Cervical adenosquamous carcinoma: detailed analysis of morphology, immunohistochemical profile, and clinical outcomes in 59 cases.

Although 2014 World Health Organization criteria require unequivocal glandular and squamous differentiation for a diagnosis of cervical adenosquamous carcinoma, in practice, adenosquamous carcinoma diagnoses are often made in tumors that lack unequivocal squamous and/or glandular differentiation. Considering the ambiguous etiologic, morphological, and clinical features and outcomes associated with adenosquamous carcinomas, we sought to redefine these tumors. We reviewed slides from 59 initially diagnosed adenosquamous carcinomas (including glassy cell carcinoma and related lesions) to confirm an adenosquamous carcinoma diagnosis only in the presence of unequivocal malignant glandular and squamous differentiation. Select cases underwent immunohistochemical profiling as well as human papillomavirus (HPV) testing by in situ hybridization. Of the 59 cases originally classified as adenosquamous carcinomas, 34 retained their adenosquamous carcinoma diagnosis, 9 were reclassified as pure invasive stratified mucin-producing carcinomas, 10 as invasive stratified mucin-producing carcinomas with other components (such as HPV-associated mucinous, usual-type, or adenosquamous carcinomas), and 4 as HPV-associated usual or mucinous adenocarcinomas with benign-appearing squamous metaplasia. Two glassy cell carcinomas were reclassified as poorly differentiated usual-type carcinomas based on morphology and immunophenotype. There were significant immunophenotypic differences between adenosquamous carcinomas and pure invasive stratified mucin-producing carcinomas with regard to HPV (p < 0.0001), PAX8 (p = 0.038; more in adenosquamous carcinoma), p40 (p < 0.0001; more in adenosquamous carcinoma), p63 (p = 0.0018; more in adenosquamous carcinoma) and MUC6 (p  < 0.0001; less in adenosquamous carcinoma), HNF-1beta (p = 0.0023), vimentin (p = 0.0003), p53 (p = 0.0004), and CK7 (p = 0.0002) expression. Survival outcomes were similar between all groups. Adenosquamous carcinomas should be diagnosed only in the presence of unequivocal malignant glandular and squamous differentiation. The two putative glassy cell carcinomas studied did not meet our criteria for adenosquamous carcinoma, and categorizing them as such should be reconsidered.

Introduction

Invasive cervical adenosquamous carcinoma is a relatively uncommon histologic subtype of cervical malignant neoplasms classified by the World Health Organization Classification of Tumors of Female Reproductive Organs as a separate entity distinct from both squamous and glandular malignant tumors of the cervix (1). Adenosquamous carcinoma was first described as a “mixed carcinoma” by Glucksmann and Cherry in 1956, and later as “adenosquamous adenocarcinoma” by Greene in 1963 (2,3). The World Health Organization, since 2014, defines adenosquamous carcinoma as a malignant epithelial tumor composed of a mixture of invasive adenocarcinoma and squamous cell carcinoma (1). Historically, both glassy cell carcinoma (2,4,5) and “mucoepidermoid carcinoma” (6,7) have been considered malignancies lying within the spectrum of adenosquamous carcinoma. The 2014 World Health Organization classification system considers glassy cell carcinoma a subtype of adenosquamous carcinoma, but recommends the term “mucoepidermoid carcinoma” only for the extremely rare lesions identical to those occurring in the salivary glands, particularly those that contain squamous (epidermoid), intermediate, and mucin-producing cells (1).

Tumors historically diagnosed as adenosquamous carcinoma in practice appear to represent a spectrum of lesions, some of which do not exhibit definitive malignant squamous and/or glandular differentiation. This degree of heterogeneity has led to variable reported estimates of adenosquamous carcinoma prevalence (2% to 50% of all invasive cervical carcinomas) (8,9). The principle rationale for classifying adenosquamous carcinomas separately from both squamous cell carcinomas and adenocarcinomas relates not only to morphology but also to reported differences in associated rates of human papillomavirus (HPV) (10–15) and clinical outcomes (9,11,16,17), with some of the older literature emphasizing the clinically aggressive nature of adenosquamous carcinoma. Given the enigmatic etiologic, morphological and clinical features of adenosquamous carcinomas, we sought to better define these tumors by examining a large series of cases originally diagnosed as adenosquamous carcinoma.

