The incidence of cervical adenocarcinoma (ADC) has

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1 Cervical Adenocarcinoma of Human Papillomavirus Positive and Human Papillomavirus Negative Tumors Edyta C. Pirog, MD, PhD Context. Cervical adenocarcinomas span a diverse group of tumors with several distinct histologic tumor types, which include endocervical, endometrioid, intestinal, villoglandular, gastric, signet ring, serous, clear cell, and mesonephric. of cervical adenocarcinoma, especially early diagnosis, poses a significant challenge. Objective. To review the pathogenesis, diagnostic criteria, immunohistochemical markers, and differential diagnosis of various subtypes of human papillomavirus (HPV) positive and HPV-negative cervical adenocarcinomas. The paper presents a concise summary of the issues that may be particularly difficult in histopathologic diagnosis, such as differentiating neoplastic lesions from benign mimics, determining the tumor type, differentiating early invasive lesions from adenocarcinoma in situ, measuring the depth of invasion, and, finally, differentiating primary cervical adenocarcinoma from uterine endometrioid adenocarcinoma and tumors metastatic from other primary sites. Data Sources. The study employed a PubMed search of recently published reports. Conclusions. Early detection of HPV-positive tumor types may be aided with the expansion of HPV testing; however, early diagnosis of HPV-negative cervical adenocarcinomas will continue to pose a challenge and may require the development of additional molecular testing techniques. (Arch Pathol Lab Med. 2017;141: ; doi: /arpa RA) The incidence of cervical adenocarcinoma (ADC) has been increasing in recent decades, both relative to cervical squamous cell carcinoma as well as in absolute numbers. 1 Because of this increase pathologists more frequently encounter in situ and invasive adenocarcinoma in the pathology practice. Cervical adenocarcinomas span a diverse group of tumors with several distinct histologic tumor types, which include endocervical (usual), endometrioid, intestinal, villoglandular, gastric, signet ring, clear cell, serous, and mesonephric. There is a growing body of literature indicating that these tumor types have typespecific pathogenesis, clinicopathologic characteristics, and prognosis. This review will summarize the basic characteristics of the individual tumor types in conjunction with novel findings. The etiology of squamous cell carcinoma is firmly linked to infection with high oncogenic risk human papillomaviruses (HPVs). Human papillomavirus DNA is detected in virtually 100% of cervical squamous cell carcinoma; however, studies of adenocarcinoma showed a considerably lower prevalence of HPV, from approximately 62% to 100%, depending on the geographic region and tumor subtype. 2 6 Detection of HPV in ADC poses a technical difficulty because of a low viral load in these lesions. Glandular cells do not support productive HPV infection, and HPV DNA in adenocarcinomas is present in low copy numbers. Only recently have thoroughly designed broad-spectrum polymerase chain reaction primers facilitated amplification of HPV DNA with high sensitivity and allowed for widespectrum HPV genotyping. 7 Using highly sensitive polymerase chain reaction methods, HPV was shown to be involved in the pathogenesis of the 3 most common histologic subtypes of ADC, namely endocervical, endometrioid, and intestinal. Human papillomavirus DNA is detected in 80% to 100% of tumors showing these differentiation patterns. 2 Human papillomavirus DNA is only very rarely detected in the nonmucinous types of ADC, such as clear cell, serous, and mesonephric adenocarcinoma. 2,8 In addition, gastric type, which includes minimaldeviation adenocarcinoma, was shown to be unrelated to HPV infection. 2 5,8 The pathogenesis of these malignancies is not well understood, and their precursor lesions are still hypothetical. 2 Accepted for publication December 8, Published as an Early Online Release June 23, From the Department of Pathology, Weill Medical College of ADENOCARCINOMA IN SITU Cornell University, New York, New York. The author has no relevant financial interest in the products or companies described in this article. Reprints: Edyta C. Pirog, MD, PhD, Department of Pathology, Weill The average age of the patients is approximately 36 years, Medical College of Cornell University, 525 E 68th St, Starr-1041, slightly less than a decade before the peak of invasive New York, NY ( ecpirog@med.cornell.edu). adenocarcinoma. 2 The progression and regression rates of Arch Pathol Lab Med Vol 141, December 2017 Cervical Adenocarcinoma Pirog 1653

2 adenocarcinoma in situ (AIS) are not known. Approximately half of the lesions are associated with high-grade squamous intraepithelial lesion. 6,9 All cases of AIS were shown to be positive for high oncogenic risk HPVs. Human papillomavirus 16 and HPV 18 are the most common genotypes detected and together account for 93% of cases. 2,6 The diagnosis of AIS is based on identification of 3 main histologic features: nuclear atypia, increased mitotic activity, and apoptosis. On low-power examination, AIS is identified as endocervical glands with normal architectural contours but with dark-staining epithelium contrasting with the background of paler-appearing benign endocervical epithelium (Figure 1). The dark appearance of the neoplastic cells is caused by nuclear enlargement, hyperchromasia, crowding, and stratification, in addition to variable degrees of cytoplasmic mucin depletion, resulting in a high nuclear to cytoplasmic ratio. On high-power examination the nuclear atypia becomes apparent. The nuclei show enlargement with dark, coarse, stippled chromatin. The mitotic activity and apoptotic bodies are readily seen. Several subtypes of AIS have been described, including endocervical, intestinal, endometrioid, and tubal. 9 The subtype classification is based on the appearance of the cytoplasm, whereas the atypical nuclear features, mitoses, and apoptosis are common to all subtypes. There is no clinical significance to the specific subtypes, but it is important for the pathologist to be familiar with various morphologies of AIS for accurate diagnosis. The most common, endocervical type of AIS, resembles benign endocervical epithelium, with vacuolated, granular, pale to basophilic, or lightly eosinophilic cytoplasm. The intestinal type is characterized by the presence of goblet cells. The endometrioid type is composed of cells with scant, deeply eosinophilic cytoplasm. Tubal-type AIS is identified as a mixture of ciliated, secretory, and intercalated cells showing nuclear atypia and mitotic figures. 