Oncogenic Activation of the Wnt/β-Catenin Signaling Pathway in Signet Ring Stromal Cell Tumor of the Ovary.

A 79-year-old woman with abnormal uterine bleeding was admitted to the Holycross Cancer Centre, Kielce, Poland. There was no relevant family medical history. The dilation and the curettage were negative for endocervical and endometrial pathology. A pelvic examination revealed no palpable masses. However, an ultrasound examination showed a slightly enlarged (5×3×4.5cm) uterus. There was no significant elevation of serum tumor markers [carcinoembyonic antigen (CEA), CA125, SCC, and CA19.9]. Dysfunctional uterine bleeding was diagnosed, and therefore, a total abdominal hysterectomy and bilateral salpingo-oophorectomy were performed. The patient has been well 11 months after the surgery.

The left ovary was slightly enlarged with a smooth and glistening outer surface and measured 3 × 1.5 × 1.5-cm. Sectioning revealed a poorly circumscribed, yellow-ish, firm, solid tumor that was 1.1 cm in diameter. No cystic, hemorrhagic, or necrotic foci were noted. A thin rim of ovarian parenchyma was present at the periphery of the tumor. The right ovary and the fallopian tubes were unremarkable. Microscopically, the tumor was circumscribed, but unencapsulated, surrounded by a rim of normal ovarian stroma of varying thickness. It showed a multinodular growth pattern composed of nests and trabeculae of tumor cells separated by spindled ovarian stromal cells with occasional focal stromal luteinization. The cellular nodules were primarily composed of ovoid to epithelioid cells without cellular atypia or mitotic figures. A large single cytoplasmic vacuole compressed and displaced the nucleus in a significant subset of tumor cells, creating a signet ring cell-like appearance. The nuclei were rather uniform, with slightly irregular contours and an open chromatin pattern ( Figs. 1A – C ). The cytoplasmic vacuoles of the signet ring cells were negative for PAS, alcian blue, mucine, and oil-red O. PAS-positive cytoplasmic hyaline globule-like structures were observed focally, and they remained PAS-positive after diastase treatment ( Fig. 1D ). Strong nuclear and cytoplasmic labeling for β-catenin and nuclear positivity for cyclin D1 was noticed in all tumor cells ( Fig. 2 ). The tumor cells were also uniformly positive for vimentin ( Fig. 2 ) and CD10, but were negative for keratins (CK7, CK20, AE1/AE3), calretinin, EMA, inhibin, α-SMA, and S-100 protein. Tumor cell nuclei expressed SF-1 and SRC-1 ( Fig. 2 ). Immunohistochemical studies for ER, PR, CD56, CD99, fibronectin, MYC, and CEA were negative. Ki-67/MIB1-labeling was lower than 1%. Analysis of NGS data revealed an in-frame deletion of 30 nucleotides (c.68_97del) in the glycogen serine kinase-3 beta (GSK3β) phosphorylation region in the tumor DNA ( Fig. 3 ). This deletion at the protein level would lead to a p.Ser23_Ser33delinsThr mutation with deletion of 10 amino acids, including 2 serine residues. In addition, the germline missense mutation c.2164G>A in the Janus kinase-3 gene ( JAK3 ) was identified. This mutation would lead to p.Val722Ile substitution at the protein level. NGS data were comfermed by Sanger sequencing ( Fig. 4 ).

