Extrauterine mesonephric-like carcinoma: a tumor with a cold lymphocytic immune microenvironment

Extrauterine mesonephric-like carcinoma (ExUMLC) is a rare female genital tract tumor associated with advanced stage disease and distant recurrences. Although there is a histologic and mutational overlap with extrauterine low grade endometrioid carcinoma (ExULGEC), the distribution of the main components of the tumor microenvironment (TME) in these two neoplasms is not known. In this study we evaluated the clinicopathologic characteristics and the main lymphocyte populations in different compartments of the TME in cases of ExUMLC (18) and ExULGEC (17) - diagnosed at a single institution. A multiplex immunofluorescence panel including CD3, CD4, CD8, CD20, CD56, pancytokeratin, Foxp3, and Ki-67 was applied in all cases. The median age of ExUMLC was 60.5 years (range: 37–69 years), while in ExULGEC was 53 years (range: 22–83 years). The tumor compartment and total area analyzed of ExUMLC were significantly enriched in CK + Ki-67 + cells [(tumor: median 478 vs. 37.4 cells/mm2, p = 0.029) and (total: median 304.5 vs. 33.2 cells/mm2, p = 0.044)], CD3 + CD4+ T-cells [(tumor: median 0.6 vs. 0 cells/mm2, p = 0.01) and (total: median 2.6 vs. 0.3 cells/mm2, p = 0.022)], and CD3 + CD4+Foxp3 + T-cells [(tumor: median 0 vs. 0 cells/ mm2, p = 0.035) and (total: median 0.8 vs. 0 cells/mm2, p = 0.007)]. Importantly, distances from CK + to CD3 + CD4+ cells were also significantly lower in the ExUMLC group compared to ExULGEC. ExUMLC has a unique lymphoid microenvironment characterized by an overall low concentration of lymphocytes with a higher concentration of CD4 + T-cells and classic Tregs when compared to ExULGEC. This “cold” and immunosuppressive TME may be implicated in the more aggressive biology, and potentially pose a challenge for targeted treatment options in patients with ExUMLC. Similar content being viewed by others

Mesonephric-like carcinoma (MLC) is an uncommon tumor recently incorporated in the 2020 World Health Organization Classification of Female Genital Tumors. This neoplasm is characterized by histologic and immunohistochemical features similar to mesonephric carcinomas but lacking associated mesonephric hyperplasia or remnants [1,2,3]. This tumor may arise in the endometrium or extra-uterine sites and its immunohistochemical profile typically shows a negative or reduced estrogen and progesterone receptor expression and positive TTF-1 and/or GATA3 expression [1, 4]. Heterogeneous histology, morphological and molecular overlaps with other neoplasms, and its common synchronous or metachronous association with other tumor histologic types, contribute to the diagnostic challenge of extrauterine mesonephric-like carcinoma (ExUMLC) [3]. Due to the propensity for histologic overlap, extrauterine low-grade endometrioid carcinoma (ExULGEC) is a frequent diagnostic pitfall. As ExUMCL has been reported to have a worse outcome when compared to ExULGEC, the correct distinction of these two entities is essential as it may have an impact on therapeutic options for these patients [5]. The tumor microenvironment (TME) is represented by the non-neoplastic component that, through the interactions with the tumor cells, plays a decisive role in several factors related to the clinical behavior and outcomes of a given tumor, such as tumor progression, metastasis, and response to treatment [6]. Although some studies exploring the main characteristics of the immune landscape of ovarian tumors have pointed out the importance of some populations of lymphocytes, macrophages, and myeloid cells, there is no data specifically addressing TME in ExUMLC and ExULGEC [7]. A proper understanding of the immuneprofiling of these neoplasms is essential to provide a better rationale for the possible targeting of immune-checkpoints and improvement of efficacy of these therapeutic options. Furthermore, any existing differences in the TME between ExUMLC and ExULGEC could explain the differences in the behavior of these two tumors. In this study, we present an analysis of the lymphocytic component from the TME of a cohort of ExUMLC and ExULGEC in addition to their clinicopathological features.