9 Adenocarcinoma in situ shows blocklike diffuse and strong positivity with p16. 10,11 It is negative for progesterone receptor (PR) and is negative or only weakly positive for estrogen receptor (ER). 12 The Ki-67 labeling index is usually higher than 45%. 13 Of other markers, the lesion is positive for IMP3 and PAX8, and negative for PAX2. 11,14 On the benign end of the spectrum, AIS must be distinguished from regenerative/inflammatory changes, tuboendometrioid metaplasia, and endometriosis. On the malignant end it has to be differentiated from early invasive adenocarcinoma (see the section for endocervical ADC). Regenerative and inflammatory changes of endocervical epithelium are characterized by mild variation of nuclear sizes in addition to scattered, rare mitotic figures. The nuclear chromatin is typically delicate and lacy, and nucleoli are seen. The nuclei in tuboendometrioid metaplasia are larger and more hyperchromatic than the nuclei of endocervical epithelium; in addition, the cytoplasm may be scant, resulting in a high nuclear to cytoplasmic ratio, and therefore such glands may appear suspicious for neoplasia. High-power examination helps to identify benign ciliated, Figure 1. Adenocarcinoma in situ, endocervical type. Dark-appearing neoplastic glands (black arrow) contrast with adjacent, paler benign endocervical epithelium (red arrow). The nuclei of adenocarcinoma in situ are enlarged and elongated, with dark, coarse chromatin, whereas the cytoplasmic mucin shows relative depletion; mitotic activity is present. All of these features impart a dark appearance to the neoplastic epithelium (hematoxylin-eosin, original magnification 3200). secretory, and intercalated cells characteristic of tuboendometrioid metaplasia. Of note, tuboendometrioid metaplasia may show mitotic activity during the proliferative phase of the cycle; however, no nuclear atypia is seen in such glands. 13 Cervical endometriosis is easily identified when both endometrial glands and endometrial stroma are present in the same focus. The diagnosis is more difficult when there is only a thin rim of stromal cells and when the glands show mitotic activity in the proliferative phase. Highpower examination helps to verify that the nuclei have a bland appearance, and deeper tissue sectioning may uncover more obvious stromal cuff. Immunohistochemistry can be of assistance in differentiating AIS from reactive or metaplastic changes. Benign reactive and metaplastic endocervical epithelium, as well as endometriosis, shows positivity for ER and PR, whereas AIS typically lacks staining with these markers. 12 Further, AIS shows a high proliferative index with the Ki-67 marker, with an average of 57% of positive cells (range, 45% 72%). For comparison, the average proliferative index in reactive/ regenerating endocervical epithelium with a history of recent biopsy, or in tuboendometrioid metaplasia during the proliferative phase is 7% (range, 0% 32%) and 5% (range, 0% 31%), respectively. 13 p16 is recognized as a sensitive marker of AIS; however, it is not entirely specific. Tuboendometrioid metaplasia and endometriosis may show positivity with p16; however, the staining has patchy appearance in contrast to blocklike staining in AIS. 10,11 ENDOCERVICAL, USUAL-TYPE ADC Adenocarcinoma of endocervical type is the most common type of adenocarcinoma arising from the mucinous endocervical epithelium, and for that reason it is also referred to as usual type. 15 The tumor accounts for approximately 75% of all ADCs. 2 The average age of patients is 45 years (range, years). Human papillomavirus DNA is identified in 80% to 100% of tumors Arch Pathol Lab Med Vol 141, December 2017 Cervical Adenocarcinoma Pirog

3 Figure 2. Usual adenocarcinoma of the cervix. A, Medium-power appearance of infiltrating neoplastic glands. B, High-power appearance of the neoplastic glands demonstrating nuclear atypia, stratification, coarse chromatin, and apoptotic bodies. Pale mucin is evident in the top portion of the cytoplasm. C, Low-power view of exophytic tumor positive for p16 immunostain. Underlying benign endocervical glands are negative for the stain, with exception of short stretches of adenocarcinoma in situ. D, The same exophytic tumor immunostained for progesterone receptor (PR). The tumor nuclei are negative. Underlying benign endocervical glands show positivity for PR, with the exception of short stretches of adenocarcinoma in situ (hematoxylin-eosin, original magnifications 3200 [A] and 3400 [B]; original magnification 340 [C and D]). Human papillomavirus 16 and HPV 18 together account for 95% of cases.2,6 Endocervical ADC exhibits a variety of architectural patterns and ranges from well differentiated to poorly differentiated. The well-differentiated tumors may show exophytic papillary growth or infiltrative growth, or both. The invasive tumor may be composed of irregular cystic and tubular glands, glands with intraluminal papillary infoldings, or cribriform glands (Figure 2, A and B). In moderately differentiated tumors the growth is more confluent with sheets of small cribriform glands. Poorly differentiated adenocarcinomas show solid areas of undifferentiated cells that may be undistinguishable from poorly differentiated squamous cell carcinoma. Arch Pathol Lab Med Vol 141, December 2017 Cytologically, the tumors show intracytoplasmic mucin that is usually abundant but may be scant and focal in some cases. The invasive glands may have more cytoplasm than adjacent AIS; however, nuclear atypia (enlargement, hyperchromasia, variation of sizes and shapes, coarse chromatin) is usually more marked than that seen in AIS. Apoptotic bodies are common, but tumor necrosis is relatively rare. For staging purposes the measurement of tumor dimensions starts with the gross exam. If the tumor is seen grossly, all 3 measurements, including the width, length, and depth, should be recorded. In microscopic tumors the measurement of the depth of invasion could be problematic, especially in early invasive ADC. The main issues are differentiation of AIS from invasive glands (see the section below) and establishing the point of origin of invasion. In most cases, the depth of Cervical Adenocarcinoma Pirog 1655

