Abstract
Background/Aim: Aberrant glycosylation is recognized as a trait of tumorigenesis. Paucimannosidic glycoepitopes (PME), a class of small mannosidic N-linked glycans have recently been linked to pathophysiologic conditions such as cancer and infection. This study aimed to investigate whether paucimannosylation is present in ovarian cancer and to explore its prognostic relevance.
Patients and Methods: We investigated the presence of PMEs in 176 patients with high-grade serous ovarian cancer using the research Mannitou antibody for immunohistochemistry. Immunohistochemical staining was assessed using eight different scoring systems and correlated with survival using the Kaplan–Meier method.
Results: Positive immunohistochemical staining of PMEs in immune cells was associated with improved 5-year disease-free survival (16.06% vs. 6.16%; p=0.032) and a trend toward improved 5-year overall survival (42.20% vs. 36.70%; p=0.052). Focal hot spot PME staining affecting more than 50% of tumor cells was associated with reduced 5-year overall survival (46.23% vs. 23.68%; p=0.029). Clinical factors such as tumor load, tumor distribution or FIGO-Stage showed no association with any PME staining pattern.
Conclusion: Paucimannosylation, indicated by the presence of PMEs, is present in both tumor and immune cells in high-grade serous ovarian cancer. The prognostic implication of PMEs seems to be cell type-dependent. While immune cell paucimannosylation was associated with improved survival, tumor cell paucimannosylation was associated with reduced survival.
Introduction
High grade serous ovarian cancer represents the most lethal gynecologic malignancy (1). Despite dedicated surgery, adjuvant chemotherapy and additional maintenance therapies, such as PARP-inhibitors or VEGF-antibodies, high-grade serous ovarian cancer is characterized by an evolving resistance to carboplatin and PARP-inhibitors (2-4). Therefore, it is of imminent interest to gain further insights into tumorigenesis of this disease.
Glycosylation is a frequent post-translational modification defining protein functions. Aberrant glycosylation has long been linked to carcinogenesis. Unfortunately, the functional role of altered glycosylation remains poorly understood in the clinical context (5, 6).
Paucimannose, a short mannosidic glycan has just recently been identified in humans and its presence seems to be linked to pathophysiologic conditions such as cancer and inflammation (7-10). New N-glycan datasets for lung adenocarcinoma show differences in the N-glycan profile of healthy and cancerous tissue. Paucimannose was increased at all tumor stages of lung adenocarcinoma (10). Paucimannose has also been shown to be released by activated neutrophils in pathogen-infected lungs (11). Furthermore, paucimannosidic epitopes (PMEs) seem to be enriched in cancer tissue when compared to normal tissue (10, 12). PMEs are associated with key oncogenic processes such as cell migration, invasion and proliferation. All of these processes can be blocked successfully in vitro by the Mannitou antibody. Therefore, PMEs may have a potential for diagnostic and therapeutic use (7). In the present study, we investigated the presence of PMEs in high grade serous ovarian cancer by immunohistochemistry using the research monoclonal Mannitou antibody (13, 14).
To the best of our knowledge, no data are available on the immunohistochemical detection of PMEs in high-grade serous ovarian cancer. Therefore, we aimed to address several key questions. First, we evaluated the most appropriate scoring method for assessment of staining with the Mannitou antibody. Second, we examined the potential association between Mannitou staining and disease-free survival (DFS) and overall survival (OS) in high-grade serous ovarian cancer. Last, we compared our immunohistochemical findings with clinically known prognostic factors such as FIGO-Stage, tumor mass described by the peritoneal cancer index (PCI) and tumor distribution (15, 16).