Following approval by the Institutional Review Board at MD Anderson Cancer Center (MDACC), we performed a retrospective search of the MDACC Department of Pathology database using the key words “ovary,” “peritoneum”, “mesonephric-like,” “low grade endometrioid” between 2002 and 2021. As one of the major aims of this study was to explore the TME in the ExUMLC and its possible role in the difference in behavior relative to ExULGEC, cases of ExULGEC were selected for comparison. In cases of ExUMLC associated with other histotypes, the TME was analyzed only in the ExUMLC component. ExULGEC cases were selected without knowledge of the FIGO stage or patient outcome. All hematoxylin and eosin (H&E) slides of ExUMLC and ExULGEC were reviewed by 1 or more gynecologic pathologists at MDACC, and those with available formalin-fixed paraffin-embedded (FFPE) tissue were selected for the study. The clinicopathologic features were collected from the patients’ electronic medical records, pathology reports and molecular testing reports and included: patient age, date of diagnosis, extrauterine site of origin, FIGO staging, presence of endometriosis, presence of other histotypes in ExUMLC immunophenotype, molecular profile by next generation sequencing, treatment, site of metastasis (if applicable), date of last follow-up, and patient status at last follow-up. The cases of ExUMLC in this study were included as part of a larger study by our group [5]. Multiplex immunofluorescence imaging assay and spatial analysis Four-micron thickness FFPE tissue sections from surgical specimens were used for the analysis of the lymphoid landscape. We previously optimized and validated a multiplex immunofluorescence panel using CD3, CD4, CD8, CD20, CD56, Foxp3, pancytokeratin (CK), and Ki-67. Each antibody was assessed by a uniplex immunofluorescence panel using the Opal 9 kit (catalog #NEL797001KT; Akoya Biosciences), according to the following clones and dilutions: εCD3 (D7A6E, CST, 1:100), CD4 (EPR6855, Abcam, 1:200), CD8 (C8/144B, Thermo Scientific, 1:25), CD20 (L-26, Dako, 1:50), CD56 (123C3, Dako, 1:25), Foxp3 (D2W8E, CST, 1:100), CK (AE1/AE3, Dako, 1:50), and Ki-67 (MIB-1, Dako, 1:100). All cases were imaged by Vectra Polaris spectral imaging system (Akoya Biosciences) using the fluorescence protocol at 10 nm λ from 420 nm to 720 nm. Lymph nodes with reactive lymphoid hyperplasia were used as a positive control. Five regions of interest (ROI, 931µ x 638 µ) of an intratumoral area were selected in each case. Each marker was analyzed at a single-cell level, and a supervised algorithm for phenotyping was built for each marker. Cell density for each marker and all possible combinations were consolidated by using Spotfire software (TIBCO Spotfire) and the nearest neighbor analysis was performed using R version 4.2.1. Statistical analysis Chi-square or Fischer’s exact test was used to calculate association between categorical variables, while the differences between continuous variables were evaluated using Mann-Whitney test. Overall survival (OS) was defined as the time interval from diagnosis to death or last follow-up, and it was calculated for all patients in the study with greater than 5 months of follow up by using Kaplan-Meier estimates. The differences in OS between subgroups were compared by using log-rank (Mantel-Cox) test. Statistical analyses were completed by using GraphPad Prism version 9.0.0 for Windows (GraphPad Software, San Diego, CA, USA, www.graphpad.com). The nearest neighbor analysis was performed using R version 4.2.1. A P value of ≤ 0.05 (two-tailed) was considered statistically significant.