4 invasion is measured from the epithelial surface rather than from the point of origin in AIS. 16 Ostör 16 recommended that in doubtful cases the entire tumor thickness should be measured because, as he remarked, it is very difficult for the pathologist to determine the precise point at which a sea of AIS ends and invasion begins. In addition, the horizontal dimension should be reported as the greatest continuous span of the invasive area measured parallel to the surface. For cases with multifocal invasion the width of each focus should be measured individually and reported, and the Federation of Gynecology and Obstetrics (FIGO) stage should be determined based on the measurement of the largest focus. 17,18 In cases in which the invasive lesion spans adjacent blocks of tissue it is recommended to estimate the third measurement of circumference by multiplying the number of involved blocks by the estimated block thickness. It is important that such measurement only be taken from invasive areas that colocalize in the contiguous sections. 18,19 There is a single study (reported consecutively in 3 publications ) in which the histologic pattern of invasion in usual-type adenocarcinoma was correlated with the rate of lymph node metastasis and recurrences. In that study the tumors with well-demarcated glands and lacking destructive stromal invasion or lymphatic invasion were classified as pattern A. Tumors with diffuse destructive stromal infiltration or solid tumors were classified as pattern C. Tumors with early invasion from well-demarcated glands were classified as pattern B. The study consisted of 352 cases, and all pattern A and B tumors as well as 83% of pattern C tumors were stage I. No lymph node metastases or recurrences were seen in any of the pattern A cases, whereas metastases and recurrences were recorded in 4.4% and 1.2% of tumors with pattern B and 23.8% and 22.1% of tumors with pattern C, respectively. It will be interesting to learn from future studies if in the multivariate analysis the pattern-based classification is an independent predictor of outcome after accounting for the depth of invasion, tumor stage, and presence of lymphvascular invasion. The terminology of microinvasive adenocarcinoma is imprecise because of its multiple definitions. The term has not been included in the most recent World Health Organization (WHO) 2014 tumor classification. Instead of a descriptive diagnosis of microinvasive adenocarcinoma, it is recommended that the pathology report include the exact measurements of the invasive lesion as well as information on the presence of lymphvascular involvement and margin involvement. 9 Endocervical ADC shows diffuse and strong cytoplasmic and nuclear positivity for p16 (Figure 2, C), 10,23 25 cytoplasmic or membranous carcinoembryonic antigen (CEA) positivity, 23,24 and strong to weak nuclear PAX8 positivity. 23,24 The tumor cells are negative for PR (Figure 2, D) and vimentin, and negative or only weakly positive for ER. 23,24 The Ki-67 labeling index is usually higher than 25%, with average 52% staining. 13 In a study of phosphatase and tensin homolog (PTEN) expression in ADCs, 88% of cases showed at least focal positivity. 26 However, because of marked variation of staining intensity and staining distribution within the same tumor section, the stain is not useful for diagnostic purposes. Distinguishing early invasive adenocarcinoma from AIS may be very difficult, and there is poor interobserver reproducibility in equivocal cases. A definite feature of invasion in ADC is infiltration of the stroma by single cells or small clusters of malignant cells. Unfortunately, such a pattern of growth is rare because invasion in adenocarcinoma usually involves the entire gland. Several appearances were described as useful in identification of invasion and include: (1) the presence of small, densely packed, confluent glands that appear more crowded than adjacent benign glands; (2) the presence of architecturally complex, branched, jagged glands with shapes that are more irregular than adjacent normal glands; (3) the presence of cribriform growth of neoplastic epithelium devoid of stroma within dilated gland profiles; (4) papillary exophytic growth; (5) the presence of neoplastic glands below the deep margin of normal glands; and (6) desmoplastic stromal response around neoplastic glands. 9,16,27 The assessment of normal gland architecture in cervical mucosa is very important for identifying early invasive glands because they may stand out from the background with their markedly abnormal shapes or crowded, confluent growth. Caution should be taken, however, because benign conditions, such as tunnel clusters, laminar endocervical hyperplasia, and mesonephric duct hyperplasia, may also show crowded, confluent growth; however, such lesions are usually circumscribed and lack the cytologic features of malignancy. Determining the deep margin of normal glands may be difficult, because on occasion benign endocervical glands, nabothian cysts, and mesonephric duct remnants may be found deep in the stroma and extend to the outer third of the cervical wall. Desmoplasia around the glands may develop as a result of a benign process, such as reaction to gland rupture and mucin extravasation. Special stains may be of help in highlighting stromal desmoplasia in invasive lesions. Three studies have reported that stromal fibroblasts immediately adjacent to foci of tumor invasion show positivity for smooth muscle actin; the finding does not have perfect sensitivity or specificity, but in the proper context the staining may be helpful in delineating the invasive areas Differentiating endocervical ADC from uterine endometrioid adenocarcinoma may be very difficult in a biopsy or curettage specimen. In addition, stage II endometrial adenocarcinoma with deep cervical stromal involvement may mimic primary cervical malignancy. 31 Accurate diagnosis may not be possible without a panel of immunostains. 24,25,31 The Table summarizes histopathologic features and immunohistochemical markers that are helpful in differentiating endocervical from uterine endometrioid adenocarcinoma. The histologic features that favor uterine endometrioid adenocarcinoma include tumor cells with dark eosinophilic cytoplasm and mild cytologic atypia (Figure 3, A). The nuclei are typically round to oval, with pale vesicular chromatin and nucleoli. Bland-appearing squamous morulae, as well as stromal foamy macrophages, may be present. The mitotic activity and apoptosis are relatively less frequent than in ADC. In addition, the uterine tumor may be seen in a background of complex atypical endometrial hyperplasia. In equivocal cases a panel of immunostaining is performed, and a staining pattern of ER /PR /vimentin /diffuse p16 þ supports the diagnosis of endocervical adenocarcinoma. Of 1656 Arch Pathol Lab Med Vol 141, December 2017 Cervical Adenocarcinoma Pirog

