Two Cases of Steroid Cell Tumour, Not Otherwise Specified, of the Ovary with Distinct Exon 3 CTNNB1 Hotspot Mutations (p.D32V and p.S45P): Further Evidence of Wnt/β-Catenin-Pathway Involvement

Abstract

Background and Clinical Significance: Steroid cell tumours of the ovary, not otherwise specified (SCT-NOSs), are rare sex cord–stromal neoplasms with a poorly characterised molecular landscape, in which only exceptional CTNNB1 mutations have so far been reported and no recurrent driver alteration is firmly established. A better characterisation of their molecular spectrum has clinical significance for accurate diagnostic categorisation of ovarian sex cord–stromal tumours and for the identification of potentially targetable pathway alterations in this rare entity. Case Presentation: We report two consecutive SCT-NOSs of the right ovary, retrieved from the archives of the Department of Pathology of the Hôpital Universitaire de Bruxelles and of Curepath. Both underwent comprehensive sex cord–stromal and differential immunohistochemistry and targeted next-generation sequencing on a 168-gene panel with a mean coverage of 2690× (Case 1) and a 17-gene panel (Case 2) (MGI DNBSEQ-T7 for Case 1; Ion GeneStudio S5 for Case 2). A 56-year-old post-menopausal woman (Case 1) and a 50-year-old immunosuppressed woman with a history of renal transplantation and lymphoma (Case 2) both presented with rapidly progressive virilisation. The two right ovarian tumours (20 to 25 mm, no Reinke crystals) displayed an unambiguous sex cord–stromal immunophenotype (α-inhibin, calretinin, SF-1 and Melan-A positive; CD10, WT1, EMA, AE1/AE3 and PAX8 negative), with aberrant nuclear and cytoplasmic β-catenin staining. Sequencing identified a pathogenic CTNNB1 c.133T>C p.(Ser45Pro) variant in Case 1 and a pathogenic CTNNB1 c.95A>T p.(Asp32Val) variant in Case 2, with wild-type FOXL2 in both. Conclusions: Three of the four molecularly characterised CTNNB1-mutated SCT-NOSs converge on the two principal GSK-3β phosphorylation residues of β-catenin, identifying Wnt/β-catenin-pathway dysregulation as a potentially recurrent event and providing additional evidence for the involvement of the Wnt/β-catenin pathway in an emerging molecular subset of SCT-NOS. In a tumour with the canonical sex cord–stromal immunophenotype, an exon 3 CTNNB1 hotspot mutation should not be regarded as evidence against the diagnosis of SCT-NOS and may help define a distinct molecular subset.

1. Introduction

Steroid cell tumours of the ovary, not otherwise specified (SCT-NOSs), are rare pure stromal neoplasms within the sex cord–stromal category, with fewer than one thousand cases reported worldwide and an estimated incidence below 0.1% of all ovarian neoplasms [1,2]. In their seminal clinicopathological analysis of 63 cases, Hayes and Scully introduced the NOS descriptor for steroid cell tumours that lack the topographic and morphological features of either stromal luteoma or Leydig cell tumours [1]. The disease occurs at any age but is most often diagnosed between the second and fifth decade, and the most frequent presentation is virilisation, observed in approximately 41% of patients in the original series; oestrogenic manifestations and Cushing’s syndrome occur less commonly [1,2].

Histologically, SCT-NOSs are composed of polygonal cells with abundant eosinophilic to vacuolated cytoplasm arranged in solid sheets, nests, or cords, without granulosa, Sertoli, or thecal differentiation, and characteristically without Reinke crystals. The diagnosis rests on a combination of morphology and a sex cord–stromal immunophenotype, with positive expression of α-inhibin, calretinin, and SF-1 (steroidogenic factor 1) in over 80% of cases, variable positivity for Melan-A, androgen receptor, and CD10, and absence of EMA expression [2]. Approximately one fifth of cases follow a malignant clinical course; the histopathological correlates of malignant behaviour proposed by Hayes and Scully include a mitotic count of two or more per 10 high-power fields, tumour necrosis, a greatest dimension of 7 cm or more, haemorrhage, and grade 2 or 3 nuclear atypia [1,2].