Patients and Methods
Data collection. This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the ethics committee of the Faculty of Medicine at the University of Bonn, Germany (Nr: 428/22). The institutional record database was screened for patients with high-grade serous ovarian cancer treated at the Department of Gynecology and Gynecological Oncology between January 2012 and December 2020. Tissue collection was conducted within the Biobank initiative of the University of Bonn. The only inclusion criterion was the histologically confirmed diagnosis of a high-grade serous ovarian cancer. The only exclusion criterion was any histology other than high-grade serous ovarian cancer. All patients provided written informed consent before tissue collection. Baseline characteristics, pathology, therapeutic course and peritoneal cancer index were recorded from patient’s charts and surgery reports (15). Follow up data were updated in July 2022. Tumor stage was based on the 2013 revised International Federation of Gynecology and Obstetrics (FIGO) system (16).
Patients. A total of 176 patients were included in the study. A board-certified pathologist (M.M.) performed Immunohistochemical evaluation of whole-tumor slides.
Immunohistochemistry (IHC). Whole tumor slides were prepared by the Institute of Pathology of the University Hospital Bonn. Hematoxylin and eosin (HE) slides of all 176 patients were re-examined by a pathologist to confirm the diagnosis of high-grade serous ovarian cancer. In all cases, the main tumor-containing slide was identified, and fresh tissue sections (2-3 μm) were prepared from the corresponding formalin-fixed paraffin-embedded (FFPE) tissue block.
For paucimannose staining, the Mannitou monoclonal mouse IgM antibody was prepared in RPMI medium as previously described (13, 14). IHC staining was performed using VECTASTAIN Elite ABC-HRP system and ImmPACT Nova Red chromogen (Vector Laboratories, Burlingame, CA, USA). Deparaffinized and rehydrated FFPE tissue sections were treated with Dako REAL peroxidase blocking solution (Dako, Glostrup, Denmark) followed by 5% bovine serum albumin (BSA; Neolab, Einhausen, Germany) in phosphate-buffered saline (blocking). Mannitou antibody (hybridoma supernatant diluted 1:50 in blocking solution) was added overnight at 4°C. Following incubations with secondary antibody (biotinylated anti-mouse IgM, 1:200 in blocking) and Vectastain ABC Elite reagent, paucimannose was stained with chromogen and slides were counterstained with hematoxylin. Control staining included secondary antibody control where Mannitou antibody was omitted and complete RPMI medium (1:50 in blocking) was used instead as well as preabsorption of Mannitou antibody with 0.5 M α-methyl mannoside (Sigma-Aldrich, Taufkirchen, Germany) as previously described (13, 14).
Evaluation of IHC. Tumor tissue presence was validated through visual examination of hematoxylin and eosin (HE) stained slides. At present, no validated scoring methods are available for the evaluation of paucimannosidic epitope staining using the Mannitou antibody. Therefore, we applied the following set of scoring methods to analyze the heterogeneous staining patterns. 1) Dichotomous scoring: positive versus negative staining. 2) Staining intensity graded as none [0], weak [1], moderate [2] and strong [3]. 3) Percentage of stained tumor cells. Classified by cut offs of <10% and >10%. 4) Immunoreactive score (IRS): following the method of Remmele and Stegner, staining intensity was graded as none (0), weak (1), moderate (2) and strong (3) and was multiplied with the percentage of positive cells (1:<10%, 2:11-50%, 3:51-80%, 4:>80%). Therefore, IRS score values ranged from 0-12 and were categorized into four groups: negative staining [0-1], mild staining [2-3], moderate staining [4-8] and strong staining [9-12] (17). 5) Immune cell staining: present versus absent staining in immune cells. 6) Detailed percentage of stained tumor cells categorized into 0%, 1-4%, 5-10%, 11-29%, ≥30%. 7) H-Score method: The percentages of weak, moderate and strong staining intensities are multiplied by 1 (weak staining), 2 (moderate staining) or 3 (strong staining) and subsequently summed up to the H-score with a range from 0-300 (18). 8) Hot-Spot method: Percentage of positively stained tumor cells within an area of notably higher staining intensities compared to the surrounding tumor cells (19).