5 Histologic features Nuclei Differentiation of Adenocarcinoma of the Endocervix From Adenocarcinoma of the Endometrium Usual Endocervical Adenocarcinoma Moderate to marked atypia, hyperchromatic, elongated nuclei with sharp contours, coarse chromatin Uterine Endometrioid Adenocarcinoma Mild atypia, round to oval nuclei, pale vesicular chromatin and nucleoli Mitoses Abundant Scattered or rare Apoptosis Common Rare Intracellular mucin Abundant, pale, clear cytoplasm Scant, mostly eosinophilic cytoplasm In situ lesion Adenocarcinoma in situ Complex atypical hyperplasia Immunostaining of tumor cells Estrogen receptor Negative, rarely focally positive Positive Progesterone receptor Negative Positive Vimentin Negative Positive on lateral cell borders p16 Diffusely positive Patchy positivity Carcinoembryonic antigen Positive Negative Immunostaining of stromal cells CD10 Negative Positive CD34 Positive Negative Molecular testing Human papillomavirus DNA Positive Negative note, p16 positivity in endocervical ADC is strong and diffuse. In contrast, uterine endometrioid adenocarcinoma typically shows less intense, patchy p16 staining (Figure 3, B), in addition to patchy vimentin staining and diffuse ER and PR positivity (Figure 3, C). Less frequently used immunostains include CD34 and CD10; negative CD34 and positive CD10 staining of stromal cells is in support of an endometrial origin of the lesion. 32 Human papillomavirus testing may be performed; however, only polymerase chain reaction based HPV detection is sensitive enough to reliably detect HPV in endocervical ADC. 25 ENDOMETRIOID ADC Cervical endometrioid ADC is a rare tumor variant thought to develop from cervical tuboendometrioid metaplasia or endometriosis. The reported average age of patients is between 43 and 50 years. 2,33 Human papillomavirus DNA is identified in 100% of endometrioid ADCs that arise from the cervical squamocolumnar junction. 2,6,24 However, endometrioid ADCs arising from the upper endocervix and lower uterine segment are typically HPV negative. 3,5,25 The tumor cells are characterized by a lack of mucin and scant, deeply eosinophilic cytoplasm, resembling endometrial-type epithelium. Architecturally, these tumors typically grow as small, round, or tubular glands with either smooth luminal contours (Figure 4, A and B) or intraluminal cribriform growth. Some tumors show papillary architecture with thick fibrovascular cores that are either exophytic or infiltrate the cervical wall. In some institutions the tumor is classified as endometrioid if the predominant pattern resembles endometrioid adenocarcinoma of the endometrium, whereas the remaining glands have a moderate to significant amount of intracellular mucin. Most authorities do not recognize such tumors as endometrioid but consider these cases as less-differentiated endocervical ADCs with a decreased capacity to produce mucin. The variation in diagnostic criteria results in a different reporting rate of the tumor prevalence, from 5% to 50%. 2,15,33 Although new WHO classification does not give clear guidance, most experts agree that true endometrioid adenocarcinomas of the cervix are rare, and only tumors with scant eosinophilic cytoplasm without apparent intracytoplasmic mucin should be classified as endometrioid. 15 Cervical endometrioid ADC of minimal-deviation type has been described The tumor is exceedingly rare. The reported cases occurred in women ages 32 to 64 years. The tumor is composed of bland, tubular, or cystically dilated endometrial-type glands infiltrating the cervical wall, with no or only minimal stromal response (Figure 4, C). The cytologic atypia is minimal and mitoses are rare (Figure 4, D). The tumors were reported to be positive for CEA and negative with vimentin stain, similar to usual ADC. 36 The pattern of immunostaining of cervical endometrioid ADC is similar to that of usual ADC, with tumor cells demonstrating strong and diffuse p16 positivity (Figure 4, B) and negative staining for hormone receptors. 24 However, rare cases originating in the upper endocervix and lower uterine segment may show an immunostaining pattern that is overlapping with endometrioid adenocarcinoma of the endometrium; in particular, such tumors may show ER and PR positivity, patchy p16 staining, and test negative for HPV. 25 Differentiating cervical from uterine endometrioid adenocarcinoma may require use of immunostains (ER /PR / diffuse p16 þ result supports the diagnosis of cervical primary). 24,25 Rare cases may show mixed/overlapping immunostaining patterns, and in such instances only complete staging procedure may determine the primary site. 25 Arch Pathol Lab Med Vol 141, December 2017 Cervical Adenocarcinoma Pirog 1657

6 Figure 3. Uterine endometrioid adenocarcinoma. A, Well-differentiated endometrioid adenocarcinoma with squamous morulae. B, A p16 immunostain of uterine endometrioid adenocarcinoma shows faint- to moderate-intensity patchy positivity. C, Progesterone receptor immunostaining of uterine endometrioid adenocarcinoma shows strong diffuse positivity (hematoxylin-eosin, original magnification 3200 [A]; original magnification 3200 [B and C]). INTESTINAL ADC This tumor accounts for approximately 8% of ADCs. The average age of patients is 47 years (range, years). 2 Human papillomavirus DNA is identified in 83% to 100% of tumors. 2,6 Recent reports have described cases of intestinal AIS that were HPV negative and occurred in older patients. 37,38 The tumor is remarkable for the presence of goblet cells resembling intestinal-type epithelium (Figure 5). Goblet cells metaplasia is virtually not seen in benign endocervical epithelium, and therefore a finding of goblet cells in endocervical mucosa should be a concern for a neoplastic lesion. Intestinal ADC displays an immunohistochemical profile similar to usual ADC. 39 In addition, the malignancy may show an enteric immunophenotype with at least focal positivity for CDX2 and/or CK It has been observed, however, that CK7 positivity in these lesions is always stronger than the staining for enteric markers. 41 The differential diagnosis includes direct extension or metastasis from colonic adenocarcinoma. The presence of large, garland-shaped glands with incomplete lining and intraluminal necrosis is highly suggestive of a spread from the gastrointestinal tract. In equivocal cases immunohistochemistry should be used; negative immunostaining for PAX8 and CK7, and positive enteric markers are indicative of spread from colonic adenocarcinoma. VILLOGLANDULAR ADC Villoglandular ADC is a well-differentiated variant of endocervical, endometrioid, or intestinal ADC. The tumor accounts for approximately 3% to 6% of all adenocarcinomas. 