In contrast with other ovarian sex cord–stromal tumours, where recurrent driver alterations are well established—FOXL2 p.C134W in adult granulosa cell tumours, DICER1 hotspot mutations in Sertoli–Leydig cell tumours, and CTNNB1 exon 3 mutations in microcystic stromal tumours [3,4]—the molecular landscape of SCT-NOSs remains poorly defined. The first comprehensive next-generation sequencing series of SCT-NOSs, recently reported by Chao and colleagues in seven sequenced patients, identified an enrichment of hypoxia-pathway alterations (HIF1A, VHL, SDHB, SRC, IDH2, and FOXO4) with no CTNNB1 alteration featured among the identified drivers [5]. A complementary clinicopathological and molecular series of 25 SCT/Leydig cell tumours by Mendoza and colleagues, in which next-generation sequencing was performed on a subset of cases, identified a single tumour harbouring a CTNNB1 mutation [6], and two earlier reports had separately described Wnt-pathway dysregulation in single steroid cell tumours: a benign right ovarian SCT in a patient with familial adenomatous polyposis, in whom biallelic APC inactivation drove nuclear β-catenin accumulation [7], and a single case with a somatic CTNNB1 codon 32 missense mutation (p.D32Y), although that case displayed an atypical immunoprofile (α-inhibin, calretinin and Melan-A negative; CD10 and β-catenin nuclear positive) more reminiscent of microcystic stromal tumour [8]. To date, no recurrent CTNNB1 hotspot has been described in SCT-NOS with the canonical sex cord–stromal immunophenotype.

Here, we report two cases of steroid cell tumours, NOS, of the right ovary, both diagnosed and molecularly characterised at the Department of Pathology of the Hôpital Universitaire de Bruxelles and at Curepath, in which targeted next-generation sequencing identified two distinct exon 3 CTNNB1 hotspot mutations, at codon 32 and codon 45. The detailed clinical, morphological, immunohistochemical and molecular findings are presented in Section 2 and Section 3, together with a discussion of the differential diagnosis and of the molecular relationship of these cases with the previously reported CTNNB1-mutated SCT-NOSs and the broader Wnt/β-catenin-driven ovarian sex cord–stromal spectrum.

2. Case Presentation

2.1. Case 1

A 56-year-old post-menopausal woman (G1P1) was referred for the work-up of a rapidly progressive virilisation that had developed over the preceding six months. Clinical signs included androgenic alopecia, marked hirsutism involving the cheeks, chin, and upper lip, and clitoral hypertrophy (“mega-clitoris”). Her relevant medical history included autoimmune (Hashimoto) thyroiditis on stable levothyroxine substitution, a metabolic syndrome with a body mass index of 33 kg/m², endometriosis treated by hysteroscopic endometrectomy in May 2025, and chronic kidney disease stage G3a (estimated glomerular filtration rate 55 mL/min/1.73 m²)—the latter, together with a documented iodinated-contrast intolerance, accounting for the use of a non-contrast computed tomography scan in the work-up detailed below. She was not taking any androgenic medication.

A formal modified Ferriman–Gallwey (mFG) score was not calculated at initial presentation in either patient. It should be noted that in rapidly progressive virilising ovarian tumours such as SCT-NOSs, hirsutism is often anatomically restricted (e.g., facial hirsutism confined to the upper lip, temples, chin or cheeks, in the present cases) rather than following a diffuse pattern, so that the mFG score—which requires scoring of nine defined body zones—may under-estimate the clinical severity of the androgen excess. Case 1 also presented androgenic alopecia and clitoromegaly, both of which are not captured by the mFG score.

The endocrine work-up demonstrated an isolated, severe ovarian hyperandrogenism. Total testosterone was 8.73 nmol/L (approximately twelve-fold the upper post-menopausal limit), bioavailable testosterone 3.528 nmol/L, free androgen index 22.1%, androstenedione 6.10 nmol/L, and dehydroepiandrosterone sulphate (DHEA-S) 1.79 µmol/L, the latter remaining within the lower normal range and effectively excluding an adrenal source. Luteinising hormone (11.6 IU/L), follicle-stimulating hormone (25.8 IU/L) and oestradiol (51.7 ng/L) were consistent with the post-menopausal status. Plasma cortisol, 24 h urinary free cortisol and 17-hydroxyprogesterone were within normal limits, ruling out hypercortisolism and a 21-hydroxylase enzymatic block. Serum CA 125 was 11 U/mL and inhibin B was below 10 ng/L. Transvaginal ultrasonography (December 2025) showed an anteverted uterus with a thin endometrium and a stable anterior fibroid but only marginally visualised ovaries, with no clearly identifiable adnexal mass. A non-contrast abdomino-pelvic computed tomography (CT) scan (November 2025), performed without intravenous contrast in view of the iodine allergy, demonstrated no adrenal mass, a right adnexal area measuring 26 × 30 mm, a left ovary obscured by adjacent bowel loops, the clinically suspected clitoromegaly, and no ascites or peritoneal carcinomatosis.

