Multi-institutional study of mesonephric-like adenocarcinoma: its clinicopathological characteristics and histogenetic association with ectopic endometrium

To explore the clinicopathological features and origin of mesonephric-like adenocarcinomas (MLAs), 83 cases diagnosed or suspected to be MLAs were collected from various institutions in Japan. We clearly classified 78 as MLAs (uterus: 47, ovary: 31) and 5 as non-MLAs (all ovary) based on our morphological and immunohistochemical criteria. In uterine MLAs (uMLAs), lymphovascular space invasion was an independent prognostic factor for progression-free survival (PFS) (P = 0.03). Patients with uMLAs had significantly shorter PFS and overall survival (OS) than those diagnosed with endometrial endometrioid carcinomas (EECs) (P < 0.0001 and P < 0.001, respectively) and comparable PFS and OS to those with copy number-high tumors. PFS and OS of ovarian MLAs (oMLAs) were similar to those of ovarian endometrioid carcinomas (OECs) overall but worse for patients with stage II-IV. Endometriosis was observed with oMLAs as often as with OECs (94% vs 93%, respectively). Adenomyosis was more frequently observed with uMLAs than with EECs (62% vs 28%, respectively, P = 0.0005). Six uMLAs were confined to the myometrium and adjacent to adenomyosis. In an analysis of molecularly speculated origin, among 29 oMLAs harboring a KRAS hotspot mutation, 23 (79%) instances of endometriosis in the background had the same mutation. Among 36 uMLAs carrying the KRAS hotspot mutation, common mutations were observed in 12 (33%) instances of adjacent adenomyosis (12/21 [67%] of adenomyosis). These findings suggest a histogenetic link between MLAs and ectopic endometrium, implicating both ovarian endometriosis and adenomyosis in MLA pathogenesis, with potential clinical significance.

Mesonephric-like adenocarcinomas (MLAs) originate in the female genital system, including the uterus or ovary, and may also arise in the peritoneal cavity. [1,2,3,4] First reported by McFarland in 2016 [1] and officially recognized as a distinct entity in the 2020 World Health Organization classification, [5] awareness of MLAs has increased, leading to more frequent diagnoses. They are defined as adenocarcinomas displaying a mesonephric-like phenotype with tubular, glandular, papillary, and solid pattern morphology. Immunohistochemically, they are typically positive for GATA3 and/or TTF1 and negative for estrogen receptor (ER) and progesterone receptor (PR); they also often express luminal CD10 and occasionally calretinin [6, 7]. Recently, grey-zone tumors that are morphologically and immunohistochemically reminiscent of MLA, but cannot entirely be excluded as other histotypes, have been identified. This diagnostic overlap highlights the importance of distinguishing MLA from other histotypes, such as endometrioid carcinoma (EC), given the differences in prognosis. Ovarian (oMLAs) and uterine (uMLAs) MLAs exhibit clinically aggressive behavior [2, 3, 8]. However, in our recent study, we found that the International Federation of Gynecology and Obstetrics (FIGO) I ovarian endometrioid carcinomas (OECs) with mesonephric-like differentiation (MLD) do not differ from conventional OEC regarding prognosis [9]. MLAs are presumed to be of Mullerian origin, and several studies have reported ovarian or extra-ovarian MLAs associated with endometriosis [4, 8, 10]. Based on our own experience, we have encountered cases of uMLA coexisting with adenomyosis, including tumors localized entirely within the myometrium. Nonetheless, no molecular studies have yet confirmed a direct etiologic link between MLA and ectopic endometrium. To molecularly investigate the tumor origin, we focused on KRAS hotspot mutations frequently observed in MLA [6, 11, 12]. KRAS hotspot mutations have also been identified in non-cancerous endometriosis [13, 14], with one study reporting higher variant allele frequency accompanied by chromosomal arm-level allelic imbalances [15]. KRAS mutations are also detected in over one-third of adenomyosis [16]. Moreover, Inoue et al. reported that adenomyosis co-occurring with endometriosis was associated with KRAS mutation and low PR expression [16, 17]. As ectopic endometrium undergoes malignant transformation in certain contexts, [18] we hypothesize that KRAS mutations act as an early driver event in the pathogenesis of MLA, implicating KRAS-mutated ectopic endometrium as a precursor lesion. In this study, to identify clinicopathological differences between MLA and EC and explore the origins of MLA, we collected resected specimens from 83 cases across multiple institutions in Japan, which were diagnosed as MLA or suspicious for MLA in the uterus or ovary. We aimed to categorize cases into MLA and non-MLA based on our morphological and immunohistochemical algorithm. Consequently, 78 and 5 of the collected cases were classified as MLAs and non-MLAs, respectively. For the 78 MLAs, we investigated the clinicopathological characteristics and origin. Whole-exome sequencing (WES) was conducted for each component in four ovarian cases (three MLAs and one non-MLA) that presented with mixed histotypes to explore the progression mechanisms of MLA.