Representative 4-μm-thick sections from FFPE blocks were used for histochemical and immunohistochemical studies. Periodic acid-Schiff (PAS) reaction with and without digestion, alcian blue, mucine, oil-red O, and silver staining were performed after the standard procedures. Immunohistochemistry was performed using a DAKO Autostainer Link 48 (Dako Denmark A/S, Glostrup, Denmark) or a Leica Bond-Max automatic immunostainer (Leica Microsystems Inc., Buffalo Grove, IL). The following antigenes were evaluated: β-catenin, CD10 (Neprilysin), CD56 (Neural Cell Adhesion Molecule), CD99 (singlechain type-1 glycoprotein), CEA (carcinoembyonic antigen), cyclin D1, cytokeratins (CK7, CK20, CKAE1/AE3), calretinin, CMYC, epithelial membrane antigen (EMA), fibronectin, inhibin, Ki-67/MIB-1, steroidogenic factor-1 (SF-1), α-smooth muscle actin (α-SMA), steroid receptor coactivator-1 (SRC-1), S100, vimentin and estrogen and progesterone receptors (ER, PR). Specific information regarding antibodies and conditions of immunohistochemical reactions are provided in the supplementary data (Supplemental Digital Content 1, http://links.lww.com/AIMM/A97.doc ). DNA was isolated from the FFPE tumor and the normal tissue as reported previously. 12 NGS of 50 oncogenes and tumor suppressor genes was performed using the Ion AmpliSeq Cancer Hotspot Panel v2 Kit (Life Technologies/Thermo Fisher Scientific, Waltham, MA) following the manufacturer instructions. The findings were verified by Sanger sequencing. Technical details of the NGS and the Sanger sequencing are provided in the supplementary data (Supplemental Digital Content 2, http://links.lww.com/AIMM/A98.doc ).

SRSCT of the ovary is a very rare benign neoplasm with characteristic morphologic, histochemical, and immunohistochemical features. The occurrence of a prominent signet ring cell change out of the context of carcinoma is a hallmark of SRSCT of the ovary. These signet ring cells can also intermingle with a fibroma-like spindle cell proliferation. A recent review of 12 cases of well-documented SRSCT suggested that SRSCT may originate from ovarian fibroma or directly from ovarian stroma. The latter variety occurred in significantly younger patients. 2 The tumor reported in this study lacked a fibroma-like component and developed in a 79-year-old patient. In the SRSCT reported in this study, NGS identified deletion (c.68_97del) in CTNNB1 exon 3 encoding the β-catenin GSK3β phosphorylation region. At the protein level, such a mutation would lead to the deletion of 2 serine residues (Ser23 and Ser33) critical for β-catenin phosphorylation, ubiquitination, and subsequent degradation by a dedicated cytoplasmic destruction complex. The failure of β-catenin degradation leads to its accumulation and nuclear transmigration, and drives oncogenic Wnt-signaling. 13 A similar CTNNB1 mutation, p.S23_S33del, has been reported previously in childhood hepatoblastoma and correlated with nuclear β-catenin accumulation and overexpression of target genes, cyclin D1, and fibronectin, but not MYC. 14 The SRSCT reported in this study revealed nuclear accumulation of both β-catenin and cyclin D1, indicating the activation of the Wnt-signaling pathway, although neither fibronectin nor MYC expression was seen, as reported previously upon mutation activation of β-catenin. 15 Previous studies have shown that CTNNB1 mutations trigger the activation of β-catenin signaling and lead to the oncogenic activation of cyclin D1 in different types of cancers. 13 In addition to β-catenin mutations, NGS detected the germline missense mutation c.2164G>A in JAK3 . This mutation would lead to p.Val722Ile substitution at the protein level. Recently, an association between JAK3 germline mutations and the development of renal clear cell carcinoma has been documented. 16 Although more cases of SRSCT of the ovary should be studied to understand the significance of the germline p.Val722Ile mutation, it is well documented that some of the mutations identified by NGS studies are not directly related to the tumorigenesis. Animal studies have shown that activated WNT/β-catenin signaling in Sertoli cells causes Sertoli cell tumorigenesis in mice. 17 CTNNB1 mutations (single-nucleotide substitutions in codons: 32, 33, 34, and 37), nuclear β-catenin accumulation, and cyclin D1 overexpression have been reported in Sertoli cell tumor of the testis. 