Serum androgens (total testosterone, androstenedione, and dehydroepiandrosterone sulphate) were measured by the standard chemiluminescent immunoassay methods routinely used at the local reference laboratories. Calculated free testosterone and bioavailable testosterone were derived from total testosterone and SHBG according to the Vermeulen equation, using a standard serum albumin concentration of 43 g/L.

The patient underwent a bilateral salpingo-oophorectomy by laparoscopy in February 2026, complemented by indirect peritoneal cytology. The procedure was uneventful and the patient was discharged on the first post-operative day. Peritoneal cytology was negative for neoplastic cells.

Macroscopically, the right ovary measured 40 × 25 × 25 mm with an intact capsule and contained a well-circumscribed solid yellow-orange tumour. The right fallopian tube measured 70 × 4 mm. The left ovary (28 × 14 × 11 mm) showed no macroscopic lesion, and the left fallopian tube (75 × 5 mm) was haemorrhagic and contained a 0.2 to 0.4 cm paratubal cyst.

On microscopic examination (Figure 1), the right ovarian tumour was well-circumscribed and located within the ovarian cortex. At low magnification, it consisted of a proliferation arranged in nests and cords of tumour cells, with scant intercellular stroma. At higher magnification, the cells were polygonal, with abundant finely granular eosinophilic to focally vacuolated cytoplasm and well-defined membranous outlines; the nuclei were round and central, with prominent nucleoli. No Reinke crystals were identified. Mitotic activity was low (one mitosis per 10 high-power fields), with only rare focal mild atypia. No tumour necrosis, no haemorrhage and no lymphovascular invasion were identified; the capsule was intact and the excision was complete.

Immunohistochemistry was performed on 4 µm formalin-fixed paraffin-embedded sections on an automated immunostaining platform, using the following validated antibody clones: α-inhibin (clone R1, Dako/Agilent, Santa Clara, CA, USA), calretinin (DAK-Calret 1, Dako/Agilent), SF-1 (EP434, Cell Marque, Rocklin, CA, USA), Melan-A (A103, Dako/Agilent), CD10 (56C6, Dako/Agilent), β-catenin (β-catenin-1, Dako/Agilent), WT1 (6F-H2, Roche/Ventana, Tucson, AZ, USA), PAX8 (MRQ-50, Roche/Ventana), EMA (E29, Dako/Agilent), AE1/AE3 (AE1/AE3, Dako/Agilent), E-cadherin (NCH-38, Dako/Agilent), ER (SP1, Roche/Ventana), PR (PgR 1294, Dako/Agilent), AR (SP107, Roche/Ventana) and Ki-67 (MIB-1, Dako/Agilent). The tumour cells showed strong, diffuse positivity for α-inhibin (×20), strong diffuse positivity for calretinin (low- and high-power confirmation at ×4 and ×40), moderate nuclear positivity for steroidogenic factor 1 (SF-1, ×20), and positivity for Melan-A. The androgen receptor was moderately positive with an Allred score of 6 (intensity 4 + proportion 2). The Ki-67 proliferation index was below 10%. β-catenin showed an aberrant pattern combining membranous, cytoplasmic and focal nuclear staining, the latter involving more than 30% of tumour cells (×20). Conversely, epithelial membrane antigen (EMA), CD10, E-cadherin and AE1/AE3 were negative.