Table 1 summarizes the main clinicopathologic features of the 35 cases included in this study. Of these, 18 (51%) were ExUMLC, and 17 (49%) as ExULGEC. The median age of the whole cohort was 60 years (range: 22–83 years) and was similar to the median age of the ExUMLC (median: 60.5 years, range 37–74) and ExULGEC (median: 53, range: 22–83 years) groups. In both groups, the most common extra-uterine site was ovary representing 83% of the ExUMLC and 100% of ExULGEC, and only the former had cases primary in the peritoneum. ExUMLC had a higher number of advanced stage cases (FIGO III/IV) when compared with ExULGEC. ExUMLC group was composed by 9 (50%) cases of Stage I, 3 (16%) cases of Stage II, 4 (23%) cases of Stage III, and 2 (11%) cases of Stage IV, while ExULGEC was composed by 3 (18%) cases of Stage I, 8 (47%) cases of Stage II, 4 (24%) of Stage III, and 2 (11%) cases of unknown stage. Among the cases, which had association with other histotypes, ExUMLC was associated with other histotypes in 10 cases and it was distributed as follows: serous/mucinous borderline tumors [4], endometrioid carcinoma [3], clear cell carcinoma [2], and low grade serous carcinoma [1]. In one case the MLC represented the predominant component of a carcinosarcoma. Of note, none of the peritoneal primary tumors were mixed with other histotypes. The ExULGEC selected in this study were all pure. Distant metastasis was present at diagnosis in two cases of ExUMLC, one in lung/pleura and another one in liver, while it was absent in the ExULGEC group. Immunohistochemical and molecular results are presented in Supplementary Tables 1 and 2, respectively. Cases of ExUMLC were characterized by positivity for GATA-3 (16/18, 88%), TTF-1 (14/18, 77%), PAX-8 (14/14, 100%), calretinin (8/9, 88%, usually focal), estrogen receptor (6/18 [10–60% nuclear staining], 33%), progesterone receptor (4/17 [5–10%, nuclear staining], 23%), wild-type p53 expression pattern (9/10, 90%), and negativity for WT-1 (0/12, 0%). For the ExUMLC with hormone receptor expression 4 had only ER expression, 2 had only PR expression and 2 expressed both ER and PR. All of these cases, including the one outlier with 60% weak nuclear expression of ER, had diffuse expression of GATA-3 and/or TTF-1, combined with the classic morphologic H&E features of mesonephric-like carcinoma. In contrast, all cases of ExULGEC were positive for hormone receptors (ER, 6/6 and PR, 7/7) and negative for GATA-3 (0/1, 0%), TTF-1 (0/2, 0%), and calretinin (0/2, 0%). In addition, they also had a wild-type p53 expression pattern (3/3, 100%), and were usually positive for PAX-8 (3/4, 75%), and negative for WT-1 (0/2). Molecularly, cases of ExUMLC were characterized by frequent KRAS mutations (6/9, 66%) which were absent in all tested ExULGEC cases (0/6). All cases with available information received adjuvant paclitaxel and carboplatin in both groups (13/18 of ExUMLC and 14/17 of ExULGEC cases). Follow-up (FU) information was available in 17/18 (94%) of ExUMLC group and in 17/17 (100%) of the ExULGEC with a median FU of 30 months (range: 1–219 months) for the former and 38 months (range: 5–225 months) for the latter. At last follow-up patients in the ExUMLC group were: alive with disease (AWD) (9/18, 50%), alive with no evidence of disease (ANED) (7/18, 38%), died of disease (DOD) (1/18), and lost to follow-up (1/18, 6%). In the ExULGEC: patients were ANED (15/17, 90%), AWD (1/17, 5%) or DOD (1/17, 5%). The median OS was not reached in both subgroups due the small number of events in the cohort. However, the mean OS was slightly lower in the ExUMLC group (175 months, 95%CI: 101.6–249.3 months) compared to ExULGEC (204 months, 95%CI: 165.8–242.6 months), although this did not reach statistical significance. In the whole cohort, FIGO staging (p = 0.437), patient’s age (p = 0.124), presence of metastasis at the time of diagnosis (p = 0.183), association with other histotypes (p = 0.492), and type of treatment (p = 0.390) had no correlation with outcome, which could be related to the small number of patients in this cohort. Multiplex immunofluorescence imaging assay A similar mean and median total area [ExUMLC (mean: 2.98 mm2, median: 3.05 mm2, range: 2.04–3.02 mm2), ExULGEC (mean: 2.78 mm2, median: 2.92 mm2, range: 1.93–3.11 mm2)] and number of cells [ExUMLC (mean: 10021 cells, median: 10255 cells, range: 5898–14954 cells), ExULGEC (mean: 9250 cells, median: 8528 cells, range: 6643–14169 cells) were analyzed for each group of cases. The same was observed when different compartments (tumoral and stromal) were evaluated separately. Based on the biological rationale of the combination of different markers, we predefined different phenotypes that were assessed in different compartments (tumor, stroma and total area) of the cases in both groups (Fig. 1). A detailed cell density per compartment for each case is shown in Supplementary Table 3, while the median cell densities are summarized in Table 