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Immunohistochemical evaluation of gastric-type markers in primary extramammary Paget disease: Differential MUC5AC expression between in situ and invasive lesions

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Abstract

Primary extramammary Paget disease (EMPD) is a rare cutaneous adenocarcinoma characterised by intraepithelial proliferation of mucin-containing carcinoma cells, with invasive carcinoma occasionally present. A characteristic immunohistochemical feature of primary EMPD is expression of MUC5AC, a gastric-type mucin core protein; however, the expression of MUC5AC in invasive EMPD remains controversial. Furthermore, the expression of other gastric-type markers, including hepatocyte nuclear factor 4α (HNF4α) and claudin 18, has not been fully investigated in primary EMPD. The present study aimed to clarify the expression profiles of MUC5AC in both in situ and invasive EMPD lesions, as well as to evaluate HNF4α and claudin 18 expression in primary EMPD. Consecutive patients with primary EMPD who underwent surgical resection were included. Immunohistochemical staining for MUC5AC, HNF4α and claudin 18 was performed, and the results were semi-quantitatively assessed using a four-tier scoring system (0, <5%; 1, 5-25%; 2, 26-50%; 3, >50% positive neoplastic cells) in in situ and invasive components, when present. A total of 40 patients (9 female patients and 31 male patients) were included, comprising 25 cases in situ and 15 invasive cases. MUC5AC expression was observed in 24 in situ and 14 invasive EMPD cases. No significant difference in MUC5AC expression scores in in situ lesions was observed between in situ-only and invasive EMPD cases (P=0.35). By contrast, MUC5AC expression scores in invasive components were significantly lower than those in the corresponding in situ lesions of invasive EMPD (P=0.0015). Neither in situ nor invasive EMPD lesions showed immunoreactivity for HNF4α or claudin 18. These findings obtained from the second-largest cohort study to date suggested that MUC5AC expression was reduced in invasive lesions. Furthermore, MUC5AC expression in EMPD was not associated with the expression of HNF4α, despite its role in inducing MUC5AC expression in normal gastric tissues.

Introduction

Extramammary Paget disease (EMPD) is a rare cutaneous adenocarcinoma characterised by predominant intraepithelial growth of carcinoma cells (1). Primary EMPD originates from skin appendages, whereas secondary EMPD represents intraepithelial involvement by an underlying adenocarcinoma, most commonly originating from the lower gastrointestinal or urinary tract (1). The most common sites of primary EMPD include the vulva, perianal region, scrotum, and penis (1,2). The characteristic histopathological features of this rare carcinoma include intraepithelial proliferation of neoplastic cells with abundant amphophilic cytoplasm, mucin, and vesicular nuclei with prominent nucleoli, known as Paget cells. These cells appear as solitary cells, solid nests, or occasionally glandular structures, and may involve hair follicles and eccrine ducts (1). The origin of primary EMPD remains controversial, as this adenocarcinoma is hypothesised to arise from pluripotent germinative cells within the epidermis or skin adnexa. Alternatively, Toker cells, mammary gland-related intraepithelial cells present in the epithelium of the normal external genital region, have been proposed as a potential cell of origin (3-5).

The hallmark immunohistochemical characteristic of primary EMPD is positive immunoreactivity for MUC5AC (6-10), a gastric-type mucin core protein (10,11). MUC5AC is expressed in normal mucous cells of the surface foveolar epithelium of the stomach but not in the pyloric and fundic glands (11,12). MUC5AC expression has been reported in several types of carcinomas, including gastric, lung, and pancreatobiliary carcinomas (11). Its expression has also been described in non-digestive system carcinomas, such as primary EMPD and endocervical adenocarcinoma (6-10,13). However, whether MUC5AC expression correlates with the invasiveness of EMPD remains controversial. Two studies reported higher MUC5AC expression in in situ lesions than in invasive lesions (6,9), whereas another study demonstrated higher MUC5AC expression in invasive lesions compared with in situ lesions (7).