Material and methods

Case selection

Slides from 462 endocervical adenocarcinomas and adenosquamous carcinomas (adenosquamous carcinomas and glassy cell carcinomas) were collected and reviewed by an international panel of pathologists from 8 institutions (USA: Memorial Sloan Kettering Cancer Center, New York, and Massachusetts General Hospital, Boston, MA; Romania: University of Medicine and Pharmacy of Targu Mures and Regional Institute of Oncology, Iasi; Japan: Jikei University School of Medicine, Tokyo; Mexico: Hospital de Oncología Mexico City, Mexico City; Israel: Sheba Medical Center, Tel-Hashomer, Ramat Gan; and Italy: Ospedale Sacro Cuore Don Calabria, Negrar). Only invasive tumors with at least 5-years’ follow-up were included. The following tumors were excluded: in situ carcinomas; squamous carcinomas; tumors with a neuroendocrine component; carcinosarcomas; any tumor demonstrating clinical, macroscopic, or microscopic features suggesting a lower uterine segment, uterine corpus, or adnexal primary; tumors represented by only biopsies and curettings; excisions lacking lymph node assessment; and specimens from patients treated with neoadjuvant chemotherapy and/or radiation therapy. Specimens from 70 loop electrosurgical excision procedures, 8 trachelectomies, 41 conizations, and 343 hysterectomies were collected. Fifty-three of the 462 total cases were excluded due to failure to meet entry criteria, missing blocks, or concern that the available slides were not representative of the lesion, leaving 409 cases for study. Institutional review board approval was obtained.

Morphological assessment

All microscopic subtypes of endocervical adenocarcinomas and adenosquamous carcinomas were included in the study. We required examination of all hematoxylin and eosin slides containing tumor (average of 12 slides per case). A consensus diagnosis was reached in every case, with at least 2, and as many as 4, study pathologists reviewing slides at a multi-head microscope. The 409 study cases were classified according to the new endocervical adenocarcinoma classification system (International Endocervical Adenocarcinoma Criteria and Classification system) (18).

Cases were classified as adenosquamous carcinoma only when unequivocal invasive malignant glandular and squamous differentiation was present, each component representing at least 10% of the tumor. Cases were classified as pure glassy cell carcinoma when all tumor cells had sharp cytoplasmic margins, “ground glass” eosinophilic cytoplasm, and large round or ovoid nuclei with prominent nucleoli. The International Endocervical Adenocarcinoma Criteria and Classification system recognizes invasive stratified mucin-producing carcinomas as part of the spectrum of HPV-associated adenocarcinomas, while noting histologic similarity to lesions historically diagnosed as adenosquamous carcinoma (19). For the purposes of this study, pure invasive stratified mucin-producing carcinomas were classified separately from HPV-associated usual type, mucinous, or adenosquamous carcinomas with >10% but <90% invasive stratified mucin-producing carcinomas components (invasive stratified mucin-producing carcinomas with components). HPV-associated adenocarcinomas with a benign squamous component resembling squamous metaplasia were not considered adenosquamous carcinomas. Adenosquamous carcinomas were classified as high grade when either the glandular or squamous component was high grade. Otherwise, they were considered low grade. Solid architecture (>50%) or diffusely distributed high nuclear grade was considered “high grade” for the glandular component, while high nuclear to cytoplasmic ratios without keratinization was considered “high grade” for the squamous component.

Clinical information on type of surgical treatment, tumor size, stage, follow-up, lymph node metastasis, distant metastasis, and recurrence and survival status was collected.