2. Cases of ExUMLC had a significantly higher cell density of CK + Ki-67 + cells (median: 435.6 vs. 37.4 cells/mm2, p = 0.032), CD4 + T-cells (median: 0.6 vs. 0 cells/mm2, p = 0.017), and CD3 + CD4+Foxp3 + T-cells (median: 0 vs. 0 cells/mm2, and mean: 0.4 vs. 0.05 cells/mm2, p = 0.035) in the tumoral compartment when compared to ExULGEC. No significant differences were found in the stromal compartment. Interestingly, when the total area of analysis was assessed, cases of ExUMLC had a significantly higher median cell density of CK + Ki-67 + cells (median: 304.5 vs. 33.2 cells/mm2, p = 0.044) (Fig. 2A-B), CD4 + T-cells (median: 2.6 vs. 0.3 cells/mm2, p = 0.022) (Fig. 2C-D), and classic T-regulatory (Tregs) cells (CD3 + CD4+Foxp3+) (median: 0.8 vs. 0 cells/mm2, p = 0.007) (Figure E-F) when compared to ExULGEC cases. No statistical differences were found in the CD8/CD4 of the different compartments. Spatial analysis The distances between neoplastic cells (CK+), and main immune populations (CD3+, CD20+, CD3 + CD4+, CD3 + CD8+, CD56 + CK-, CD3 + CD4+Foxp3+) within the tumor and/or the stroma are summarized in the Supplementary Table 4. Except for the distances from CD3 + CD4+Foxp3 + cells, cases from the ExUMLC group had an overall shorter median distance from the tumor cells to the immune populations when compared to cases of ExULGEC (Table 3). However, only the median distances from CK + to CD3 + CD4+ cells were statistically significant (p = 0.013) (Fig. 3).

In this study, we describe the main clinicopathologic differences between 18 cases of ExUMLC and 17 cases of ExULGEC, and for the first time we report the differences in the lymphocytic component of the TME, mainly represented by B-, T-helper, T-cytotoxic, and Treg cells, and their spatial landscape in these tumors. Most ExUMLC and ExULGEC were FIGO stage I/II, and cases of ExUMLC had a slightly higher proportion of patients with FIGO III/IV. In fact, the reported literature is heterogeneous regarding the predominant stages in ExUMLC, but in most case series, early-stage disease is more common [2, 8]. The association with endometriosis or endometriotic cyst, was observed in both groups in this series and it was not significantly associated with any of the groups, most likely due the limited number of patients included in the cohort, since the literature describes a positive association of these lesions with ExUMLC [9, 10]. Though less than 50% of the cases in both groups had molecular testing available, it is important to note that more than 70% of ExUMLC cases had mutations in KRAS, which were absent in all tested ExULGEC cases. Similar findings were also described in other case series that evaluated the molecular profile of ExUMLC and observed a high frequency of mutations in KRAS, mainly p.G12D, providing additional support for the implication of this molecular alteration in the pathogenesis of these tumors [3, 8, 10,11,12,13,14]. The normal immune microenvironment of the ovary in ExUMLC is usually composed of low cell densities of normal B/T-cell lymphocytes, dendritic cells, macrophages, and myeloid-derived suppressor cells (MDSC) [7]. The same cold or “non-inflamed” microenvironment is observed in other ovarian tumors, such as serous carcinoma, mucinous carcinoma, and clear cell carcinoma. They are usually characterized by a lack of T-cell infiltration within the tumor bed and generates not only a more immunosuppressive TME, but also a great challenge in the election of a precise and effective immunotherapy treatment [15]. Overall, in cases of ovarian tumors, especially serous carcinoma, there is a shift to a more immunosuppressive and cold microenvironment, mainly characterized by an upregulation of regulatory B- and T-cells, MDSC, and tumor-associated macrophages (TAM) with pro-tumor activity. Due to the limited number of studies on ExUMLC, there are no described implications of the molecular aspects in the immune landscape of these tumors. In our analysis of ExUMLC, the cell densities of the lymphocytic population (CD3 + and CD20 + cells), when compared with the total number of tumor cells, was extremely low in all compartments confirming the cold and poor immune landscape in these tumors. The total T-cell component was predominant in both groups over the B-cell component. The T-helper population was statistically significantly more present in the ExUMLC group when compared to ExULGEC. Although the median cell density of cytotoxic cells was higher in the ExUMLC, no statistical difference was found between the groups. In fact, the proportion of T-helper and cytotoxic cells was similar in ExUMLC, while the cytotoxic component was more abundant in the ExULGEC. Specific subsets of T-cells, such as classical Tregs (CD3 + CD4+Foxp3+) have been reported to be associated with tumor angiogenesis, metastasis, progression, and consequently, an aggressive behavior in different stages of ovarian carcinoma [7, 16]. Activated by the myeloid