Next-generation sequencing was performed at the H.U.B. Department of Molecular Pathology on a 168-gene panel (BELAC accreditation B-727 MED) using the MGI DNBSEQ-T7 platform on macrodissected tumour material with 70% tumour cellularity and a mean overall coverage of 2690×. Three findings were reported: a pathogenic CTNNB1 exon 3 hotspot mutation, NM_001904.4:c.133T>C, p.(Ser45Pro), at a variant allele frequency (VAF) of 29% with a variant coverage of 1047×; a KMT2D variant, c.12889T>C, p.(Ser4297Pro), at a VAF of 48%, classified as a variant of unknown significance; and a wild-type FOXL2.

The mean overall coverage of the panel was 2690× (above the 1500× minimum required by the assay for reliable variant reporting), the sequencing depth at the CTNNB1 variant position was 1047× (variant allele frequency 29%), and the tumour purity (estimated on the H&E slide adjacent to the macrodissected block) was 70%. The assay reliably detects single-nucleotide variants at a variant allele frequency of at least 5%.

The final integrated diagnosis was a steroid cell tumour of the right ovary, not otherwise specified, harbouring a pathogenic CTNNB1 exon 3 hotspot mutation at codon 45 (p.Ser45Pro), with no histopathological criterion of malignant behaviour. Endocrine follow-up at approximately two months post-operatively (April 2026) confirmed a complete normalisation of the androgen profile, with total testosterone falling from 8.73 to 0.31 nmol/L, calculated free testosterone from 0.151 to 0.008 nmol/L, bioavailable testosterone from 3.528 to 0.096 nmol/L, and the free androgen index from 22.1% to 0.6%—corresponding to a roughly 30-fold decrease for the main androgenic parameters. Clinically, the patient reported a clear improvement of the virilising symptoms and remains under endocrine follow-up.

2.2. Case 2

A 50-year-old post-menopausal woman (G4P4, menopause approximately three years before presentation) was referred for the work-up of newly developed hirsutism involving the upper lip and the temples. Her past medical history was particularly noteworthy. She had undergone a renal transplantation in 2007 for a nephropathy of unknown origin and had been receiving long-term immunosuppressive therapy since that time. In 2017, she was diagnosed with a high-grade diffuse large B-cell lymphoma, which was treated by single-agent rituximab followed by R-CHOP polychemotherapy; complete remission was achieved and maintained at the time of the current presentation.

At the time of presentation, she was not receiving hormone replacement therapy, and her ongoing medication regimen (related to her renal transplantation and associated comorbidities) did not include any drug known to interfere with androgen metabolism.

The endocrine work-up similarly demonstrated an isolated ovarian hyperandrogenism. Total testosterone was 3.81 nmol/L (laboratory reference range 0.4–1.42 nmol/L), calculated free testosterone 76.20 pmol/L (reference 3–37 pmol/L), and androstenedione 5.7 ng/mL (reference 0.1–3 ng/mL). DHEA-S was decreased at 0.55 µmol/L (reference 0.96–6.95 µmol/L), again effectively excluding an adrenal source. Luteinising hormone was mildly elevated at 63.7 IU/L, follicle-stimulating hormone was within the post-menopausal range at 98.5 IU/L, and prolactin was normal at 14.3 µg/L. Pelvic imaging identified a right adnexal mass measuring 30 mm in its greatest dimension, located high in the pelvis at the level of the renal allograft hilum, in keeping with the surgically anteposed position of the right adnexa imposed by the prior transplantation.

Serum androgens were measured using the same chemiluminescent immunoassay methodology as described for Case 1 (Section 2.1), with calculated free and bioavailable testosterone derived from the Vermeulen equation.

Given the patient’s age and post-menopausal status, the multidisciplinary tumour board recommended a total hysterectomy with bilateral salpingo-oophorectomy, complemented by a parietal peritoneal biopsy and a peritoneal washing for cytology. The procedure was performed by laparoscopy in September 2022, without intra-operative complications.

Macroscopically (Figure 2), the right ovary harboured a well-circumscribed nodular tumour measuring 25 × 22 mm, with a uniform yellow-brown cut surface. The ovarian capsule was intact, allowing a complete excision.

Microscopically, the tumour was well-circumscribed and located within the ovarian cortex. At low magnification, it consisted of a proliferation arranged in nests and cords of tumour cells, with scant intercellular stroma. At higher magnification, the cells were polygonal, with abundant finely granular eosinophilic cytoplasm and well-defined membranous outlines; the nuclei were round and central, with prominent nucleoli. Mitotic activity was extremely low (<2 mitoses per 10 high-power fields at ×40 magnification), with only rare focal mild atypia. No Reinke crystals, calcifications, necrosis, haemorrhage or lymphovascular invasion were identified, the capsule was not breached, and the excision was complete.