Tissue microarray construction and immunohistochemical reactions

Tissue microarrays were constructed using previously described methods (20,21). These included 25 putative adenosquamous carcinoma cases from New York, Boston, Mexico, Japan, and Romania for analysis of p16, p53, progesterone receptor, androgen receptor, Vimentin, HER2, HIK1083, MUC6, CAIX, SATB2, HNF-1beta, PAX8, CK7, CDX2, GATA3, p63, and p40 expression (Table 1). Each of the tumors from the New York, Mexican, and Romanian centers were represented by three 0.6-mm cores; those from Japan were represented by single 3-mm cores. Stains were scored by consensus among 2 study pathologists (RAS and SS). Disagreements were extremely rare (approximately 2–3%) and were adjudicated by re-reviewing the stated criteria for positivity, as described below. p16 was interpreted as positive if diffuse block-like staining was found in all cores, and negative if there was no or patchy staining. p53 was interpreted as positive if ≥75% of tumor cell nuclei were strongly positive or if no staining was present in the background of an intact internal control. progesterone receptor, androgen receptor, PAX8, CK7, and HNF-1beta were interpreted as positive if >25% (Score 3 or 4) of tumor cell nuclei or cytoplasm were stained. Scoring was as follows: Score 0: <5%; Score 1+: 5–10%; Score 2+: 11–25%; Score 3+: 26–75%; and Score 4+: >75%. Vimentin was interpreted as positive if ≥50% of tumor cells showed membranous/cytoplasmic staining. HER2 was scored using the College of American Pathologists guidelines for gastric carcinoma: 3+ membranous positive (22). HIK1083, MUC6, CAIX, SATB2, GATA3, p63, p40, and CDX2 were considered positive if any nuclear staining was noted in >5% of tumor cells. HIK1083 is currently not available in the United States.

Table 1:

Immunohistochemical Antibodies

Antibody	CLONE	VENDOR	Instrument (dilution)
Vimentin	V9	Roche	Roche Discovery XT
p53	D07	Roche	Roche Benchmark Ultra
p16	E6H4	Roche	Roche Benchmark Ultra
PAX8	Poly	Protein Tech	Roche Benchmark Ultra
AR	Poly	Santa Cruz Biotechnology	Roche Discovery XT
PR	1E2	Roche	Roche Discovery XT
HER2	4B5	Roche	Roche Discovery XT
HIK1083	HIK1083	Kanto	Manual (1/20)
MUC 6	CLH5	Novocastra	Manual (1/200)
CA IX	Poly	Novus	Roche Benchmark Ultra
SATB2	EP281	Cell Marque	Roche Benchmark Ultra
HNF1beta	CLO374	Sigma	Leica Bond III
CK7	OV-TV12/30	DAKO	Roche Benchmark Ultra
CDX2	CDX2–88	Biogenex	Roche Benchmark Ultra
p63	4A4	Roche	Roche Benchmark Ultra
p40	BC28	Biocare	Roche Benchmark Ultra

HPV detection

HPV detection for high-risk HPV subtypes was performed on adenosquamous carcinomas in the tissue microarray that had sufficient tissue to score and had not been improperly fixed or stored (n=23). HPV in situ hybridization with a chromogen was performed using the Advanced Cell Diagnostics (Hayward, CA) RNAscope® system (catalogue no.312598). The RNAscope® Probe “HPV HR18” contains probes targeting E6 and E7 mRNA for the following high-risk subtypes: HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82. The methodology and interpretation were discussed in detail in a previous paper (18). Positive and negative control cases were used for optimal results. A full range of cytoplasmic and nuclear signals were encountered. Cases were interpreted to be HPV-positive if any brown signal appeared (nuclear, cytoplasmic) in the presence of a negative control.

Statistical analysis

GraphPad Prism 6 for Windows was used for statistical analyses. The log-rank (Mantel-Cox) test and Kaplan-Meier curves were used for survival analyses. p<0.05 was considered statistically significant.