Cases Eighty-three cases (uterus, 47; ovary, 36), which were diagnosed as MLA or suspicious for MLA in 17 institutions, were included from 2008 until 2024. Three cases were previously reported as MLA (Cases 10–12) [19], along with 10 cases of OEC with MLD (Cases 28–37) [9]. Whole section hematoxylin and eosin slides of formalin-fixed paraffin-embedded (FFPE) samples were reviewed at Saitama Medical University International Medical Center (SMUIMC). Clinical information was collected from medical records at each institution. Diagnostic criteria of MLA MLAs are defined as adenocarcinomas with a mesonephric-like phenotype. In this study, to distinguish MLA from other histotypes, cases were conclusively diagnosed as MLA based on specific diagnostic criteria regarding morphology and IHC (Fig. 1). Initially, cases were stratified into an MLA-favoring group and an MLA-indeterminate group based on morphology. We recorded several structural patterns with predominance (Online Resource Materials and Methods and Online Resource Fig. 1). MLA typically exhibits various histological patterns: small tubules with luminal eosinophilic colloid-like material predominate, along with an admixture of papillary-ductal, retiform, solid, or spindled architecture [5, 10]. Such morphology predominated in MLA-favoring cases. Specifically, MLA-indeterminate cases showed either MLA morphology but exhibited predominant ductal and papillary architecture, which are also characteristic of EC, or an admixture of other architecture not characteristic of MLA, such as micropapillary. Two gynecological pathologists, YM and MY, independently performed histological assessments, blinded to the IHC results. Discrepancies were resolved with discussion under a multi-head microscope, and assessments were finalized. For indeterminate MLA morphology cases, other histotypes to be excluded were noted. Subsequently, cases with TTF1 and/or GATA3 positivity and ER and PR negativity were considered typical IHC with a positive cutoff value of ≥5%. Cases with typical IHC were classified as MLA. In addition, WT1 negativity was confirmed in the diagnosis of oMLA to exclude low-grade serous carcinoma. Those combining favoring MLA morphology with atypical IHC were classified as non-MLA. For cases with favoring MLA morphology and atypical IHC, IHC was reviewed, and those with any GATA3 and TTF1 expression with inverse patterns [6], calretinin and/or CD10 positivity, and ER and PR negativity were classified as MLA. When a distinct histotype or component from MLA was evident, the diagnosis was “mixed MLA and distinct histotype,” detailing proportions within the carcinoma. Other pathological findings Tumor diameter, gross pattern, stromal amount (predominance vs minor), pathological staging, lymphovascular space invasion (LVSI), and necrosis were morphologically assessed. IHC Details on the assessment of expression for each marker are provided in the Online Resource Materials and Methods, and the primary antibodies used are listed in Online Resource Table 1. Morphological speculation of origin Uterus We morphologically speculated the origin of the tumor based on the adjacent preexisting lesion and tumor location (Online Resource Fig. 2). We identified preexisting lesions from which carcinoma could develop, including endometrioid intraepithelial neoplasia (EIN), endometrial polyp (EP), and adenomyosis. Among these, only the lesions adjacent to the tumor were considered. Tumor involvement in the endometrium and/or myometrium was assessed based on predominance. Finally, the morphologically speculated origin was determined. Ovary Possible origins of the tumor included the following epithelial structures: endometriosis, mesonephric remnant, Walthard nest, and parafallopian cyst. Among them, only sites of endometriosis were found adjacent to MLA, which was considered a morphologically speculated origin. KRAS mutation analysis For MLA, other coexisting histotypes, and morphologically speculated origins, KRAS codon 12 hotspot mutation analysis was conducted. As the suspected origins generally comprised only a minor portion of the sample, laser-capture microdissection was performed using an LMD 7000 instrument (Leica Microsystems, Wetzlar, Germany) on the 10 µm-thick FFPE samples to extract DNA. The estimated targeted cell ratio was 60–90%. The procedure of DNA extraction and KRAS hotspot mutation analysis using Sanger sequencing is described in the Online Resource Materials and Methods. The primers used for sequencing are listed in Online Resource Table 2. WES Four ovarian cases coexisting with other histotypes were selected. The procedures of DNA extraction, WES, and data analysis are described in the Online Resource Materials and Methods. Control cases We utilized clinicopathological data from 100 endometrial endometrioid carcinomas (EECs) and 100 OECs from SMUIMC, along with 232 ECs from The Cancer Genome Atlas [20]. Detailed information is described in the Online Resource Materials and Methods. Statistics Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan–Meier method. Survival analyses were conducted separately for uMLA and oMLA. For each organ, univariate Cox proportional hazards models were fitted. Variables with P < 0.20 in univariate analysis were included in multivariate models. Statistical significance was set at P < 0.05. FIGO stage comparisons (I vs II, I vs III, and I vs IV) were modelled independently to avoid instability. All analyses were conducted using Python (lifelines 0.30.0) and JMP Pro 17 (SAS Institute Inc., Cary, NC, USA). Ethics All participants provided informed consent. The ethics review board of SMUIMC approved the study design as a centralized review (approval number: 2022-067).

Case selection All 83 cases were classified into 78 MLA and 5 non-MLA cases based on the diagnostic criteria shown in Fig. 1. Representative micrographs of MLA and non-MLA are shown in Fig. 2 and Online Resource Fig. 3, respectively. One non-MLA, an OEC, had a high-grade (HG) component and was previously reported as an OEC with MLD coexisting with dedifferentiated carcinoma [9]. Online Resource Table 3 compares pathological differences between MLA and non-MLA. Full clinicopathological data are included in Online Resource Table 4. Clinicopathological characteristics and prognostic factors Table 1 presents the clinicopathological characteristics of MLAs. Nine of the 31 (29%) oMLAs harbored mixed distinct histotypes or components with a variable range (10–95%). However, only one uMLA coexisted with heterologous sarcoma. Among 47 patients with uMLA, PFS was 1 to 92 months (median = 15 months) and OS was 1 to 127 months (median = 25 months). During the follow-up period, 20 patients (42.6%) progressed and 11 (23.4%) died. Among 31 patients with oMLA, PFS was 1 to 107 months (median = 21 months) and OS was 1 to 107 months (median = 26 months). During the follow-up period, seven patients (22.6%) progressed and four (12.9%) died. Among 100 patients with EEC, PFS was 1 to 75 months (median = 62 months) and OS was 4 to 76 months (median = 62.5 months). During the follow-up period, seven patients (7.0%) progressed and five (5.0%) died. Among 100 patients with OEC, PFS was 0 to 87 months (median = 35.5 months) and OS was 0 to 97 months (median = 57). During the follow-up period, 14 patients (14.0%) progressed and 7 (7.0%) died. Prognostic factors of progression are summarized in Table 2. In uMLA, there were 20 progression events among 47 patients. LVSI was an independent and significant poor prognostic factor in multivariate analysis (P = 0.003 [HR: 11.33, 95% CI: 1.26–101.69]). However, factors other than LVSI did not reach significance in the multivariate analysis, and the wide confidence intervals warrant cautious interpretation. In oMLA, there were only 7 progression events among 31 patients. Accordingly, multivariate analysis using the Cox proportional hazards model was not performed. Clinicopathological comparison between uMLA and EEC uMLA occurred in older patients (P = 0.01), presented at a more advanced stage (P < 0.0001), was associated with more frequent LVSI (66%, P < 0.0001), and had a greater presence of adenomyosis (62%, P = 0.0005) than EEC (Table 3). The PFS and OS for uMLA were worse than those for EEC (Fig. 3a, b). The OS did not vary between uMLA and EEC when divided into stage I and II–IV (Online Resource Fig. 4a, b). Although the