Recently, several gastric-type markers have been utilised in pathological diagnosis through immunohistochemical analysis and explored for targeted therapeutic applications. Claudin 18 is a member of the claudin family and a major component of tight junction complexes (14). Its expression is restricted to non-neoplastic gastric mucosa and alveolar epithelial cells (14) and is frequently observed in gastric carcinoma, oesophageal and pancreatobiliary tract adenocarcinomas, as well as mucinous carcinomas of the female genital tract (15-18). Accordingly, analysis of this tight junction protein may aid in identifying the primary origin of metastatic carcinoma (17). Moreover, claudin 18 has recently attracted attention as a promising therapeutic target in claudin 18-expressing malignant tumours (15,16). Zolbetuximab, a monoclonal antibody targeting claudin 18.2, a gastric mucosa-specific isoform, has been shown to improve survival in patients with claudin 18.2-positive advanced gastric cancer (15). In addition, hepatocyte nuclear factor 4α (HNF4α) is a nuclear transcription factor involved in liver and kidney development and intestinal functions; accordingly, its expression is observed in normal liver, kidney, stomach, and intestinal tissues (19,20). Although HNF4α is not a specific gastric-type marker, it has been shown to induce MUC5AC expression in the normal stomach (21), and most gastric adenocarcinomas demonstrate positive immunoreactivity for this marker (22). To date, the expression profiles of claudin 18 and HNF4α in primary EMPD have not been fully investigated. Therefore, the present study aimed to examine the expression profiles of MUC5AC, claudin 18, and HNF4α in EMPD, as well as the differences in MUC5AC expression between in situ and invasive lesions.

Materials and methods

Patient selection

Consecutive patients with primary EMPD who underwent surgical resection at Osaka Medical and Pharmaceutical University Hospital between January 2016 and December 2024 were included. Patients who underwent biopsy without subsequent surgical resection were excluded.

This retrospective, single-institution study was conducted in accordance with the tenets of the Declaration of Helsinki, and the study protocol was approved by the Institutional Review Board of Osaka Medical and Pharmaceutical University Hospital (Approval No.: #2025-030). All data were anonymised. Informed consent was obtained using the opt-out method because of the retrospective study design, in which medical records and archived samples were used and posed no risk to participants. In addition, the present study did not include minors. Information regarding the study, including the inclusion criteria and the opportunity to opt out, was provided on the institutional website (https://www.ompu.ac.jp/u-deps/path/img/file36.pdf).

Histopathological analysis

Surgically resected specimens were fixed in 10% neutral buffered formalin, sectioned, and stained with haematoxylin and eosin. Two researchers (NK and MI) independently evaluated the histopathological features of the slides. When invasive neoplastic growth was identified (defined as the growth of carcinoma cells over the basement membrane of the epidermis or skin appendages into the dermis), the tumours were classified as invasive EMPD, regardless of the depth of tumour invasion.

Immunohistochemical analysis

Immunohistochemical analysis was performed using an automated immunostainer (Discovery Ultra System; Roche Diagnostics), according to the manufacturer's instructions. The OptiView DAB Universal Kit (cat. no. 518-111427; Roche Diagnostics) was used for immunostaining. The primary antibodies used in the present study were mouse monoclonal antibody against claudin 18 (43-14A; Abcam, Cambridge, UK, diluted 1:8,000), rabbit monoclonal antibody against HNF4α (C11F12; Cell Signaling Technology, Danvers, MA, USA, diluted 1:200), and mouse monoclonal antibody against MUC5AC (45M1; Thermo Fisher Scientific Inc., MA, USA, diluted 1:4,000). Non-neoplastic gastric and colon mucosae and skin tissues were concurrently stained as the positive and negative controls, respectively, for these three markers.

Expression of these markers was assessed semi-quantitatively as follows: 0, <5%; 1, 5-25%; 2, 26-50%; 3, >50% of positive neoplastic cells, using the same method as in a previous study (7). Immunohistochemical expression in in situ and invasive lesions (when present) was evaluated separately.

Two researchers (NK and MI) independently evaluated the immunohistochemical findings. In cases of discrepancy, a final consensus was reached through reassessment using a multi-headed microscope.

Statistical analysis

Differences between the two groups were analysed using the Mann-Whitney U test or the Wilcoxon signed-rank test. Statistical significance was set at P<0.05.