Results

Composition of study group and pathologic findings

Of the 409 cases, 57 were originally diagnosed as adenosquamous carcinoma and 2 as glassy cell carcinomas. After review for this study, 34 of 57 cases retained their diagnosis as pure adenosquamous carcinoma (Figures 1 and 2). The other cases were reclassified as pure invasive stratified mucin-producing carcinomas (n=9) (Figure 3) or invasive stratified mucin-producing carcinomas with components such as HPV-associated, usual-type adenocarcinoma (n=4) (Figure 4), adenosquamous carcinoma (n=3), or mucinous adenocarcinoma, not otherwise specified (n=3). There were also 4 mimickers of adenosquamous carcinoma—3 HPV-associated adenocarcinoma usual-type and 1 mucinous adenocarcinoma with benign-appearing squamous metaplasia (Figure 5). The 2 glassy cell carcinomas (Figure 6) were reinterpreted as poorly differentiated carcinoma, NOS after morphological evaluation (no overt squamous or glandular components) and after immunohistochemical results showed p63 and p40 negativity.

Figure 1.

Distribution of endocervical adenocarcinomas, adenosquamous carcinomas, and mimickers after microscopic evaluation.

Figure 2.

Adenosquamous carcinoma.

A: Malignant glandular and squamous differentiation is present; B: Poorly differentiated squamous component; C: p63 expression; D: p40 expression; E: Positive high-risk human papillomavirus in situ hybridization; F: Block-like p16 expression

Figure 3.

Invasive stratified mucin-producing carcinoma.

A: Invasive nests of cells with stratified mucinous cells surrounded by a palisade; B: p63 expression in the peripheral palisade; C: Block-like p16 expression

Figure 4.

Invasive stratified mucin-producing carcinoma combined with usual-type adenocarcinoma

Figure 5.

Usual-type adenocarcinoma with benign-appearing squamous differentiation

Figure 6.

Glassy cell carcinoma (A) with positive high-risk human papillomavirus in situ hybridization (B)

None of the 34 patients with adenosquamous carcinoma was pregnant or had a recent history of pregnancy. Mean and median patient ages were 46 and 44 years, respectively (range, 24–68 years). International Federation of Gynecology and Obstetrics stage distribution was as follows: stage I, 65%; stage II, 32%; and stage III, 3%. Regional lymph nodes were assessed in all 34 adenosquamous carcinomas, with over 380 lymph nodes examined. Ten adenosquamous carcinomas had metastatic lymph node involvement (29%); one of the 10 had 5 involved lymph nodes. Pelvic metastasis occurred in 1 patient (to the ovary).

Fifty percent of the adenosquamous carcinomas were high grade. Twenty-two (65%) of the 34 adenosquamous carcinomas had precursor lesions—high-grade squamous intraepithelial lesions, adenocarcinoma in situ, or stratified mucin-producing carcinomas. Lymph-vascular invasion was present in 26 adenosquamous carcinomas (76%). The immunohistochemical profiles of the adenosquamous carcinomas and pure invasive stratified mucin-producing carcinomas, the most common mimicker of adenosquamous carcinoma, are shown in Table 2. Adenosquamous carcinomas were block-like positive for p16 in 72% of cases and positive for HPV in 82.6% of cases. The pure invasive stratified mucin-producing carcinomas were positive for p16 in 62.5% of the cases, similar to the rate in adenosquamous carcinomas, while 100% of them were positive for HPV. Significant differences in immunophenotype between the adenosquamous carcinomas and pure invasive stratified mucin-producing carcinomas were observed for HPV (p<0.0001), PAX8 (p=0.038; more in adenosquamous carcinoma), p40 (p<0.0001; more in adenosquamous carcinoma), p63 (p=0.0018; more in adenosquamous carcinoma) and MUC6 (p<0.0001; less in adenosquamous carcinoma), HNF1beta (p=0.0023), vimentin (p=0.0003), p53 (p=0.0004), and CK7 (p=0.0002) expression (Table 2).

Table 2.

