Identifying the Carcinogenic Mechanism of Malignant Struma Ovarii Using Whole-Exome Sequencing and DNA Methylation Analysis

Background: Since malignant struma ovarii is a very rare disease, its carcinogenic mechanism has not been elucidated. Here, we sought to identify the genetic lesions that may have led to the carcinogenesis of a rare case of malignant struma ovarii (follicular carcinoma) with peritoneal dissemination. Methods: DNA was extracted from the paraffin-embedded sections of normal uterine tissues and malignant struma ovarii for genetic analysis. Whole-exome sequencing and DNA methylation analysis were then performed. Results: Germline variants of RECQL4, CNTNAP2, and PRDM2, which are tumor-suppressor genes, were detected by whole-exome sequencing. Somatic uniparental disomy (UPD) was also observed in these three genes. Additionally, the methylation of FRMD6-AS2, SESN3, CYTL1, MIR4429, HIF3A, and ATP1B2, which are associated with tumor growth suppression, was detected by DNA methylation analysis. Conclusions: Somatic UPD and DNA methylation in tumor suppressor genes may be associated with the pathogenesis of malignant struma ovarii. To our knowledge, this is the first report of whole-exome sequencing and DNA methylation analysis in malignant struma ovarii. Genetic and DNA methylation analysis may help elucidate the mechanism of carcinogenesis in rare diseases and guide treatment decisions.


Introduction
Struma ovarii is defined as a mature cystic teratoma that contains more than 50% thyroid tissue. Struma ovarii accounts for approximately 5% of mature cystic teratomas [1]. The malignant transformation of struma ovarii occurs in only 5-10% of struma ovarii cases. Additionally, the frequency of distant metastases is very low, occurring in only 5-6% of cases [2,3]. Given its rarity, malignant struma ovarii's carcinogenic mechanism has not been elucidated and no standard treatment has been established. Surgery and 131 I therapy have been reported as the main treatment for malignant struma ovarii [4]. However, there have been few reports on the treatment of residual tumors after surgery and 131 I therapy.
Here, we report a case of malignant struma ovarii with peritoneal dissemination. The patient was treated with surgery, 131 I therapy, and chemotherapy. However, the peritoneal dissemination gradually increased. Therefore, genetic analysis was performed to elucidate the cause of malignant struma ovarii and provide the guidance for treatment.

Materials and Methods
This study was conducted according to the ethical standards of national and international guidelines as well as the Declaration of Helsinki and was approved by the institutional review board (Shimane University Hospital). Tumor specimens were collected after obtaining written consent from the patient with the approval of the Facility Ethical Committee (Shimane University Hospital; approval no. 2004-0381).
Genetic analysis was performed using the PleSSision exome ® test (MITSUBISHI SPACE SOFTWARE, Tokyo, Japan) [5]. Hypermutated and ultra-hypermutated statuses were defined as ≥10 and ≥100 SNVs/Mbp, respectively, as previously described [6]. For genetic analysis, DNA was extracted from the paraffin-embedded sections of normal uterine tissues and malignant struma ovarii. The annotation and curation of the sequencing data were performed using the bioinformatics pipeline GenomeJack (MITSUBISHI SPACE SOFTWARE).
We used the Infinium MethylationEPIC BeadChip ® (Illumine, San Diego, CA, USA) to analyze the methylation rates of normal uterine tissues and malignant struma ovarii. DNA methylation levels were expressed as β values in normal tissues and malignant struma ovarii. The β value ranges from 0 to 1, and approaches 1 as the methylation rate increases. The difference in methylation rate between normal tissues and malignant struma ovarii for the genes was obtained by the log ratio. The higher the methylation rate in malignant struma ovarii compared to that in the normal tissue, the higher the log ratio. We extracted the top 50 genes with large differences in methylation rates and picked up genes that may have been involved in tumor growth due to methylation changes in malignant struma ovarii.