Results

Patient characteristics

This study included 40 patients with EMPD, comprising nine women (22.5%) and 31 men (77.5%). The median age at the time of surgery was 74 (range: 45-89) years. Tumour locations were as follows: scrotum (26 patients), external genitalia (10 patients), penis (2 patients), and anus and vulva (1 patient each). Lymph node dissection was performed in seven patients. No follow-up analysis including recurrence was performed in the present study.

Histopathological characteristics

This cohort included 25 in situ and 15 invasive EMPD cases. In all invasive EMPD cases, in situ lesions were present overlying and/or surrounding the invasive components. Typical histopathological features of in situ and invasive EMPD are shown in Fig. 1A and B. Lymph node metastasis was identified in two patients with invasive EMPD; among the seven patients who underwent lymph node dissection, five had only in situ lesions, and no metastasis was observed in these cases.

Immunohistochemical characteristics

Table I summarises the expression scores of MUC5AC in both in situ and invasive EMPD. MUC5AC expression was observed in 23 in situ and 15 invasive EMPD cases, whereas no MUC5AC expression was observed in one in situ case and one invasive case. Representative immunohistochemical features of in situ and invasive EMPD are shown in Fig. 1C and D.

In patients with invasive EMPD, MUC5AC expression scores in the invasive EMPD lesions were significantly lower than those in the corresponding in situ lesions, with mean expression scores of 2.07 (range, 0-3) for in situ lesions and 0.33 (range, 0-2) for invasive lesions (P=0.0015) (Fig. 2B).

Moreover, MUC5AC expression scores in lymph node metastasis were 0 in both patients. Neither HNF4α nor claudin 18 expression was observed in any in situ or invasive EMPD lesions. Notably, claudin 18 and HNF4α positivity were observed in the non-neoplastic gastric and colon mucosae used as positive control (Fig. 1E and F).

Discussion

This study clearly demonstrated that MUC5AC expression scores were significantly lower in invasive lesions than in the corresponding in situ lesions of invasive EMPD, and that MUC5AC expression in in situ lesions did not differ significantly between in situ-only and invasive EMPD. Moreover, the present study is the first to demonstrate the absence of immunohistochemical expression of HNF4α and claudin 18 in primary EMPD.

It is well recognised that MUC5AC expression is present in primary EMPD, with reported positive expression rates ranging from 42.1 to 89% (6-10). The findings of the present study are consistent with previous reports (6-10), as 95% (38 of 40) of primary EMPD cases showed positive immunoreactivity for MUC5AC. In normal gastric mucosa, MUC5AC expression is induced by HNF4α (21), and most gastric adenocarcinomas also demonstrate positive immunoreactivity for this transcription factor (22). Although MUC5AC expression is observed in normal endocervical glandular cells and respiratory epithelium (13,23), HNF4α expression is not detected in these normal epithelial tissues (24). These findings suggest that the mechanisms regulating MUC5AC expression may differ among organs. In the present study, no immunohistochemical expression of HNF4α was detected in primary EMPD, including both in situ and invasive tumours. Further studies are required to elucidate the hitherto unknown molecular mechanisms underlying MUC5AC expression in patients with primary EMPD.

The expression profiles of MUC5AC in in situ and invasive lesions of primary EMPD remain controversial. Table II summarises the expression profiles of MUC5AC in invasive EMPD reported in previous studies, as well as in the present study (6,7,9). Three of four studies, including the present one, demonstrated that MUC5AC expression was significantly lower in invasive lesions than in situ lesions, although the methods used to evaluate MUC5AC expression differed among these studies. Specifically, the cut-off value for positivity was set at 5% of carcinoma cells in three studies including the present one (6,7), whereas the tumour was considered to be positive for MUC5AC expression when more than one MUC5AC-positive cell was present in one study (9). Moreover, expression scores were evaluated separately for in situ and invasive lesions in two studies, including the present study (9); however, the methods for evaluating MUC5AC expression scores in invasive EMPD were not fully described (whether only invasive lesions or both in situ and invasive lesions were evaluated) in the remaining two studies (6,7). The present study represents the second-largest cohort of both total and invasive EMPD cases (Table II) (6,7,9) and included a statistical comparison of MUC5AC expression scores between in situ and invasive lesions. Accordingly, MUC5AC expression may be decreased in invasive EMPD lesions, although further analyses in larger cohorts are required to clarify the expression profiles of MUC5AC in EMPD. In the present cohort, both lymph node metastases showed loss of MUC5AC expression implying that metastatic EMPD cells might lack MUC5AC expression; however, this preliminary observation was seen in only two patients. Finally, the molecular mechanisms underlying these changes remain unresolved, and it is still unclear whether these changes are the cause or the effects of invasion. Therefore, further studies are required to elucidate the mechanisms responsible for altered MUC5AC expression in invasive EMPD.