Statistical analysis. Statistical analyses were performed using Minitab Version 18 (Minitab LLC., State College, PA, USA). The survival analyses for DFS and OS are based on the Kaplan–Meier method. The time-to-event intervals are described in months from the date of primary diagnosis until the date of the event. Data were censored at the date of the last follow up if there was no event. Using the log-rank test, 5-year survival curves were compared at the 95% confidence level. Mannitou staining intensity was correlated by Spearman test to FIGO stage, tumor mass (expressed as the peritoneal cancer index, PCI), tumor distribution (small bowel with/without carcinosis) and to the result of surgery (macroscopically tumor-free/residual disease).
Results
General patient characteristics. A total of 176 patients with high-grade serous epithelial ovarian cancer were included in this study. The median PCI was 13 (range=2-30). Five-year OS was 100% in stage I, 85.71% in stage II, 42.68% in stage III and 19.64% in stage IV. All general patient characteristics are depicted in Table I.
General patient characteristics.
Staining results. In 146 patients, there was a cytoplasmic staining, in two patients there was cell membrane staining and in three patients there was a cell membrane and cytoplasmic staining. All eight sets of scoring methods described in the methods section were applied. Details are depicted in Table II. Examples of staining results are depicted in Figure 1.
Detailed staining results according to the applied scoring method.
Representative immunohistochemical staining for paucimannose in high-grade serous ovarian cancer using the Mannitou antibody. (A, B) Negative controls. (A) Tumor cells only: Pretreatment with 0.5 M α-methyl-mannoside abolished Mannitou staining (magnification ×400). (B) Tumor and immune cells: Pretreatment with 0.5 M α-methyl-mannoside abolished Mannitou staining (magnification ×200). (C, D) Immune cell staining patterns. (C) Scattered Mannitou-positive immune cells (magnification ×400). (D) Clustered Mannitou-positive immune cells (magnification ×200). (E) Tumor cell staining pattern. Hot-spot staining of >50% of tumor cells showing clustered Mannitou-positive tumor cells (magnification ×400).
Immune cell staining was associated with improved DFS and a trend toward improved OS. Only the hot spot staining of more than 50% of tumor cells could be correlated with a reduced OS. No effect on DFS was observed. None of the alternative scoring approaches were able to detect statistically significant survival differences (Table III).
Five-year overall survival (OS) and disease-free survival (DFS) according to different staining scores applied to the IgM Mannitou immunocytochemical staining.
None of the scoring methods was able to show an association with clinical prognostic factors such as tumor load measured by the PCI or FIGO stage, or tumor distribution or surgical outcome as detailed in Table IV.
Association of Mannitou staining with clinical factors.
Discussion
Generally, PMEs are present in high-grade serous ovarian cancer. The findings from this study indicate that PME presence in immune cells is linked to favorable outcomes, but their presence in tumor cells correlates with reduced survival. Previous research linked aberrant glycosylation to carcinogenesis decades ago. Up until today, it is still unclear whether these changes are the cause of cancer development or just a consequence of cancer development (20-23).
Paucimannose, referring to a group of N-linked glycans consisting of mannose1-3fucose0-1N-acetylglucosamine2, is a rare and uncommon type of glycosylation in humans. A potential association with cancer development was recognized just recently (8). In some tumor types, increased paucimannose levels in tumor cells were observed compared to matched normal tissues (8, 10, 12). As immunohistochemistry is the most economical and widely available method to analyze tumor materials in daily practice, we decided to screen for PME in high grade serous ovarian cancer using the research Mannitou antibody (13, 14). Generally, PMEs are linked to key cellular processes such as cell migration, invasion and proliferation. In vitro all of these processes are inhibited effectively by the Mannitou antibody (7).
Given the lack of standardized evaluation methods for immunohistochemical staining of PMEs using the Mannitou antibody, we applied various scoring systems to analyze our heterogeneous staining results. Among the patients in our cohort, tumor staining was cytoplasmic in 146 patients, membranous only in two patients and both cytoplasmic and membranous in three patients. No tumor cell staining was observed in 25 patients. In general, we observed no case of nuclear or extracellular PME-staining.