The immunohistochemical profile of pure invasive stratified mucin-producing carcinomas in comparison with adenosquamous carcinomas

Marker	Pure invasive stratified mucin-producing carcinomasn (%)	Adenosquamous carcinomasn (%)	p
HPV*	8/8 (100)	19/23 (82.6)	< 0.0001
P16	5/8 (62.5)	18/25 (72)	0.1757
PAX8*	2/7 (28.5)	10/23 (43.4)	0.0382
p40*	2/7 (28.5)	15/23 (65.2)	< 0.0001
p63*	3/8 (37.5)	15/25 (60)	0.0018
PR	2/8 (25)	6/25 (24)	1.0000
AR	0/8 (0)	0/25 (0)	1.0000
CAIX	4/7 (57.1)	16/23 (69.5)	0.4022
MUC6*	4/7 (57.1)	5/23 (21.7)	< 0.0001
HIK1086	0/8 (0)	1/23 (4.3)	0.1212
HNF1beta*	1/7 (14.2)	7/23 (30.4)	0.0023
GATA3	1/7 (14.2)	2/23 (8.7)	0.2747
Vimentin*	1/8 (12.5)	0/25 (0)	0.0003
HER2	1/8 (12.5)	1/25 (4)	0.0652
SATB2	0/8 (0)	0/23 (0)	1.0000
CDX2	0/8 (0)	0/23 (0)	1.0000
p53*	2/7 (28.5)	2/25 (8)	0.0004
CK7*	8/8 (100)	20/23 (86.9)	0.0002

statistically significant differences

Clinical findings and survival results

Follow-up data were available for 26 of the patients with adenosquamous carcinoma. Mean and median times to follow-up were 87 months and 74 months, respectively (range, 6–189 months). Only 1 patient with adenosquamous carcinoma died of disease. She was 24 years old and had an International Federation of Gynecology and Obstetrics stage II, grade 3 adenosquamous carcinoma treated with radical hysterectomy, bilateral salpingo-oophorectomy and lymph node dissection followed by chemotherapy. She died 24 months after initial diagnosis with lung metastases. Two patients, both of whom presented with International Federation of Gynecology and Obstetrics stage II disease, are alive with disease. One had a histologically confirmed para-aortic nodal recurrence 14 months from diagnosis, and the other developed 2 pulmonary nodules, interpreted as metastases, 57 months after the initial diagnosis. Both patients were originally treated with radical hysterectomy and lymph node dissection followed by chemotherapy and radiation therapy.

There was no difference in OS and DFS between pure bona fide adenosquamous carcinomas and HPV-associated adenocarcinomas (p=0.33 and p=0.83, respectively); originally diagnosed adenosquamous carcinomas and usual-type HPV-associated adenocarcinomas (p=0.8 and p=0.32, respectively); pure invasive stratified mucin-producing carcinomas and invasive stratified mucin-producing carcinomas with components (p=0.55 and p=0.75, respectively), and invasive stratified mucin-producing carcinomas (pure and with components) and HPV-associated mucinous carcinomas exclusive of invasive stratified mucinous carcinoma (p=0.89 and p=0.87, respectively) (Figures 7 and 8).

Figure 7.

Analysis of overall survival between cervical adenosquamous carcinomas, adenosquamous carcinomas with invasive stratified mucin-producing carcinomas components, invasive stratified mucin-producing carcinomas, and usual-type human papillomavirus HPV-associated adenocarcinomas

Figure 8.