33 The entity was established as a separate diagnostic category based on patients excellent prognosis. 42 The average age of patients is reported at 33 to 41 years (range, years), with most patients younger than 40 years. 33,42,43 The prognosis for villoglandular ADC is significantly better than that for usual ADC. 42,43 Because the tumor occurs at a young age and many of the patients still desire fertility, it has been suggested that this lesion may be treated with cervical cone excision or trachelectomy, provided that the tumor is superficial and has been completely excised with negative margins, there is no lymphatic space invasion, and follow-up is possible. 42 Human papillomavirus DNA is identified in 100% of villoglandular ADC. 44 The tumor is characterized by exophytic papillary growth and only minimal cytologic atypia (Figure 6). The papillae are elongated, thin, and fingerlike, and are lined by uniform columnar cells with a smooth apical border. At the base of the tumor an invasive component may be present that is typically composed of elongated interanastomosing glands separated by fibromatous stroma. The tumor is usually well 1658 Arch Pathol Lab Med Vol 141, December 2017 Cervical Adenocarcinoma Pirog

7 Figure 4. Cervical endometrioid adenocarcinoma. A, Medium-power appearance of cervical endometrioid adenocarcinoma showing glands with deep eosinophilic cytoplasm. B, A p16 immunostain of cervical endometrioid adenocarcinoma shows strong diffuse positivity. C, Cervical endometrioid adenocarcinoma, minimal deviation type; low-power appearance of haphazard cervical infiltration by bland-appearing glands. D, Cervical endometrioid adenocarcinoma, minimal deviation type; higher-power appearance of glands with scant, deeply eosinophilic cytoplasm. The cytologic atypia is minimal and mitoses are not seen (hematoxylin-eosin, original magnifications 3200 [A and D] and 3100 [C]; original magnification 3200 [B]). circumscribed with only small foci of invasion. Cytologically, the tumor cells are of endocervical, endometrioid, or intestinal morphology. The nuclei show stratification, hyperchromasia, and coarse chromatin, but they lack significant variation in sizes and shapes and nuclear atypia. Scattered mitotic figures are present. The stroma of the fibrovascular cores may show prominent inflammation. The diagnosis should be reserved only for the tumors that are not associated with underlying conventional adenocarcinoma. The pattern of immunostaining is similar to that of usual endocervical ADC.24,44,45 Of note, the tumor shows wildtype p53 immunostaining.44,45 Figure 5. Intestinal adenocarcinoma of the cervix. Medium-power appearance of infiltrating neoplastic glands. Goblet cells are evident (hematoxylin-eosin, original magnification 3200). Arch Pathol Lab Med Vol 141, December 2017 The tumor is differentiated from usual endocervical ADC based on predominant papillary architecture and a lack of significant cytologic atypia. In addition, it must be differentiated from papillary serous ADC. In contrast to serous tumor, villoglandular ADC is characterized by the presence Cervical Adenocarcinoma Pirog 1659

8 Figure 6. Villoglandular adenocarcinoma of the cervix. A, Low-power appearance of papillary exophytic growth. B, High-power appearance of tumor papillae. Nuclear atypia is present, but the nuclei appear blander than that in usual-type adenocarcinoma (hematoxylin-eosin, original magnifications 3100 [A] and 3400 [B]). of regular, more elongated papillae, smooth apical borders instead of hobnail cellular contours, and a lack of highgrade nuclear features. Immunostaining with p53 may be helpful. GASTRIC ADC Gastric ADC is a recently described histopathologic entity (Kojima et al 46 ) that was included in the updated WHO 2014 tumor classification. Minimal deviation adenocarcinoma of mucinous type is now considered a well-differentiated form of gastric ADC. 46 The true incidence of gastric ADC is not currently known because this tumor has been previously misclassified as usual, intestinal, and clear cell ADCs. In Japan the tumor may account for more than 20% of all ADCs. 46 The pathogenesis of gastric ADC is being elucidated gradually. Notably, gastric ADC is not related to HPV infection. 4,5,8,47 Some cases of gastric ADC were reported to be associated with Peutz-Jeghers syndrome, an autosomal dominant disorder caused by germ line mutation of STK11, a serine threonine kinase gene. 48 In addition, somatic mutations of STK11 are identified in more than half of the sporadic cases. 49 The average age of the patients is between 42 and 50 years (range, years), 46,48,50 which is similar to that of patients with usual ADC. Many patients present with profuse watery vaginal discharge, or enlarged, barrel-shaped cervix. Cytologic screening has low sensitivity for minimal-deviation adenocarcinoma, and cytologic features of gastric ADC were described only recently. 51 In the era of conversion to HPV testing as the primary screening modality it has been proposed that molecular testing for gastric mucin should be added to HPV testing in order to provide coverage for gastric ADC. With that in mind, a latex agglutination test using monoclonal antibody against gastric mucin (HIK1083, Kanto Chemical Co Inc, Tokyo, Japan) was developed as a screening tool to be used on cervical/ vaginal discharge. Positive result was shown to correlate with the presence of benign or neoplastic gastric-type lesion in 44 tested patients. 52 The test, however, has not been approved by the US Food and Drug Administration. The prognosis for patients with gastric ADC, including that of minimal-deviation adenocarcinoma, is significantly worse in comparison with usual ADC. The overall 5-year diseasespecific survival rate was reported as 30% to 42% for gastric ADC compared with 74% to 91% for usual ADC. 46,50 Lobular endocervical gland hyperplasia (LEGH) is a benign proliferation of endocervical glands with gastric phenotype that is considered to be a putative precursor of gastric ADC The reported average age of patients with LEGH is similar to that of patients with gastric ADC (45 49 years). 