component regulation, the classical Tregs play an important role in several other components of the TME, such as the suppression of tumor-associated antigens and effector T-cell function, promoting a disruptive and more immunosuppressive TME [16]. Despite the fact that both groups studied had minimal lymphoid infiltration, total T-helper (CD3 + CD4+) cells represented 18.5% and 6.2% of the T-cell component in ExUMLC and ExULGEC, respectively. Importantly, when comparing the amount of Tregs in relation to all T helper cells, this proportion goes to around 50% and 20%, respectively, in keeping with a cold TME We also found a statistically significant shorter distance from tumor cells to T-helper cells in ExUMLC. This finding also reinforces that local immunosuppression is related to the closer relationship besides the higher cell density of this immune population. Cytotoxic T-cells (CD3 + CD8+) are key factors in the anti-tumor activity and act through the recognition of MHC class I, promoting direct tumor killing [7, 17]. Sato and colleagues evaluated the TME by immunohistochemistry of 117 cases of epithelial ovarian tumors and showed a potential role of T-helper, Tregs, and T-cytotoxic populations. In their analysis, most of the cases were composed of serous carcinoma and a correlation between higher CD8 + tumor-infiltrating lymphocytes and better overall survival was found in these patients [17]. Interestingly, their results support that the cell density of T-helper cells influences the anti-tumoral role of the cytotoxic component, especially in such cases with an upregulation of Tregs. In our study, due the limited size of the sample, the correlation of the cell densities and outcomes within the groups was not performed to avoid biases of interpretation. Nevertheless, there was a higher cell density of the cytotoxic immune component in ExUMLC group when compared to ExULGEC, but it was not significant. Different proportions of the CD3 + CD8+/CD3 + CD4+ ratio also support the differences in the TME of these two groups of neoplasms, since cases of ovarian carcinomas with higher ratios have better outcomes than those with lower ratios [17]. We acknowledge some limitations of the current study, its small sample size due the rarity of the disease and cases obtained from a single center, absence of whole tissue analysis, and lack of functional analysis to corroborate the translational findings. It is also important to note that similar analyses need to be performed using assays in which other different immune subsets, such as different subtypes of myeloid and B/T-cells, are analyzed to determine the real influence of each population in the immune landscape of ExUMCL. In particular, the comparison with the TME of other types of extra-uterine tumors is also needed to provide further biological and clinical implications. In conclusion, cases of ExUMLC are characterized by tumor cells with a higher proliferation index and a unique cold microenvironment, which may impact the possibilities of targeted immune therapies as also observed in other types of tumors [13, 14]. Importantly, we found a higher concentration of CD4 + T-cells and classic regulatory T-cells in ExUMLC, and a shorter distance from tumor to immune cells, providing additional biological rationale for the immunosuppressive landscape and worse outcomes of these tumors. Data availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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The authors thank the technical support of the Translational Molecular Pathology Immunoprofiling Laboratory (TMP-IL). Funding This work was funded in part by the Cancer Center Support Grant (NCI Grant P30 CA016672). Author information Authors and Affiliations Contributions M.L.M.-P. and E.D.E. performed study concept and design; M.L.M.-P., C.F.P.L, R.P., S.B., D.E.D, E.R.P., S.R., and I.I.W., performed multiplex studies, data interpretation and statistical analysis; B.C.L., P.R., A.M., and E.D.E, provided histopathological assessment. All authors read and approved the final paper. Corresponding author Ethics declarations Ethics approval and consent to participate The study was approved by the Institutional Review Board at The University of Texas MD Anderson Cancer Center (MDACC). Conflict of interest The authors have no other sources of funding or conflicts of interest to disclose. Additional information Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Presented in part at the 110th Annual Meeting of The United States and Canadian Academy of Pathology, Los Angeles, CA, March 19-24 2022. 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 Marques-Piubelli, M.L., Lima, C.F.P., Lawson, B.C. et al. Extrauterine mesonephric-like carcinoma: a tumor with a cold lymphocytic immune microenvironment. Virchows Arch (2026). https://doi.org/10.1007/s00428-026-04566-3 Received: Revised: Accepted: Published: Version of record: DOI: https://doi.org/10.1007/s00428-026-04566-3