Only the hot spot scoring method was suitable to detect survival differences in our cohort (19). In detail, a hot spot staining of tumor cells of more than 50% was associated with a worse OS. Kaprio et al. linked elevated Paucimannose levels to a poor prognosis in advanced colorectal carcinoma (24). Therefore, we investigated a potential association between established clinical prognostic factors in high-grade serous ovarian cancer – such as tumor load, FIGO-Stage or tumor distribution – and the levels of paucimannose in tumor cells and immune cells. Furthermore, we included carcinomatosis of the small bowel which is considered a clinical marker for poor prognosis, indicating an aggressive tumor biology (25, 26). Unfortunately, we were not able to detect any significant association of paucimannosylation and clinical prognostic factors by applying the eight different scoring methods.
In prostate cancer, glycosylation gene signatures and glycosylation-specific antibodies seem to be closely associated with tumor biology and may serve as potential biomarkers for risk stratification (22). Generally, glycosylation contributes to many hallmarks of cancer such as avoiding immune destruction, promoting tumor inflammation and resistance to growth suppression (22). One example is the highly selective macrophage mannose receptor CD206, which plays a significant role within the innate immune system promoting tumor growth and metastases when recognizing glycosylated structures on tumor cells (23). In this context, our finding of a paucimannosidic epitope staining in immune cells in109 patients (61.93%) was very interesting. Especially its association with improved survival.
According to our findings in high-grade serous ovarian cancer, paucimannosylation in immune cells, defined by immunohistochemical staining, may be used as biomarker and indicator of a more favorable prognosis, while paucimannosylation of tumor cells is indicative of a poor prognosis.
However, the specific cell origin and specific function of PMEs has yet to be determined. Furthermore, the underlying mechanism of the differential presence of paucimannosylation in certain cells is still unknown. It is assumed that PMEs are present in and can be released from lysosomes or lysosome-like departments (11, 12). They may derive either from tumor cells or other surrounding cells such as immune cells. Further studies elucidating the specific cellular origin of PMEs are therefore of high interest and may contribute to obtaining further insights into tumor biology.
Our study has several limitations such as its retrospective design and the single center analysis. Nevertheless, as our study only focused on patients with high-grade serous ovarian cancer, a very homogenous cohort of patients was analyzed. Furthermore, this is the first study presenting a potential scoring method for the analysis of paucimannosylation in high-grade serous ovarian cancer using immunohistochemistry. In addition, this study is the first to demonstrate the cell-dependent prognostic implication of PMEs in high-grade ovarian cancer.
Conclusion
In conclusion, our data provides evidence that paucimannosylation is present in high-grade serous ovarian cancer and its prognostic impact seems to depend on its presence in a specific cell type. Our results indicate that PMEs in high-grade serous ovarian cancer may represent a prognostic biomarker. PMEs might serve as potential target for future studies to improve therapeutic strategies.
Acknowledgements
Mannitou hybridoma cells provided by Dr. Simone Diestel; Mannitou generation with the help of Yvonne Becker (both Institute of Nutrition and Food Sciences, University of Bonn, Bonn, 53127, Germany). Tissue slides preparation: Carsten Golletz and Christiane Esch (both Institute of Pathology, University Hospital Bonn).
Footnotes
Authors’ Contributions
Conceptualization: Sarah Foerster, Methodology: Michael Muders. Formal analysis: Eva K. Egger. Investigation: Sarah Föerster, Lina Adloff, Michael Muders, Thore Thiesler. Validation: Michael Muders. Writing Original Draft Preparation: Eva K. Egger. Writing-Review and Editing Preparation: Sarah Foerster, Milka Marinova, Alexander Mustea, Luzia A. Otten, Carolin Schröder; Michale Muders, Dominique Könsgen. Supervision: Michael Muders. All Authors read and approved of the final manuscript.
Conflicts of Interest
All Authors declare no conflicts of interest in relation to this study.
Funding
There was no external funding.
Artificial Intelligence (AI) Disclosure
No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.
- Received August 21, 2025.
- Revision received October 7, 2025.
- Accepted October 9, 2025.
- Copyright © 2026 The Author(s). Published by the International Institute of Anticancer Research.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
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