PFS for uMLA was inferior to that for EEC in stage I or I–II, no differences were observed in stage II–IV or III–IV (Fig. 3c, d and Online Resource Fig. 4c, d). The PFS and OS of uMLA were comparable to those of copy number-high tumors in integrated subtypes of EC (Online Resource Fig. 5). Clinicopathological comparison of oMLA and OEC FIGO stage, gross pattern, and LVSI were similar between oMLA and OEC (Online Resource Table 5). oMLA had a worse PFS and OS than OEC in stage II–IV, but not across all stage I, early (I–II), or advanced (III–IV) stages (Fig. 4 and Online Resource Fig. 6). This tendency was not different in the 22 pure oMLAs without other histotypes (Online Resource Fig. 7). Origin of MLA In uMLAs, the origin was speculated using an algorithm illustrated in Online Resource Fig. 2, with two representative cases shown in Online Resource Fig. 8. Table 4 summarizes the results of KRAS hotspot mutation analysis of uMLA. Among the 36 uMLAs that harbored a KRAS mutation, endometriosis specimens shared the same mutation with the tumor most frequently (12 [33%]), followed by the endometrium (4 [11%]) and EIN (3 [8%]). In total, 29 of the 31 (94%) oMLAs harbored endometriosis (Table 5), and 23 of these endometrioses (79%) shared the same KRAS mutation with the tumor.

In this study, we aimed to elucidate the clinicopathological characteristics of MLA. Moreover, to explore the origins of MLA, we reviewed the whole section slides of each case and analyzed KRAS hotspot mutations in the morphologically speculated origins and MLA. To the best of our knowledge, this is the first study to molecularly substantiate the link between MLA and ectopic endometrium. The putative tumor progression of MLA is depicted in Fig. 5. We set the diagnostic criteria for MLA based on morphological and immunohistochemical evaluations. It is crucial to morphologically suspect the MLA and conduct IHC. Specifically, MLA frequently shows GATA3, TTF1, calretinin, and CD10 positivity, and ER negativity [1, 3, 7, 8, 21, 22]. Among them, several researchers have suggested that hormone receptor negativity and a combination of TTF1 and GATA3 expression are useful diagnostic markers [3, 8]. Although the cutoff value of positivity is controversial, we set the positive cutoff at ≥5% for initial assessment. However, for cases with favoring MLA morphology, ER and PR negativity, and an inverse pattern of TTF1 and GATA3, even if they were expressed in <5%, were rendered significant because Euscher et al. reported this pattern to be characteristic of MLA [6]. We also confirmed calretinin and CD10 expression. Consequently, three cases with favoring MLA morphology and atypical IHC were classified as MLA. Köbel et al. suggested a low threshold for morphological features of MLA to recommend an ancillary immunohistochemical marker panel, including PAX2, hormone receptor, GATA3, and TTF1 [8]. We concur with their proposal and categorized cases into typical and atypical IHC. Nevertheless, several cases exhibited both ER and GATA3 and/or TTF1 expression. Köbel et al. reported cases with both OEC morphology and TTF1 and/or GATA3 expression (≥1%) and varying levels of ER and/or PR expression, showing a prognosis similar to that of MLA [8]. However, we recommend cautious interpretation of cases exhibiting ER and/or PR expression, which aligns with prior studies indicating that “patchy” or low-level ER expression can be consistent with MLA [23, 24]. Among the five non-MLA, four were diagnosed as OEC, and the remaining was identified as high-grade serous carcinoma (HGSC), exhibiting TTF1, GATA3, and ER positivity. Although this case exhibited indeterminate MLA morphology, severe nuclear atypia and marked mitotic figures were observed (Online Resource Fig. 2d). p53 exhibited a null pattern, indicating aberrant TP53 status. Euscher et al. and Mills et al. reported two similar cases, harboring p53 abnormality and KRAS hotspot mutation [3, 24]. Although whether they should be classified as HGSC or oMLA is debatable, we categorized them as HGSC and emphasized that HGSC could also harbor a mesonephric-like phenotype. Nine of the 31 (30%) oMLAs and one uMLA presented mixed distinct histotypes. The most