Analysis of disease-free survival between adenosquamous carcinomas, adenosquamous carcinomas with invasive stratified mucin-producing carcinoma components, invasive stratified mucin-producing carcinomas, and usual-type human papillomavirus HPV-associated adenocarcinomas

Discussion

Historically diagnosed adenosquamous carcinomas appear to be a heterogeneous group of tumors, which is in accordance with our series and the published literature (1,5–7,16,23–25). They have included cases of infiltrating tumors with distinct neoplastic squamous and glandular differentiation (i.e., pure adenosquamous carcinomas), HPV-associated adenocarcinomas with benign squamous differentiation, HPV-associated adenocarcinomas with invasive stratified mucin-producing carcinoma components, adenosquamous carcinomas with invasive stratified mucin-producing carcinoma components, pure invasive stratified mucin-producing carcinomas, and glassy cell carcinomas. In our study, only 34 of 59 cases originally diagnosed as pure adenosquamous carcinoma retained that diagnosis on review, while the 2 glassy cell carcinomas studied could not be supported as adenosquamous carcinoma variants on morphological or immunohistochemical evaluation. The remaining cases were pure invasive stratified mucin-producing carcinomas (n=9), invasive stratified mucin-producing carcinomas with components (in association with usual-type, HPV-associated mucinous, or adenosquamous carcinoma [n=10]) and benign squamous metaplasia in association with usual-type and HPV-associated mucinous endocervical adenocarcinomas (n=4).

Tumor categorization used for this study overlaps significantly with that of the 2014 World Health Organization classification system. The only two notable exceptions relevant to this study are that 1) all mucinous carcinomas in this manuscript are HPV-associated carcinomas, unlike the “mucinous carcinomas” in the 2014 World Health Organization classification system, some of which are HPV-associated and others that are not, and 2) invasive stratified mucin-producing carcinomas are recognized as an entity in the International Endocervical Adenocarcinoma Criteria and Classification system but not in the 2014 World Health Organization classification system. The International Endocervical Adenocarcinoma Criteria and Classification system was specifically developed to enable practitioners to recognize HPV-associated and –unassociated variants of endocervical adenocarcinomas. In the original International Endocervical Adenocarcinoma Criteria and Classification paper, 95% of tumors demonstrating HPV-associated morphology were positive by high-risk HPV mRNA and 90% showed block-like p16 staining, indicating better performance characteristic for the in situ hybridization assay (18). In the current study, wherein only rare tumor types were studied, we report that 82.6% of adenosquamous carcinomas were HPV positive by in situ hybridization, compared to 72% with block-like p16 staining; 100% of invasive stratified mucin-producing carcinomas were HPV-positive, while only 62.5% showed block-like p16 staining. The reasons underlying these discrepancies and the existence of cases without detectable HPV are discussed in detail in a related manuscript (18). The use of an in situ hybridization probe targeting E6 and E7 mRNA of 18 different high-risk HPV types, instead of the more common DNA-based probes, underlies the superior performance of the former assay.

Invasive stratified mucin-producing carcinoma is a newly recognized subtype of endocervical adenocarcinoma that differs from in situ stratified mucin-producing carcinomas (26). Invasive stratified mucin-producing carcinomas was first described by Lastra et al (19) in 2015 as an invasive adenocarcinoma containing nests of stratified columnar epithelium with round to ovoid hyperchromatic nuclei, intracytoplasmic mucin in the form of large mucin droplets, or more delicate and collapsing vacuoles that created spacing between adjacent nuclei and peripheral palisading. In the Lastra series, the amount of mucin among the cases (of which 7 were pure invasive stratified mucin-producing carcinomas and 1 was usual-type endocervical adenocarcinoma with an invasive stratified mucin-producing carcinoma component) varied from abundant to scarce. In a subsequent publication describing 3 additional cases of invasive stratified mucin-producing carcinomas, these HPV-related tumors were reported to show stratified mucinous epithelium that mimicked the appearance of immature squamous metaplasia, presumably in mucin-poor examples (27). This likely accounts for the historical misclassification of invasive stratified mucin-producing carcinomas as adenosquamous carcinomas. Invasive stratified mucin-producing carcinomas were recently reported to show some notable immunohistochemical differences from other HPV-associated endocervical adenocarcinoma subtypes, such as a higher prevalence of p40 and p63 expression and a lower prevalence of PAX8 expression, with possibly more frequent aberrant p53 staining (28). These data suggest that invasive stratified mucin-producing carcinomas diverge from other mucinous HPV-associated adenocarcinomas and could be categorized separately. This is the first work, to our knowledge, that addresses the clinical outcomes of invasive stratified mucin-producing carcinomas or the impact of an invasive stratified mucin-producing carcinoma component within an HPV-related adenocarcinoma or adenosquamous carcinoma.

