A newly identified allelic mutation c.110C>T in the CTNNB1 gene in diffuse adenomyosis among young women—a report of two cases

Introduction

Adenomyosis (AM) is a prevalent benign gynecological condition, which is characterized by invasion of the basal endometrium (glands and stroma) into the myometrium. This diagnosis involves ectopic endometrial glands and stroma situated at least 2.5 mm below the endometrial-myometrial junction, which often elicits hyperplasia and hypertrophy of surrounding smooth muscle cells (1,2). This condition arises when the typical boundary between the endometrial basal layer and the myometrium is compromised (3,4). The main symptoms include increased menstrual volume and prolonged menstruation, periodic and progressive aggravation of dysmenorrhea, sexual intercourse pain and infertility (5). At present, there is no effective treatment, which seriously affects the quality of life of women of reproductive age. Most women are diagnosed in the later stages and typically receive different medications to treat their symptoms or hormone-based drugs to control disease progression (6). For women with severe symptoms that do not respond to medication, invasive surgical interventions such as hysterectomy are often required (7). Lately, Wang et al. proposed that distal radial artery approach for uterine artery embolization is an effective way to treat AM and is a blessing for patients who want to preserve uterine fertility (8).

Based on the imaging findings, AM can be classified into two forms: diffuse and focal. Diffuse AM is marked by the widespread presence of multiple foci within the myometrium, whereas focal AM is characterized by isolated nodules of hypertrophic myometrium and ectopic endometrial tissue, comprising uterine adenomyoma and uterine cystic AM (9-11). The severity of AM can be classified according to Morphological Uterus Sonographic Assessment (MUSA) criteria (12) in terms of the percentage of myometrium affected (mild, <25%; moderate, 25–50%; severe, >50%) (13). Despite extensive research, the pathogenesis of AM remains poorly understood. The relationship between the extent of the disease and its clinical manifestations is unclear, which complicates efforts to establish standardized treatment protocols (14,15). Two main hypotheses have been proposed to explain the development of AM. The first suggests that tissue injury and subsequent repair in the endometrium lead to stromal invagination into the myometrium, potentially influenced by environmental factors. The second hypothesis suggests that metaplasia occurs in displaced embryonic pluripotent Mullerian remnants or stem cells. A key process implicated in AM is the epithelial-mesenchymal transition (15). Recent advances in molecular pathology have led to the identification of various molecular changes associated with AM (10,11).

Driver mutations in genes like KRAS, ARID1A, PIK3CA, and PPP2R1A have been identified in deep-infiltrating endometriotic epithelial cells (16). In AM, genetic alterations often involve activating mutations in PIK3CA, KRAS, PPP2R1A, and MED12, while ARID1A mutations lead to a loss of function (17,18). The whole-exome sequencing (WES) (17) research detected 134 unique synonymous and non-synonymous single-nucleotide variations (SNVs) in 31/51 (60.8%) AM cases, reinforcing the hypothesis that AM is a clonal disorder driven by somatic mutations. At the same time, SNVs in AM occurred at low frequencies, akin to those in concurrent endometriosis, but were notably less frequent than in concurrent ovarian cancer. Inoue et al. (17) further concluded that somatic KRAS mutations are critical genomic alterations associated with AM. Endometriosis samples from certain AM patients exhibited both KRAS and PIK3CA mutations, aligning with previously reported mutational profiles of endometriosis (7,16,18,19).

Constitutively activated mutant KRAS may induce a number of downstream pathways, including PI3K-PDK1-AKT and RAF-MEK1/2ERK1/2 (20,21). Both pathways favor cell proliferation and survival. So far, efforts to therapeutically target these pathways in cancers with KRAS mutations have been generally disappointing because of their complexity. Although more promising progress has been made in directly targeting KRAS proteins (21), the number of cases that have been subjected to mutational analysis is limited, it remains to be confirmed if KRAS mutations are important in the pathogenesis of AM or if the mutations merely represent clonal markers without biological consequences. Therefore, elucidating the potential mechanism of uterine AM increases the possibility of developing targeted therapies to improve the current situation of ineffective drug treatment. This is still an urgent problem that needs to be solved.

Uterine AM is most commonly observed in women during their fourth and fifth decades of life (3). However, with advancements in imaging technologies such as transvaginal ultrasound (TVS) and magnetic resonance imaging (MRI), an increasing number of young women are being diagnosed with uterine AM (9,22). In this report, we present diffuse AM in two young females and detected a rare genetic mutation site that has not been reported before. We present this article in accordance with the CARE reporting checklist (available at https://gpm.amegroups.com/article/view/10.21037/gpm-24-42/rc).

Case presentation

All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Helsinki Declaration and its subsequent amendments. Written informed consent for publication of this case report and accompanying images was not obtained from the patients or the relatives after all possible attempts were made. The article has been sufficiently anonymized to cause no harm to the patients or their families.

