serous carcinomas show patchy PR expression and have retained PTEN. Once endometrioid histology is confirmed, immunohistochemical stain for mismatch repair proteins (MLH1, PMS2, MSH2, and MSH6) is important to exclude Lynch syndrome, an autosomal-dominant heritable cause of endometrial carcinoma that is associated with an increased risk of colon cancer.

Endometrial clear cell carcinoma

Clear cell carcinoma comprises polygonal or hobnail cells with high-grade nuclear atypia (at least focally) and clear or eosinophilic cytoplasm, displaying papillary, tubulocystic, and/or solid growth patterns. Clear cell carcinomas are typically diffusely napsin-A positive, ER and PR negative, and p53 wild-type.

Endometrial neuroendocrine carcinoma

Endometrial neuroendocrine carcinomas may be morphologically small cell neuroendocrine carcinomas or large cell neuroendocrine carcinomas. Small cell neuroendocrine carcinomas are characterized by loosely cohesive cells with scant cytoplasm, nuclear molding, frequent mitosis, apoptosis, and necrosis. Large cell neuroendocrine carcinomas comprise polygonal cells with prominent nucleoli and moderate to abundant cytoplasm displaying neuroendocrine growth patterns, that is, organoid nests, trabeculae growth, rosette-like formations, cords, and/or peripheral palisading. Neuroendocrine carcinomas stain positive for CD56, synaptophysin, and chromogranin.

Undifferentiated carcinoma

Undifferentiated endometrial carcinomas typically form large polypoid partially necrotic endometrial masses and occur in postmenopausal women. Histologic sections show discohesive small to intermediate monomorphic cells displaying diffuse growth pattern. A differential diagnosis of lymphoma, plasmacytoma, and high-grade sarcoma is often entertained as no well-formed glands, or neuroendocrine growth features such as trabeculae, cords, or organoid nests are appreciable. Classification as a carcinoma is often based on immunohistochemical stains showing EMA positivity (at least focally); cytokeratins may be negative. A diagnosis of dedifferentiated carcinoma is rendered when a component of FIGO grade 1 or grade 2 endometrioid adenocarcinoma is present in an otherwise monomorphic undifferentiated carcinoma.

Features useful in distinguishing endometrioid adenocarcinoma from the various type II tumors are summarized in Table 2.5 .