Immunohistochemistry, performed on 4 µm formalin-fixed paraffin-embedded sections on an automated immunostaining platform using the same validated antibody clones as for Case 1, confirmed a complete sex cord–stromal phenotype, with positivity for calretinin, α-inhibin, CD56, Melan-A and SF-1. Critically, CD10, WT1, EMA, AE1/AE3 and PAX8 were all negative, effectively excluding microcystic stromal tumour, müllerian-derived carcinomas including the sex cord-like variants of endometrioid carcinoma, and female adnexal tumour of probable Wolffian origin from the differential diagnosis. The progesterone receptor was positive with an Allred score of 6 (intensity 4 + proportion 2), whereas oestrogen and androgen receptors were negative. The Ki-67 proliferation index was approximately 10%.

β-catenin immunohistochemistry showed an aberrant staining pattern combining strong membranous and cytoplasmic positivity throughout the tumour cells, with focal nuclear positivity in scattered tumour cells, consistent with cytoplasmic accumulation and partial nuclear translocation of β-catenin (Figure 3f).

The pathological examination of the remainder of the surgical specimen revealed several incidental findings of interest. The contralateral left ovary displayed stromal hyperthecosis associated with Leydig cell hyperplasia, indicating a more diffuse hyperandrogenic stromal background on the non-tumoural side. A small 5 mm serous adenofibroma was identified in the left fallopian tube. and a 10 mm endometrial polyp was present at the uterine fundus, on a quiescent endometrium without hyperplasia or malignancy. Diffuse uterine adenomyosis was noted; the exo- and endocervix were unremarkable. The right parietal peritoneal biopsy showed only mild, non-specific inflammatory changes, and the peritoneal washing was negative for neoplastic cells.

Targeted next-generation sequencing was performed at the Department of Molecular Pathology of the Hôpital Universitaire de Bruxelles (H.U.B.)—Erasme site, using a 17-gene panel tailored to ovarian, endometrial and breast tumours, on the Ion GeneStudio S5 platform (Ion Torrent) with AmpliSeq library preparation, on macrodissected tumour material containing 70% tumour cells. The analysis identified a single pathogenic exon 3 hotspot mutation of CTNNB1, p.(Asp32Val) (corresponding DNA notation c.95A>T on transcript NM_001904.3), at a variant allele frequency of 27% with a variant coverage of 1162×, classified at the time of reporting as a variant of unknown clinical impact. FOXL2 and DICER1 were wild-type, strongly arguing against adult granulosa cell tumour and Sertoli–Leydig cell tumour on molecular grounds. The validated report explicitly referenced the single previously published CTNNB1-mutated SCT-NOS case [8].

The sequencing depth at the CTNNB1 variant position was 1162× (variant allele frequency 27%), and the tumour purity was estimated at 70% on the H&E slide adjacent to the macrodissected block. The Ion Torrent AmpliSeq assay reliably detects single-nucleotide variants at a variant allele frequency above 4%; only variants reported in the COSMIC database with a variant allele frequency above 4% and a variant coverage above 30× are included in the final report.

The final integrated diagnosis was a steroid cell tumour of the right ovary, not otherwise specified, harbouring a pathogenic CTNNB1 exon 3 hotspot mutation at codon 32 (p.Asp32Val), in a patient with prior renal transplantation and ongoing chronic immunosuppression. Endocrine follow-up at one month post-operatively confirmed a complete normalisation of the androgen profile, with total testosterone falling below the laboratory detection threshold of 0.42 nmol/L, calculated free testosterone below 7 pmol/L, and a normal sex hormone–binding globulin of 36.6 nmol/L. Clinically, the patient reported a clear improvement of the virilising symptoms; she remains under endocrine and oncological follow-up, in the ongoing context of chronic post-transplant immunosuppression.

2.3. Comparative Immunohistochemical and Molecular Summary

The comparative immunohistochemical, histopathological and molecular findings of Case 1 and Case 2 are summarised in

Table 1. Comparative immunohistochemical, histopathological and molecular findings of the two cases.