53,54 Gastric ADC is notable for diffuse cervical infiltration resulting in cervical enlargement (barrel cervix) without a distinct gross mass. The invasive glands show marked variation of sizes and shapes from simple, tubular glands to either cystically dilated lumina (Figure 7, A through C), or markedly complex and branched glands with intralumenal papillary infoldings. The tumor is usually deeply invasive, and desmoplastic stromal response surrounding the invasive glands may be seen. Cytologically, the glands are lined by mucinous epithelium with voluminous, clear, or pale eosinophilic cytoplasm showing distinct cellular borders (Figure 7, D through F). The nuclei appear pale in comparison with hyperchromatic nuclei of usual ADC and are typically present basally in a single row. The nuclei are round to oval, with delicate, diffuse chromatin and distinct nucleolus. Gastric ADCs may show a spectrum of differentiation from very well-differentiated areas lacking atypia and categorized as minimal-deviation adenocarcinoma to areas with moderate to marked cytologic atypia (Figure 7, C), including nuclear enlargement, variations of nuclear shapes, and the presence of macronucleoli. Mitotic figures are present but are not abundant. The tumor is frequently associated with lobular endocervical glandular hyperplasia. 55 The typical appearance of LEGH is that of distinct, well-demarcated lobules of small, tubular mucinous glands arranged around a central, cystically dilated channel (Figure 8, A and B). The central 1660 Arch Pathol Lab Med Vol 141, December 2017 Cervical Adenocarcinoma Pirog

9 Figure 7. Gastric adenocarcinoma of the cervix. A, Gastric adenocarcinoma, minimal-deviation type. Low-power appearance of diffuse cervical infiltration by irregular and cystically dilated glands. B, Gastric adenocarcinoma, minimal-deviation type. Medium-power appearance of tumor showing glands with voluminous cytoplasm, basally located nuclei, and no atypia. C, Gastric adenocarcinoma. Medium-power appearance of tumor demonstrating mixture of bland (minimal deviation) as well as markedly atypical cells with voluminous pale cytoplasm. D, Gastric adenocarcinoma. High-power appearance of the tumor from panel C showing an area with voluminous clear cytoplasm and round, markedly atypical nuclei. In small sample such tumor foci may mimic clear cell carcinoma. E, Gastric adenocarcinoma. High-power appearance of tumor with voluminous eosinophilic cytoplasm and round nuclei with prominent nuclei. F, Gastric adenocarcinoma. High-power appearance of tumor with depleted eosinophilic cytoplasm. Such a tumor may mimic usual-type adenocarcinoma; however, the appearance of nuclei demonstrating round contours, pale chromatin, and prominent nucleoli is diagnostic of gastric tumor subtype (hematoxylin-eosin, original magnifications 3100 [A], 3200 [B and C], and 3400 [D through F]). Arch Pathol Lab Med Vol 141, December 2017 Cervical Adenocarcinoma Pirog 1661

10 Figure 8. Lobular endocervical glandular hyperplasia. A, Low-power appearance of glandular proliferation in the superficial endocervical mucosa. B, Medium-power appearance showing central dilated channel surrounded by proliferation of small tubular glands. C, High-power magnification showing bland-appearing glands with round, basally located nuclei and abundant mucinous cytoplasm (hematoxylin-eosin, original magnifications 340 [A], 3100 [B], and 3400 [C]). channels may be round or show variation in shape, including undulating contours and papillary infoldings. Both tubular glands in the lobules and central cystic channels are lined by a single layer of tall columnar cells with clear, mucin-filled cytoplasm and basal, bland, round to oval nuclei with indistinct nucleoli (Figure 8, C). The cytoplasm of gastric ADC characteristically shows immunopositivity for gastric mucin markers MUC-6 (Figure 9, A) and HIK-10838,55,56; however, in some cases such staining may be focal. Of note, benign endocervical glands may be seen staining positive for HIK-1083 and MUC-6 in 2% and 8% of cases, respectively, and therefore the staining should be interpreted in the proper context.55 Similar to usual ADC, gastric ADC is negative for ER and PR8,56 and demonstrates cytoplasmic positivity for CEA.48,56 The staining for p16 is typically negative, correlating with negative HPV status; however, a small percentage of cases may show strong, blocklike p16 positivity.56 Approximately 41% of the cases show mutation-type p53 staining with either diffuse strong positivity (24%) or null pattern (17%).56 Lobular endocervical gland hyperplasia stains positive for gastrin mucin markers and negative for hormone receptors; in addition, it demonstrates apical positivity for CEA and wild-type p53 staining pattern.54,55,57 Gastric minimal-deviation adenocarcinoma has to be differentiated from benign conditions, of which the most important are LEGH, deep nabothian cysts, diffuse laminar endocervical glandular hyperplasia, and adenomyomas of endocervical type. Lack of ER/PR staining distinguishes this tumor from the benign conditions except for LEGH. Differentiation of minimal-deviation adenocarcinoma from noninvasive LEGH can be aided by smooth muscle actin immunostaining of the stromal cells. Positive smooth muscle actin staining is seen in cervical stromal cells immediately adjacent to foci of tumor invasion (Figure 9, B), but not around LEGH.28 In addition, minimal-deviation adenocarcinoma may be diffusely positive for p53, whereas LEGH exhibits wild-type p53 staining.8,56,57 Lobular endocervical gland hyperplasia was described as having apical CEA positivity, whereas adenocarcinoma typically shows Figure 9. Immunostaining in gastric adenocarcinoma of the cervix. A, Positive MUC-6 immunostain in neoplastic glands. B, Positive stromal smooth muscle actin immunostaining surrounding invasive glands of gastric adenocarcinoma (red arrow); note there is no smooth muscle actin staining underneath the cystic channel of lobular endocervical glandular hyperplasia (black arrow; original magnifications 3200 [A] and 3100 [B]) Arch Pathol Lab Med Vol 141, December 2017 Cervical Adenocarcinoma Pirog