Case one

A 29-year-old female, G0P0, presented with recurrent episodes of contact bleeding in January 2021, without additional symptoms. In August 2021, the patient experienced menorrhagia, with menstrual bleeding 2–3 times the volume of a normal period, accompanied by blood clots. The bleeding lasted for over 10 days and was associated with mild abdominal pain. A gynecological ultrasound in September 2021 identified a faint echogenic area in the uterine cavity, measuring 9.2 cm × 7.4 cm × 9.5 cm, accompanied by multiple dark regions resembling bladder cysts, with the largest reaching 12 cm.

Surgical intervention was performed in November 2021, during which extensive necrotic tissue, resembling decayed fish, was discovered in the uterine cavity. Certain areas of the myometrium exhibited nodularity, with necrotic tissue extending through more than half of the muscular layer.

Histopathological examination revealed a polypoid tumor within the uterine cavity. The tumor consisted of sparse glands, some of which showed dilation. The spiral arterioles in the stroma were densely proliferated (Figure 1). Endometrial glands and stroma were diffusely distributed within the myometrium, with certain glands situated about 4 mm from the serosa. Pathological diagnosis of diffuse AM. The surgery alleviated the symptoms and improved the quality of life. Up to now, no other adverse symptoms have been observed during follow-up.

Figure 1 Histologic features of diffuse adenomyosis in a young woman. (A) The intrauterine growths are all polypoid growths. (B) Diffuse endometrial glands and stroma can be seen between the uterine muscle walls, with some glandular cavities expanding. (C,D) Adenomyosis of the uterine wall invades the vasculature, including glands and stroma (hematoxylin-eosin stain, magnification: 100×).

Case two

A 26-year-old female, G0P0, had regular menstruation in the past. 4+ years ago, the menstruation increased to three times its previous level, without accompanying symptoms. An ultrasound in October 2021 revealed a strong echo measuring 7.7 cm × 7.8 cm × 8.8 cm within the uterine cavity. Computed tomography (CT) imaging demonstrated a space-occupying lesion within the uterine cavity, measuring approximately 10.5 cm × 7.2 cm × 9 cm, with uneven density and enhancement, and an ill-defined border with the anterior uterine wall muscle. On December 6, 2021, a partial hysterectomy via hysteroscopy was performed. During the procedure, a tumor tissue approximately 8 cm in diameter was observed on the right uterine wall, accompanied by several small polypoid protrusions nearby.

The pathological examination identified multiple endometrial polyps, characterized by widespread endometrial glands and stroma within the uterine muscle walls. Some glands were enlarged, and the condition had compromised blood vessels. The deepest part was within 1 mm of the serosa. Pathological diagnosis of diffuse AM. During follow-up, no other adverse symptoms have been observed after the surgery.

Next-generation sequencing (NGS) revealed a CTNNB1 (EX3 c.110C>T p.S37F) gene mutation at the same site in both young patients with diffuse AM. No other gene mutations were detected, as summarized in Table 1. However, this gene mutation was not found in cases of focal AM in two ordinary patients of reproductive age (the ages of the two patients are 40 and 48 years old, respectively).

Immunohistochemical analysis of β-catenin showed positive cell-membrane staining. Immunofluorescence, as shown in Figure 2, verified that the positive staining was confined to the cell membrane.

Figure 2 β-catenin showed cell-membrane staining in immunohistochemical (A) and in situ immunofluorescence (magnification 100×) (B).

Discussion

For more than a century after AM was first described, the diagnostic gold standard was histological post-hysterectomy confirmation (6,23). Since then, advancements in TVS imaging and MRI have led to early diagnosis (6). In the two young patients we reported, abnormalities were detected through TVS examination and surgery was performed, followed by further diagnosis through histology. Due to their young age and over 50% of the uterine muscle layer being affected, these two patients were classified as severe AM according to MUSA criteria. To further investigate the molecular characteristics of this type of AM, we conducted NGS testing. The results show that both young women with extensive invasive AM exhibited a CTNNB1 (EX3 c.110C>T p.S37F) gene mutation at the same site, with no other gene mutations detected. This study is the first to identify the CTNNB1 p.S37F mutation in young AM patients. Surprisingly, immunohistochemical analysis of β-catenin showed only positive staining of the cell membrane, and immunofluorescence confirmed that positive staining was also limited to the cell membrane.

These two cases of AM related immune indicators, estrogen receptor (ER) and progesterone receptor (PR), both showed diffuse strong positivity. The finding raises the question of whether mutations in β-catenin signaling in AM are similarly associated with elevated estrogen levels.