Claudin 18 is a major component of tight junction proteins and plays an important role in epithelial barrier function and cellular polarity (14). Its expression is restricted to non-neoplastic gastric mucosa and alveolar epithelial cells of the lung (14). Claudin 18 has two splice variants, among which claudin 18.2 is a gastric mucosa-specific isoform (14). Recent advances in molecular-targeted therapy have demonstrated that zolbetuximab, a monoclonal antibody against claudin 18.2, significantly improves survival in patients with claudin 18.2-positive advanced gastric cancer (15). In addition, positive immunoreactivity for claudin 18 has been reported in pancreatobiliary tract adenocarcinomas and mucinous carcinomas of the female genital tract (15-18), suggesting that patients with these malignancies may be candidates for molecular-targeted therapy. However, the present study demonstrated a lack of claudin 18 expression in all primary EMPD cases examined. Accordingly, patients with primary EMPD are unlikely to benefit from anti-claudin 18-targeted therapy. Moreover, a monoclonal antibody against claudin 18 (clone 43-14A) was used for immunohistochemical detection in the present study. This antibody reacts with both claudin 18.1 and 18.2. Although the specific antibody against claudin 18.2 might have helped retrieve more information regarding expression of gastric-type marker in EMPD, we used this claudin 18 antibody clone because it was used for detecting claudin 18 expression in cancer tissues in a clinical trial that demonstrated the significant survival benefits of zolbetuximab in patients with claudin 18-positive gastric cancer (25) and is used as a companion diagnostics tool.

The present study had a few limitations. First, although this is the second-largest cohort of EMPD to be studied so far, it included a relatively small number of patients with EMPD (25 in situ and 15 invasive EMPD). As this study was a pilot study, further study with a larger cohort is required to overcome statistical bias. Moreover, the relationship between MUC5AC expression status and survival was not analysed, because this cohort included only two patients with metastatic EMPD. Second, the methods used to evaluate MUC5AC expression profiles differed among studies, as mentioned earlier, resulting in bias. Further analysis using standardized methods for evaluation of MUC5AC expression in a larger cohort is required. Third, the present study demonstrated that MUC5AC expression in EMPD was not correlated with HNF4α expression. Although different mechanisms of MUC5AC expression may be present, molecular analysis was not performed in the present study. Thus, further molecular study is needed to clarify the mechanism underlying MUC5AC expression in EMPD.

In conclusion, the present study demonstrated that MUC5AC expression was significantly lower in invasive lesions than in in situ lesions of invasive EMPD; however, MUC5AC expression in in situ lesions did not differ significantly between in situ-only and invasive EMPD. Although HNF4α induces MUC5AC expression in normal gastric mucosa, HNF4α expression was not detected in primary EMPD. Therefore, the molecular mechanisms underlying MUC5AC expression in primary EMPD warrant further investigation.

Acknowledgements

Not applicable.

Funding Statement

Funding: No funding was received.

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

NK and MI conceived and designed the present study. NK and MI analysed histological and immunohistochemical staining. NK and MI confirm the authenticity of all the raw data. NK, MI, SO and YH analysed the data. NK and MI performed the statistical analyses. NK and MI wrote the manuscript and prepared figures and tables. All authors have read and approved the final version of the manuscript.

Ethics approval and consent to participate

The present retrospective, single-institution study was conducted in accordance with the tenets of The Declaration of Helsinki, and the study protocol was approved by the Institutional Review Board of Osaka Medical and Pharmaceutical University Hospital (approval no. 2025-030; Takatsuki, Japan). All data were anonymised. Informed consent was obtained from the patients using the opt-out methodology because of the retrospective study design, in which medical records and archived samples were used. The present study did not include minors.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Data Availability Statement

The data generated in the present study may be requested from the corresponding author.

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