Marker/Feature	Case 1 (p.Ser45Pro, 2026)	Case 2 (p.Asp32Val, 2022)
Sex cord–stromal markers
α-inhibin	+ diffuse strong	+
Calretinin	+ diffuse strong	+
SF-1	+ nuclear	+
Melan-A	+	+
CD56	—	+
Differential-diagnosis markers
CD10	−	−
WT1	—	−
EMA	−	−
AE1/AE3	−	−
PAX8	—	−
Wnt-pathway marker
β-catenin	Aberrant: membranous, cytoplasmic and focal nuclear (>30% cells)	Aberrant: membranous, cytoplasmic and focal nuclear
Hormone receptors
Androgen receptor	+ (Allred 6)	−
Oestrogen receptor	—	−
Progesterone receptor	—	+ (Allred 6)
Proliferation
Ki-67 index	<10%	~10%
Histopathology
Tumour size	20 mm	25 mm
Mitoses	1/10 HPF	<2/10 HPF
Necrosis, haemorrhage, capsular breach, LVI	All absent	All absent
Reinke crystals	Absent	Absent
Molecular
NGS platform/panel	MGI DNBSEQ-T7/168-gene BrightCore panel (H.U.B., 2026)	Ion GeneStudio S5 (Ion Torrent), AmpliSeq, 17-gene panel (H.U.B.—Erasme site, 2022)
Tumour cellularity	70%	70%
CTNNB1 variant	c.133T>C, p.(Ser45Pro)—codon 45	c.95A>T, p.(Asp32Val)—codon 32
VAF/coverage	29%/1047×	27%/1162×
Other variants	KMT2D p.(Ser4297Pro), VOUS, VAF 48%	None reported
FOXL2	Wild-type	Wild-type
DICER1	Not specifically reported	Wild-type

“+”, positive; “−”, negative; “—”, not assessed. Abbreviations: HPF, high-power field; LVI, lymphovascular invasion; NGS, next-generation sequencing; VAF, variant allele frequency; VOUS, variant of unknown significance.

.

3. Discussion

To the best of our knowledge, the two cases reported here represent the second and third single-case descriptions of a CTNNB1-mutated steroid cell tumour, NOS, of the ovary with the canonical sex cord–stromal immunophenotype and add to the small but growing body of evidence that Wnt/β-catenin-pathway dysregulation, although uncommon, is a potentially recurrent molecular event in this entity. This claim is best appreciated against the backdrop of the molecular landscape of SCT-NOSs, which has long resisted systematic characterisation [2,9] despite the morphological recognition of the entity dating back to the seminal series of Hayes and Scully in 1987 [1]. The two largest sequencing efforts published to date—the seven-tumour series of Chao and colleagues [5] and the molecular sub-cohort of Mendoza et al. drawn from 25 SCT/Leydig cell tumours [6]—converged on a hypoxia-pathway signature (HIF1A, VHL, SDHB) and on a near-complete absence of CTNNB1 alterations among the highlighted findings, with only a single CTNNB1-mutated tumour identified in either dataset [6]. Together with the lone case report of Suzuki et al. (codon 32, p.D32Y, in a tumour with an atypical microcystic-stromal-like immunoprofile [8]) and the familial-adenomatous-polyposis-associated APC-inactivated SCT of Hu et al. [7], no more than three SCT-NOSs with documented Wnt-pathway dysregulation had been published prior to this report. Our two cases more than double this count and, taken together with Suzuki [8], show that three of the four molecularly characterised CTNNB1-mutated SCT-NOSs now converge on the two principal GSK-3β phosphorylation residues of β-catenin—codon 32 (p.D32Y, p.D32V) and codon 45 (p.S45P)—thereby complementing rather than competing with the hypoxia-associated signature of Chao [5].

Case 1 harboured CTNNB1 p.(Ser45Pro) and Case 2 harboured CTNNB1 p.(Asp32Val).