11 Figure 10. Signet ring cell adenocarcinoma of the cervix. A, Low-power appearance showing extravasated, dissecting mucin with scattered tumor cells. B, High-power appearance of mucin-filled signet ring tumor cells (hematoxylin-eosin, original magnifications 3100 [A] and 3400 [B]). diffuse cytoplasmic staining. 48,54 Among the malignant lesions, gastric ADC may be difficult to distinguish from usual ADC. In such cases MUC-6 positivity (HIK-1083 is not routinely available outside of Japan) and negative p16 staining are in favor of gastric differentiation. In some cases gastric ADC may mimic clear cell ADC. The latter tumor, in contrast to gastric ADC, typically shows variable positivity for p16 and is negative for CEA and MUC-6. 8 SIGNET RING CELL ADC A proportion of signet ring cell adenocarcinomas found in the cervix are metastatic lesions, most commonly from a primary gastric carcinoma. 58 Primary cervical signet ring cell tumors of the cervix are rare. 59,60 Tumors arising in a background of usual ADC were shown to be HPV positive. 60 The tumor shows characteristic infiltration by clusters or single cells distended by a mucin vacuole (Figure 10). Pure signet ring pattern is very rare, and more often it is seen in combination with endocervical, intestinal, gastric, or neuroendocrine differentiation. Of the reported cases, the tumors were positive for p16, CK7, and CEA, with variable staining for enteric and neuroendocrine markers. 59,60 A metastasis from primary gastric, breast, colonic, appendiceal, or ovarian carcinoma has to be ruled out using immunohistochemical analysis in correlation with imaging studies and quantitation of serum tumor markers. CLEAR CELL ADC Clear cell ADCs account for 2% to 7% of ADCs and comprise a heterogeneous group of malignancies. Clear cell ADC has a bimodal age distribution. The first peak occurs between ages 17 and 37 years (mean age, 26 years); in addition, rare cases of clear cell ADC in children have been reported. 61,62 The second peak occurs in women who are ages 44 to 88 years (mean age, 71 years). 62 The pathogenesis of clear cell ADC of the cervix is still not well understood. In the past, cases occurring in younger patients were traced to diethylstilbestrol (DES) exposure in utero. In these patients the tumors were developing on the ectocervix, rather than in the endocervical canal. More recent data on patients born outside the DES exposure period yet again show a bimodal age distribution, with many cases still occurring in young, virginal women without exposure to either DES or HPV. 63 It is thought that clear cell ADC may develop from either adenosis of the ectocervix, cervical endometriosis, or cervical tuboendometrioid metaplasia. 64 Immunostaining analysis of tumors from a group of older patients showed increased expression of EGFR (75% of cases) and HER2 (25% of cases), and loss of PTEN expression (50% of cases). 65 In addition, 58% of cases demonstrated positive p-akt and 50% of cases showed p- mtor immunostaining, suggesting involvement of the PI3K-AKT pathway. 65 With rare exceptions, clear cell ADCs are negative for HPV DNA. 2,5,8,65 The histologic features of clear cell ADC are similar to those of the more common clear cell adenocarcinomas of the endometrium or ovary. Similarly, 3 architectural patterns are observed: tubulocystic, papillary, and solid (Figure 11, A through C). The fibrovascular cores in the papillary pattern may show characteristic hyalinization. Cytologically, the cells are cuboidal or flattened, hobnail-shaped, with abundant clear, glycogen-rich cytoplasm. The nuclei show moderate to marked atypia, which includes marked variation of nuclear sizes and shapes and hyperchromasia. Cervical clear cell carcinomas are positive for hepatocyte nuclear factor-1b, 8 as well as napsin-a, similar to their ovarian and uterine counterparts. The staining for both of these markers, however, is not entirely specific, because a proportion of gastric ADC also shows positivity with both stains (E.C.P., unpublished data, 2016). Despite negative Arch Pathol Lab Med Vol 141, December 2017 Cervical Adenocarcinoma Pirog 1663

12 Figure 11. Clear cell adenocarcinoma of the cervix. A, Low-power appearance of tubular pattern of architecture. B, High-power appearance of papillary architecture; marked nuclear enlargement and atypia are evident. C, Medium-power appearance of solid architecture. D, Positive p16 immunostain in clear cell carcinoma. Clear cell carcinoma of the cervix may be positive for p16 despite lack of detection of human papillomavirus in this tumor type (hematoxylin-eosin, original magnifications 3100 [A], 3400 [B], and 3200 [C]; original magnification 3200 [D]). HPV status, the tumors may show positivity for p16 with a spectrum from focal to diffuse (Figure 11, D). Clear cell ADCs are negative for CEA, ER, and PR, and most show wild-type p53 expression. 8,65 Clear cell ADC has to be differentiated from a clear cell variant of squamous cell carcinoma and a highly aggressive clear cell adenosquamous carcinoma of the cervix, 66 both of which are related to HPV infection. Further, the differential diagnosis is with HPV-negative gastric ADC and mesonephric ADC. In addition, spread from clear cell adenocarcinoma of the endometrium or ovary has to be excluded clinically. The clear cell variant of squamous cell carcinoma shows a diffuse solid growth pattern, whereas clear cell ADC displays a spectrum of various architectural patterns within the same lesion. Clear cell adenosquamous carcinoma is composed of solid sheets of clear cells in addition to glandular spaces lined by columnar cells that test positive with mucicarmine staining. 66 Clear cell ADC with tall, columnar cells may be difficult to distinguish from gastric ADC on routine sections. of clear cell ADC may be confirmed in such cases with positive p16 and negative CEA staining. Clear cell ADC with tubulocystic pattern and flattened epithelium must be differentiated from mesonephric ADC, which typically shows positivity with calretinin or GATA-3 immunostain (see below). On a benign spectrum the tumor has to be differentiated from florid or solid microglandular hyperplasia; in such cases lack of significant nuclear atypia and positive ER/PR staining will favor a benign diagnosis. SEROUS ADC Primary cervical serous ADC is exceedingly rare; most serous adenocarcinomas involving the cervix are malignancies spreading directly from the endometrium or metastases from a primary ovarian, tubal, or peritoneal serous adenocarcinoma. Cervical serous ADC has distinct clinicopathologic characteristics, including a bimodal age distribution, with the first peak occurring before age 40 years and the second peak after age 65 years, coinciding with the peak of uterine serous carcinoma. 67,68 Of the few cases reported in 1664 Arch Pathol Lab Med Vol 141, December 2017 Cervical Adenocarcinoma Pirog