18 Combining histologic, immunohistochemical, and molecular genetic features, SRSCT of the ovary may be related to Sertoli-Leydig cell-type sex-cord stromal tumors. An aberrant nuclear β-catenin expression and identical c.98C > G (Ser33Cys) CTNNB1 mutation were reported in 2 cases of microcystic stromal tumor (MCST), a recently described subtype of ovarian stromal tumors. Ovarian MCST is typically a larger mass-forming lesion characterized by macrocystic and microcystic, often hemorrhagic, changes, a solid growth pattern, and diffuse expression of vimentin, CD10, and the Wilms tumor 1 (WT1) protein. 19 The tumor described in this report showed no macroscopic or microscopic features suggesting a relationship to ovarian MCST. 20 Also, WT1 protein expression typically seen in MCST was not detected. Likewise, MCST SRSCT was strongly positive for CD10 and vimentin. CD10 and vimentin expression was documented in different types of ovarian stromal tumors, including Sertoli cell tumors, although ovarian fibromas remained negative for this marker. 21 The PAS-positive hyaline globule-like structures seen in this case were reported previously in 5 SRSCTs with fibromα-like areas. 2 Cytoplasmic hyaline globule-like structures have been reported in different types of cancers including renal carcinoma and oncocytoma, and malignant mullerian-mixed tumor. 14 , 22 More recently, heavy deposition of PAS-positive hyaline globules was identified in ovarian fibromas. 23 The biological origin of the hyaline globule-like structure in SRSCT is not well understood. They may represent secretory glycoprotein accumulated in the cytoplasm of the tumor cells. 3 , 4 The SRSCT reported in this study showed strong vimentin positivity and a lack of EMA, α-SMA, cytokeratin, and inhibin. Thus, the immunohistochemical profile of this tumor was similar to the one recounted recently for the archetypical first case of SRSCT of the ovary. 2 SF-1 and SRC-1 expression has not been extensively studied previously in SRSCT of the ovary. 2 Both SF-1 and SRC-1 were expressed in the SRSCT reported in this study. SF-1 and SRC-1 are regulators of endocrine development and functions. 24 , 25 Dysregulation of SF-1 has been linked to endometriosis and adrenocortical carcinoma, 26 whereas elevated expression of SRC-1 was reported in breast and prostate cancer. 25 Animal models documented SRC-1 expression in Sertoli, Leydig, and germ cells of mice and rat testis. 27 , 28 On the basis our immunohistochemical studies, both SF-1 and SRC-1 are often expressed in ovarian stromal tumors including fibroma/fibrothecoma, and Sertoli cell tumors, and therefore, signet ring cell stromal tumor could be viewed as an ovarian sex-cord stromal tumor related to these tumors. Signet ring cell transformation has also been reported in non-neoplastic stromal cells of serous cystadenofibroma and Brenner tumor. However, in these tumors, signet ring cells showed α-SMA and inhibin positivity, an immunophenotype not seen in authentic SRSCT. 2 In the case reported in this study, there was evidence of neither serous cystadenofibroma nor Brenner tumor. In addition, neither tumor is characterized by nuclear β-catenin accumulation (Markku Miettinen and Jerzy Lasota, unpublished data, 2015) or CTNNB1 mutation. 29 Oncogenic CTNNB1 mutation and nuclear accumulation of β-catenin similar to signet cell stromal tumor has been reported in metastatic signet ring cell carcinoma associated with endometrioid carcinoma. 30 However, the clinicopathologic and the immunohistochemical features of metastatic signet ring cell carcinoma differ significantly from SRSCT by its extensive keratin positivity. 31 In summary, this study reports a case of SRSCT of the ovary with CTNNB1 mutation and activation of the Wnt/β-catenin signaling pathway. Although more cases should be evaluated, it seems that SRSCT of the ovary belongs to the subset of stromal ovarian tumors, including MCST and Sertoli cell tumor driven by β-catenin oncogenic signaling. Also, this study shows that a search for genetic mutations underlying the tumorigenesis in archival FFPE material could be performed successfully using the NGS approach.