Mechanistically (Figure 4), exon 3 of CTNNB1 encodes the regulatory N-terminal domain of β-catenin, on which the casein kinase 1/GSK-3β tandem phosphorylates serine 33, serine 37, threonine 41 and serine 45, thereby earmarking β-catenin for proteasomal degradation via the β-TrCP/SCF ubiquitin ligase complex [10]. Missense substitutions at these residues—and at the immediately adjacent codon 32—disrupt the phosphorylation-destruction signal, allowing cytoplasmic accumulation, nuclear translocation and constitutive activation of TCF/LEF-driven transcription, with downstream upregulation of CCND1, MYC and LEF1 [10]. Crucially, both our cases displayed the corresponding β-catenin accumulation phenotype on immunohistochemistry (Figure 3e), with aberrant membranous, cytoplasmic and focal nuclear staining demonstrated in the tumour cells. This protein-level corroboration of the functional consequence of the codon 32 and codon 45 variants identified by next-generation sequencing is fully in keeping with the canonical mechanism.

Both variants affect the exon 3 β-catenin degradation motif: the codon 45 (p.S45P) variant disrupts a direct residue of the CK1/GSK-3β phosphorylation cluster, whereas the codon 32 (p.D32V) variant lies within the adjacent β-TrCP recognition motif required for β-catenin ubiquitination. In both cases, the resulting escape from proteasomal degradation leads to constitutive activation of Wnt/β-catenin signalling.

The uniform yellow-brown macroscopic colour of Case 2, well appreciated on the gross specimen (Figure 2), is most likely attributable to intracellular lipochrome pigment, which is a common feature of steroidogenically active ovarian lesions and reflects the intense endocrine activity of the tumour.

The differential diagnosis raised by a CTNNB1-mutated ovarian sex cord–stromal lesion has expanded considerably over the past two years (

Table 2. Synoptic differential diagnosis of ovarian SCT-NOS with the principal entities sharing morphological or molecular overlap.

Marker/Feature	SCT-NOS (Our Cases)	MCST	Sertoli–Leydig	Adult GCT	FATWO	Sex Cord–Like Endometrioid Ca
α-Inhibin	+ diffuse	− to focal +	+	usually + diffuse	− to focal +	−
Calretinin	+ diffuse	− to focal +	+	+	− to focal +	−
SF-1	+	−	+	+	−	−
Melan-A	+	−	+ (Leydig)	− to focal	−	−
CD10	−	usually + diffuse	− to focal +	+ variable	+ often	− to focal +
WT1	−	usually + diffuse	variable	usually + diffuse	variable	−
β-Catenin nuclear	+ focal (Case 1)	+ diffuse	−	−	+ (CTNNB1-mut cases)	+ (Lengyel 2026 [15])
EMA	−	−	−	−	+ often	+ variable
AE1/AE3	−	− to focal	− to focal	−	usually + diffuse	+
PAX8	−	−	−	−	− to focal	− (in sex cord-like variant)
FOXL2 p.C134W	wild-type	wild-type	wild-type	+ defining (>95%)	wild-type	wild-type
DICER1 hotspot	wild-type	wild-type	+ defining (~60%)	wild-type	wild-type	wild-type
CTNNB1 exon 3	+ (p.D32V and p.S45P)	+ defining (>80%)	wild-type	wild-type	+ rare	+ frequent (9/10 in Lengyel 2026)

Bold indicates the defining marker expression pattern for each entity. “+”, positive; “−”, negative; “+/−”, variable; “− to focal +”, negative to focal positive. Abbreviations: FATWO, female adnexal tumour of probable Wolffian origin; GCT, granulosa cell tumour; MCST, microcystic stromal tumour; SCT-NOS, steroid cell tumour, not otherwise specified.

). Microcystic stromal tumours remain the principal mimic—exon 3 CTNNB1 mutations are present in over 80% of cases [4,11,12], occasional APC-inactivated variants arise in the context of familial adenomatous polyposis [7,13], and the entity may relapse despite its conventionally benign reputation [14]. The defining MCST signature—CD10 diffuse, WT1 diffuse nuclear, β-catenin diffuse nuclear, with negative or focal α-inhibin, calretinin and SF-1—was unambiguously absent in both our cases. Two recently described mimics further complicate the picture: adnexal endometrioid carcinoma with sex-cord-like morphology, in which CTNNB1 activating variants are present in roughly nine of ten sequenced tumours and drive a PAX8-negative, SOX17-positive, β-catenin-nuclear immunoprofile [15], and the sertoliform variant of ovarian endometrioid carcinoma, in which the same CTNNB1 mechanism is being increasingly recognised [16]. Female adnexal tumours of probable Wolffian origin similarly harbour occasional exon 3 CTNNB1 mutations but are reliably set apart by their diffuse cytokeratin and EMA positivity [17]. The combined negativity for CD10, WT1, EMA, AE1/AE3 and PAX8 in both our cases, alongside the strong, diffuse positivity for α-inhibin, calretinin, SF-1 and Melan-A, strongly argues against each of these alternatives and, together with the negative Reinke crystals and canonical sex cord–stromal immunophenotype, supports the diagnosis of a SCT-NOS.