13 most commonly with low-grade villoglandular adenocarcinoma. 67 The tumors are positive for p16 but negative for CEA, ER, and PR. Mutation-type staining for p53 was seen in roughly half of the reported cases. 8,68 The histologic features and immunostaining pattern of cervical serous ADC are indistinguishable from serous tumors of the upper genital tract, and therefore assignment of the primary site has to be done based on complete staging procedure. Most cases of serous adenocarcinoma involving the cervix are secondary tumors extending or metastatic from the endometrium. Figure 12. Serous adenocarcinoma of the cervix. Papillary architecture with marked nuclear atypia and hobnail nuclei are evident (hematoxylin-eosin, original magnification 3100). the literature, only rare tumors were positive for HPV DNA. 2,5,8 It is speculated that cervical serous ADC may be caused by p53 gene mutations, similar to its endometrial counterpart. 68 Microscopically, the tumor may show either papillary architecture (Figure 12) or large glandular structures with irregular slitlike glandular lumina. The cells are columnar or cuboidal with relatively scant, eosinophilic cytoplasm. The nuclei display marked variation of sizes and shapes, including bizarre, multinucleated forms. Most of the nuclei show a single, prominent cherry-red nucleolus. Mitotic activity is brisk, and atypical mitotic figures are present. Because of the scant cytoplasm and marked crowding of the nuclei, some of the nuclei protrude from the epithelial surface in the characteristic hobnail appearance. Such nuclei may become discohesive and shed into the glandular lumen. In the reported series of serous adenocarcinomas, almost half of the cases were mixed with another histologic type, MESONEPHRIC ADC Mesonephric adenocarcinoma is a rare cervical tumor that develops from the mesonephric duct remnants located deep in the lateral cervical stroma. The average age of patients is 52 years (range, years). 69 There is no apparent age peak for mesonephric adenocarcinoma, and the tumor shows a similar prevalence in age groups from the third through the sixth decades. In a recent study of targeted next-generation sequencing, 81% of mesonephric ADCs had either a KRAS (n ¼ 12) or NRAS (n ¼ 1) mutation. Mutations in chromatin remodeling genes (ARID1A, ARID1B, orsmarca4) were present in 62% of cases. No mutations of PIK3CA or PTEN genes were identified. 70 In addition, 1q gain was found in 75% of cases. Human papillomavirus DNA is not detected in this tumor type. 2,8,71 The tumor develops in the lateral cervical wall and less commonly involves the entire circumference of the cervix without forming a grossly apparent mass. Numerous architectural patterns of growth were described, including tubular, ductal, papillary, retiform, sex cord like, solid, and sarcomatoid, and these patterns may be seen in various proportions within the same tumor (Figure 13). 69 The characteristic feature of tumors forming glandular spaces is Figure 13. Mesonephric adenocarcinoma of the cervix. Microphotographs show different areas of the same tumor. A, Ductal pattern with complex glands lined by tall, columnar epithelium with deep eosinophilic cytoplasm; this pattern may resemble endometrioid adenocarcinoma. B, Papillary pattern with cells displaying hobnail nuclei; this pattern may be difficult to distinguish from serous or clear cell carcinoma. C, Tubular pattern composed of small tubular glands with eosinophilic intraluminal secretions; tubular pattern is the most specific for mesonephric adenocarcinoma (hematoxylin-eosin, original magnifications 3100 [A], 3200 [B], and 3400 [C]). Arch Pathol Lab Med Vol 141, December 2017 Cervical Adenocarcinoma Pirog 1665

14 the presence of deeply eosinophilic intraluminal secretions similar to those present in benign mesonephric proliferations (Figure 13, C). The ductal pattern consists of large tubular or dilated glandular spaces with occasional intraluminal infoldings or papillae lined by tall columnar cells with large hyperchromatic nuclei. This pattern may mimic endometrioid adenocarcinoma. In the tubular pattern the tumor grows as small, round to oval, tightly packed glands lined by low columnar, cuboidal, or flattened cells. The retiform pattern is characterized by branching, zig-zag shaped glandular spaces resembling rete ovarii. The papillary pattern resembles the papillary growth of clear cell or serous adenocarcinoma; however, the nuclei are bland, uniform, and lack atypia. In the sex cord pattern the tumor grows in long cords and trabeculae of cells with scant eosinophilic cytoplasm. Cytologically, the tumors are composed of relatively uniform columnar or cuboidal cells with scant to moderate amounts of dark eosinophilic cytoplasm. The nuclei are oval and hyperchromatic, with mostly stippled chromatin showing minimal to moderate atypia. Marked nuclear atypia is not seen. The mitotic index is highly variable and may range from 1 to 50 mitoses per 10 high-powered fields. 69 Mesonephric hyperplasia of the lobular or diffuse pattern is seen adjacent to the invasive lesions in most cases. The tumor shows positivity for calretinin, which may be focal, and vimentin, seen in the basal portion of the cells, as well as CD10, seen in the apical portion of the cells. 69 It is diffusely and strongly positive for PAX8, negative for ER, and shows only patchy cytoplasmic staining for p Positive GATA-3 staining was recently reported as a sensitive and specific marker of benign and malignant mesonephric lesions; however, the staining extent and intensity show variation from case to case. 73 Mesonephric ADC has a wide differential diagnosis. On the benign spectrum it has to be differentiated from hyperplasia of mesonephric remnants. Presence of cytologic atypia and variable architectural patterns favor adenocarcinoma. In addition, Ki-67 immunostain may be helpful because it has been reported to show positivity in only 1% to 2% of cells in hyperplasia versus 5% to 36% in adenocarcinoma. 69 On the malignant spectrum the ductal variant of the tumor has to be differentiated from endometrioid adenocarcinoma. The presence of other distinct architectural patterns, intraluminal eosinophilic secretions, adjacent benign mesonephric remnants, and the pattern of ER /PR /calretinin þ /GATA-3 þ staining confirms the diagnosis of mesonephric ADC. Tubular pattern of mesonephric ADC may simulate clear cell carcinoma whereas papillary pattern may mimic either clear cell or serous adenocarcinoma; however, in contrast to these 2 latter tumors mesonephric ADC typically shows lower nuclear grade, adjacent mesonephric remnants, and positive GATA-3 staining. 73 CONCLUSIONS It is expected that HPV vaccines will reverse the trend of rising incidence of ADC. 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