frequent mixed histotype was EC, which exhibited ER positivity. One uMLA had chondroid elements, which appear to be a characteristic of mesonephric-like carcinosarcoma [25]. Although not included in this study, Yano et al. reported a case of mixed uMLA and EEC, where hormone therapy was effective only for EEC [26]. Our study showed no instances of coexisting serous tumors, germ cell tumors, or sex cord-stromal tumors previously reported in other studies [3, 27,28,29,30,31]. uMLA presented a more advanced stage and a poorer prognosis than conventional EEC, consistent with a retrospective review by Kim et al., which analyzed the seven uMLAs extracted from 237 ECs [32]. In our study, 23/47 (49%) cases of uMLAs were in stage II–IV, showing a slightly lower frequency than that previously reported by Pors et al. and Kim et al. (18/25 and 25/43, respectively) [2, 33]. Furthermore, the median PFS of the uMLAs, 30 months, was longer than that reported by Pors et al. and Euscher et al. (18–21 months) [2, 6]. Nevertheless, it is noteworthy that uMLA had worse PFS than EEC, even when limited to stage I (Fig. 3c) or I–II (Online Resource Fig. 8c). This might be explained by the fact that stage I uMLA had more frequent deep myometrial invasion (uMLA 57% vs EEC 17% [P = 0.003], Table 3) or that LVSI was an independent and significant poor prognostic factor of uMLA in multivariate analysis (P = 0.003 [HR: 11.33, 95% CI: 1.26–101.69]. In the oMLA of our study, the prognosis of stage I was favorable, similar to that of OEC. However, Köbel et al. compared disease-specific survival between 14 oMLAs and 157 OECs limited to stage I and found a considerably worse prognosis for oMLA than for OEC [8]. This discrepancy between their results and ours may be due to different MLA selection criteria, such as IHC panels or expression cutoff values. However, the small number of stage I oMLAs makes it difficult to conclude their prognosis. MLA is currently considered to be of Mullerian origin [10]. oMLAs are commonly associated with endometriosis [1, 34,35,36]. Several studies have revealed that mutation and loss of expression of ARID1A are observed in the endometriosis immediately adjacent to EC or CCC in the ovary [37, 38]. Similar to these endometriosis-associated carcinomas, we hypothesized that KRAS hotspot mutations, frequently observed in oMLA [6, 11, 12], initially occur in the tumor progression of oMLA before being recognized as a tumor. Twenty-three of the 29 (79%) oMLAs with KRAS mutations harbored a common one associated with endometriosis. One case of oMLA coexisting with a mucinous borderline tumor harbored a common KRAS G12C mutation. Although Nilforoushan et al. identified four cases of ovarian mucinous tumors coexisting with mesonephric-like lesions sharing common KRAS hotspot mutations [39], Sim et al. reported that in the ovarian mucinous tumor populations, mesonephric-like components were rarely detected [40]. Additionally, Mezzapesa et al. reported five oMLAs that were all categorized as endometriosis-correlated carcinoma, occupying 10% of this category [41]. Three EINs and four endometria were identified as the molecularly speculated origin of uMLA. Santoro et al. reported mesonephric-like lesions in the endometrium [42], whereas Lac et al. found that the KRAS codon 12 hotspot mutation appears in 28% (31/110) of normal endometrium from women lacking evidence of gynecologic malignancy or EIN [43]. Therefore, we propose that the endometrium with KRAS hotspot mutations may evolve into uMLA. Conversely, adenomyosis was noted in uMLA two times as often as in EEC (62% vs 28%). Yamamoto et al. also reported magnetic resonance imaging findings indicating that coexisting adenomyosis is present in over 50% of patients with uMLA [44]. In our molecular analysis, among 36 uMLAs harboring KRAS hotspot mutations in tumors, 12 (33%) had common mutations with adenomyosis, which also indicated 12/21 (67%) of adenomyosis had KRAS mutations. Additionally, six uMLAs were confined to the myometrium (13%). Cases confined to the myometrium without endometrial involvement might be analogous to those reported as uterine mesonephric adenocarcinoma without mesonephric remnant [45,46,47], with adenomyosis in one case [48]. Among previously reported cases of EC arising in adenomyosis, we found one case that morphologically seemed to resemble uMLA [49]. Moreover, in a systematic review of ER expression in ECs arising in adenomyosis by Machida et al., 12 of the 14 (86%) cases were ER-negative [50]. This study had some limitations. First, the number of cases and events in the oMLA cohort was small. In oMLA, progression events were limited to 7 of 31 cases overall, and the stage II–IV subgroup was even smaller. Although the uMLA cohort had a larger number of progression events (n = 20), factors other than LVSI did not emerge as independent prognostic variables in multivariate analysis, and the confidence intervals were wide. Thus, validation in larger cohorts is warranted. Second, we molecularly speculated the origin of MLA based on the presence of a common KRAS codon 12 hotspot mutation between MLA and an adjacent lesion through Sanger sequencing. However, analyses on some morphologically speculated origins were not performed owing to insufficient DNA quantity. Third, the other mutations reported to be observed in MLA, including PIK3CA, NRAS, and BRAF, were not analyzed [6, 11]. In conclusion, we revealed the clinicopathological characteristics of MLAs. Especially, among 29 oMLAs harboring a KRAS hotspot mutation, 23 (79%) instances of endometriosis in the background had the same mutation. Moreover, among 36 uMLAs carrying the KRAS hotspot mutation, common mutations were observed in 12 (33%) instances of adjacent adenomyosis. Our hypothesis regarding the close histogenetic association of MLA with ectopic endometrium harboring the KRAS mutation can be anticipated to contribute immensely to the pathological and clinical analysis of MLA. Data availability All data generated or analysed during this study are included in Online Resource Table 4.

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The study was aided by the Japanese Society of Gynecologic Pathology for case collection. We thank Koichi Kamada, Nobuyuki Suzuki, and Yusuke Hosonuma for their technical support and Hidetoshi Uenaka for quality control of statistical analyses. We would also like to thank Editage (www.editage.com) for English language editing. This study was supported by Grants-in-Aid for Scientific Research (No. 24K18493 and 21K06928) and an internal grant (No. 22-B-1-23) from Saitama Medical University. Funding Open Access funding provided by Saitama Medical University. Japan Society for the Promotion of Science, 24K18493, Yu Miyama, 21K06928, Masanori Yasuda, Saitama Medical University, internal grant 22-B-1-23, Yu Miyama Author information Authors and Affiliations Contributions All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Yu Miyama, Kousuke Uranishi, Masataka Hirasaki, Akiko Tonooka, Fumi Kawakami, Yoshiki Mikami, Takako Kiyokawa, Hiroshi Kajiwara, Junichi Shiraishi, Eiji Kudo, Kozue Ito, Morihiro Higashi, Sachiko Minamiguchi, Nasa Okazaki, Yuzuru Kondo, Tetsuya Shimada, Yoshinobu Maeda, Yumiko Yasuhara, Yoshiaki Kawano, Yohei Kawasaki, Kosei Hasegawa, and Masanori Yasuda. The first draft of the manuscript was written by Yu Miyama, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Corresponding author Ethics declarations Informed consent All participants provided informed consent. Research involving human participants and/or animals The ethics review board of SMUIMC approved the study design as a centralized review (approval number: 2022-067). Competing interests The authors declare no competing interests. Additional information Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Information Below is the link to the electronic supplementary material. Rights and permissions Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. About this article Cite this article Miyama, Y., Uranishi, K., Hirasaki, M. et al. Multi-institutional study of mesonephric-like adenocarcinoma: its clinicopathological characteristics and histogenetic association with ectopic endometrium. Virchows Arch (2025). https://doi.org/10.1007/s00428-025-04286-0 Received: Revised: Accepted: Published: Version of record: DOI: https://doi.org/10.1007/s00428-025-04286-0