Marker expression is described here in qualitative terms based on the published literature; individual cases may deviate from these typical patterns.

The take-home message of Table 2 is straightforward. In the appropriate immunophenotypic context—α-inhibin and calretinin positive, CD10 and WT1 negative—an exon 3 CTNNB1 hotspot mutation should not be regarded as evidence against the diagnosis of SCT-NOS, even though the same alteration is the molecular hallmark of a microcystic stromal tumour [4]. Whether this potentially recurrent Wnt-pathway activation carries prognostic or therapeutic weight remains an open question. The two cases reported here display low mitotic activity, small tumour size, intact capsule and absence of necrosis or lymphovascular invasion—none of the Hayes–Scully criteria of malignant behaviour [1]—and both patients have achieved post-operative biochemical normalisation. Whether Wnt activation correlates with a more indolent course in SCT-NOS, or, conversely, whether it predisposes to local recurrence as it does in a subset of microcystic stromal tumours [14], can only be addressed by a larger collaborative series. The pharmacological dimension is equally pertinent: several Wnt-targeted inhibitors, including porcupine and tankyrase inhibitors, are currently being evaluated in clinical trials for tumours with constitutive β-catenin activation [10], opening a tentative—but not yet practice-changing—therapeutic perspective for the rare malignant SCT-NOS in which Wnt activation would be identified.

Our observations carry the usual limitations of a two-case report: the small sample precludes statistical inference, no functional validation of the two variants was performed, and no germline testing was undertaken to exclude an underlying FAP-spectrum predisposition. Notwithstanding, the systematic deployment of a sex cord–stromal-oriented next-generation sequencing panel in both cases, combined with the exhaustive differential immunohistochemistry summarised in Table 1 and Table 2, provides a sufficiently robust framework to anchor our conclusion.

The short duration of post-operative follow-up (approximately two months for Case 1) limits any firm conclusion regarding the long-term biological behaviour of these tumours; both patients remain under active endocrine and clinical follow-up.

Imaging and Case 1 macroscopic findings are reported in textual form only, based on the original radiology and pathology reports.

Further studies on a larger, multicentric series are needed to clarify whether Wnt-pathway activation correlates with clinical behaviour in SCT-NOSs and to evaluate the potential therapeutic relevance of Wnt-targeted inhibition in the rare malignant variants of this entity. In the meantime, we would advocate for the systematic inclusion of CTNNB1 in any targeted panel applied to ovarian sex cord–stromal lesions, the routine performance of β-catenin immunohistochemistry whenever a CTNNB1 variant is detected, and the constitution of a dedicated multicentric registry to consolidate the molecular spectrum of this rare entity.

4. Conclusions

In summary, we report two cases of steroid cell tumours, not otherwise specified, of the ovary harbouring distinct exon 3 CTNNB1 hotspot mutations—p.(Asp32Val) and p.(Ser45Pro)—both targeting the GSK-3β phosphorylation residues of β-catenin. Together with the previously published case of Suzuki et al. [8], they bring to three of four the molecularly characterised SCT-NOSs in which Wnt/β-catenin-pathway dysregulation converges on the codon 32 and codon 45 hotspots, identifying Wnt/β-catenin-pathway dysregulation as a potentially recurrent event and providing additional evidence for the involvement of the Wnt/β-catenin pathway in an emerging molecular subset of SCT-NOS. In a tumour exhibiting the canonical sex cord–stromal immunophenotype (α-inhibin, calretinin and SF-1 positive; CD10 and WT1 negative), the detection of an exon 3 CTNNB1 hotspot mutation should not be regarded as evidence against the diagnosis of an SCT-NOS but may rather help to define a distinct molecular subset of the entity, which deserves dedicated documentation in future multicentric series.