β-catenin (CTNNB1) has dual functions in epithelial cells based on its intracellular location (24). It is crucial for cell differentiation and maintaining normal tissue architecture as part of the E-cadherin–catenin complex at the plasma membrane. In the nucleus, β-catenin serves as the main effector of the canonical Wnt signaling pathway, crucial for tissue differentiation in embryonic development. Typically, β-catenin associates with glycogen synthase kinase 3β (GSK-3β) and adenomatous polyposis coli (APC) to form a complex. GSK-3β phosphorylates β-catenin, targeting it for proteasomal degradation via ubiquitination, thereby sustaining low cytoplasmic β-catenin levels. Mutations in CTNNB1 exon 3 result in protein stabilization, accumulation in the cytoplasm and nucleus, and transcriptional activation via deoxyribonucleic acid (DNA) binding. Mutations in CTNNB1 or APC activate the Wnt/β-catenin pathway in adenocarcinomas, promoting tumorigenesis (24).

Recent studies (25,26) indicate that estrogen-induced proliferation in the endometrium is linked to activated Wnt signaling, as estrogen stimulates elements of the Wnt pathway. Nuclear β-catenin expression, indicative of canonical Wnt signaling, is present in up to 30% of estrogen-related cancers. Alterations in the Wnt/β-catenin pathway may cause abnormal activation of target genes, such as those promoting epithelial-mesenchymal transition (EMT). Oh et al. (27) utilized the PRcre/+ Ctnnb1 (ex3)/+ mouse model and human samples to highlight β-catenin activation’s role in AM. The study revealed that β-catenin activation triggers SNAIL and ZEB1 expression and suppresses E-cadherin expression, facilitating mesenchymal marker expression in certain epithelial cells during AM development.

The two primary mechanisms of β-catenin dysregulation in disease are: (I) hotspot mutations in CTNNB1, and (II) downregulation of β-catenin degradation. Most mutations identified in CTNNB1 occur as single missense mutations in exon 3, affecting serine/threonine residues, particularly at positions 33, 37, 41, and 45, as well as 32 and 34. Of these, residues 33, 37, and 41 are phosphorylation sites for GSK-3β, which are critical for initiating β-catenin degradation, while residue 44 is a phosphorylation site for casein kinase 1. These phosphorylation events are necessary to regulate β-catenin degradation (28,29).

This study is the first to identify the CTNNB1 mutation in young AM patients. Only two patients were included in our study, mainly because this kind of diffuse and extensive AM in young female patients is very rare. Although the number of cases is limited, this study describes the clinical phenotypic characteristics of CTNNB1 mutations and AM for the first time, providing a new perspective for subsequent genotype-phenotype association studies. Previous research, Polakis (30) has extensively summarized CTNNB1 mutation sites and rates in human cancers. The β-catenin S37F mutation activates Wnt signaling in various melanoma cell lines, resulting in nuclear β-catenin accumulation, protein stabilization, and tumorigenesis. Benign tumors like desmoid-type fibromatosis are linked to CTNNB1 mutations and abnormal nuclear β-catenin expression (31).

Under normal physiological conditions, β-catenin is mainly found at the cell membrane, with minimal free cytoplasmic β-catenin due to regulation by the destruction complex. When CTNNB1 is mutated or upstream regulatory genes are altered, β-catenin can accumulate in the cytoplasm and nucleus, reflecting abnormal signaling (31). Despite the discovery of point mutations in β-catenin in the two young patients with extensive invasive AM, it is only expressed on the cell membrane and not in the cytoplasm and nucleus. Although we believe that this result is not accidental, further research is needed to elucidate the specific underlying mechanisms. We will continue to monitor such cases and hope to confirm the role of this gene mutation in the pathogenesis through larger cohorts, functional studies, and broader genetic analysis.

Conclusions

It is rare for young women to develop extensive and diffuse AM, and the pathogenesis remains unclear. This study is the inaugural report of a CTNNB1 gene mutation in this type of AM. It is hoped that the new discovery will provide a new perspective for the molecular pathology and pathogenesis research of AM. However, the data on this are not conclusive, and more rigorous research employing new technologies with a larger case cohort will be needed to validate this mutation and explore its role in AM.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the CARE reporting checklist. Available at https://gpm.amegroups.com/article/view/10.21037/gpm-24-42/rc

Peer Review File: Available at https://gpm.amegroups.com/article/view/10.21037/gpm-24-42/prf

Funding: This study was supported by the Key Research and Development Project of Cadre Health Care in Sichuan Province Research on Diagnostic Strategy and Clinicopathological Study of Endometrial Cancer Molecular Typing (No. ZH2023-1701).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://gpm.amegroups.com/article/view/10.21037/gpm-24-42/coif). W.W. serves as an unpaid editorial board member of Gynecology and Pelvic Medicine from May 2023 to December 2025. All authors report support from Key Research and Development Project of Cadre Health Care in Sichuan Province Research on Diagnostic Strategy and Clinicopathological Study of Endometrial Cancer Molecular Typing (No. ZH2023-1701). The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Helsinki Declaration and its subsequent amendments. Written informed consent for publication of this case report and accompanying images was not obtained from the patients or the relatives after all possible attempts were made. The article has been sufficiently anonymized to cause no harm to the patients or their families.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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