HPV-associated adenocarcinomas with benign squamous differentiation, HPV-associated adenocarcinomas with invasive stratified mucin-producing carcinoma components, and pure invasive stratified mucin-producing carcinomas should not be regarded as adenosquamous carcinomas, because they do not contain a malignant squamous component.

There are, apparently, no differences in clinical outcomes between these categories; however, invasive stratified mucin-producing carcinomas are morphologically distinct and have a different immunophenotype from adenosquamous carcinomas. Most invasive stratified mucin-producing carcinomas and adenosquamous carcinomas are positive for p16 and HPV, but statistically significant differences were found in the expression of HPV, p40, p63, PAX8, HNF1beta, vimentin, p53, CK7, and MUC6. p40 and p63 are less often positive in invasive stratified mucin-producing carcinomas, and the positivity is patchy in the peripheral palisade of tumor cell nests, while MUC6 is more often positive in invasive stratified mucin-producing carcinomas, suggesting more glandular differentiation in invasive stratified mucin-producing carcinomas compared with adenosquamous carcinomas. Moreover, the scant p63 and p40 expression in pure invasive stratified mucin-producing carcinomas in the palisade around invasive nests and the relative lack of PAX8 suggest that these tumors may be of reserve cell origin, as has been suggested (26), compared with pure adenosquamous carcinomas.

The older literature suggested that adenosquamous carcinoma is a relatively aggressive disease type, especially in advanced stages, occurring more frequently in pregnant and younger patients than either squamous carcinomas or endocervical adenocarcinomas (16,29,30). In our study, adenosquamous carcinomas were associated with clinical outcomes similar to those of HPV-associated adenocarcinomas, including mucinous endocervical adenocarcinomas, as reported previously (8,31,32), whether or not the invasive stratified mucin-producing carcinoma present was pure or with components. Tumor grade did not appear to be correlated with clinical outcomes.

Ultrastructural studies that pointed to the presence of glandular and squamous differentiation (33,34) originally supported the idea that glassy cell carcinomas were a type of adenosquamous carcinoma. Although evidence of glandular differentiation is reportedly rather obvious using electron microscopy, only focal concentrations of tonofilaments have been construed as evidence of squamous differentiation. On examination of hematoxylin and eosin slides, however, these tumors display a uniform population of neoplastic cells, unlike adenosquamous carcinoma, although there are rare reports of glassy cell carcinomas with subtle intracytoplasmic mucin or squamous differentiation. glassy cell carcinoma has been thought of as an aggressive subtype of cervical carcinoma (35,36), although there are few studies describing this. In contrast, a recent publication reported the successful treatment of 5 glassy cell carcinoma patients with advanced-stage disease (37). Based on studying only 2 such cases, neither expressing markers associated with squamous differentiation, we conclude that glassy cell carcinomas are either not adenosquamous carcinomas or are so poorly differentiated that they can only be classified as such with difficulty. Further studies addressing this issue are therefore required for further elucidation of this entity.

In summary, adenosquamous carcinomas can be diagnosed in the presence of unequivocal evidence of malignant glandular and squamous differentiation. Mimics such as invasive stratified mucin-producing carcinoma, invasive stratified mucin-producing carcinoma with components, and HPV-associated adenocarcinomas with benign-appearing squamous metaplasia should not be diagnosed as adenosquamous carcinomas based on distinguishing morphological features and some immunohistochemical differences, despite the fact that clinical outcomes appear similar. Since the two putative glassy cell carcinomas studied did not meet our criteria for adenosquamous carcinoma and lacked evidence of squamous differentiation with immunohistochemistry, one should reconsider whether these tumors should be categorized as adenosquamous carcinomas.