Results
A 29-year-old unmarried, nulliparous woman visited a gynecological clinic due to irregular vaginal bleeding. The patient was referred to our hospital for further examination. She had undergone a laparoscopic left ovarian cystectomy 4 years prior to the visit to our hospital because of a mature cystic teratoma including thyroid tissue.
A multiple cystic mass was detected in the left ovary by transvaginal ultrasonography. To further investigate the left ovarian tumor, magnetic resonance imaging (MRI) and computed tomography (CT) were performed. MRI showed a solid component in the left ovarian tumor, and CT showed peritoneal dissemination ( Figure 1A,B). Ovarian cancer was suspected based on the medical examination and imaging findings. Therefore, open surgery was selected as the treatment.  Intraoperatively, the left ovarian tumor was approximately 7 cm in size and adhered to the rectum. Extensive peritoneal dissemination (max. 3 cm) was observed (Figure 2A,B). First, we removed the left ovary. The intraoperative pathological diagnosis of the tumor in the left ovary was malignant struma ovarii (follicular carcinoma). Since the patient elected to not preserve fertility, total hysterectomy, bilateral salpingo-oophorectomy, and omentectomy were performed. After the operation, >2 cm of peritoneal dissemination remained (suboptimal surgery).
First, we removed the left ovary. The intraoperative pathological diagnosis of the tumor in the left ovary was malignant struma ovarii (follicular carcinoma). Since the patient elected to not preserve fertility, total hysterectomy, bilateral salpingo-oophorectomy, and omentectomy were performed. After the operation, >2 cm of peritoneal dissemination remained (suboptimal surgery).  The pathological analysis of the resected tumor indicated that the histological type was a highly differentiated follicular carcinoma ( Figure 3). No findings of papillary carcinoma, such as psammoma body, intranuclear cytoplasmic inclusion, ground glass nuclei, or nuclear groove, were observed. Similar histology was observed on the posterior wall of the uterus, omentum, and mesentery. This case was diagnosed as Stage IIIC by the 2014 International Federation of Gynecology and Obstetrics (FIGO).

Curr. Issues Mol. Biol. 2023, 3, FOR PEER REVIEW
The pathological analysis of the resected tumor indicated that the histologic was a highly differentiated follicular carcinoma ( Figure 3). No findings of papillar noma, such as psammoma body, intranuclear cytoplasmic inclusion, ground glass or nuclear groove, were observed. Similar histology was observed on the posteri of the uterus, omentum, and mesentery. This case was diagnosed as Stage IIIC by t International Federation of Gynecology and Obstetrics (FIGO). Approximately 8% of patients with malignant struma ovarii are reported to h perthyroidism [7], but the patient was euthyroid (thyroid-stimulating hormo µ U/mL; free T3, 2.6 pg/mL; free T4, 1.4 ng/dL). Because the ovarian metastasis of cancer must be ruled out for the diagnosis of malignant struma ovarii, we exami removed thyroid gland but found no malignant findings. After thyroidectomy, 1 apy (100 mCi) was used to treat the residual tumor. After 131 I therapy, the periton Approximately 8% of patients with malignant struma ovarii are reported to have hyperthyroidism [7], but the patient was euthyroid (thyroid-stimulating hormone, 0.7 µU/mL; free T3, 2.6 pg/mL; free T4, 1.4 ng/dL). Because the ovarian metastasis of thyroid cancer must be ruled out for the diagnosis of malignant struma ovarii, we examined the removed thyroid gland but found no malignant findings. After thyroidectomy, 131 I therapy (100 mCi) was used to treat the residual tumor. After 131 I therapy, the peritoneal dissemination gradually increased. Therefore, chemotherapy (paclitaxel, carboplatin, and bevacizumab) was administered based on the treatment for the residual ovary tumor. However, the peritoneal dissemination increased slowly during chemotherapy.
Most cases of malignant struma ovarii are only treated with surgery and 131 I therapy, and few case reports have shown other types of treatment. Therefore, we performed the genetic and DNA methylation analyses to the identify genetic lesions that may have led to the carcinogenesis of malignant struma ovarii that was intractable even after surgery and 131 I therapy.
RecQ like heliccase4 (RECQL4), CNTNAP2, and PRDM2 variants, which are tumor suppressor genes, were detected by whole-exome sequencing. All three variants were germline variants and revealed a somatic uniparental disomy (somatic UPD). Variant positions and variant allele frequencies are shown in Table 1. However, no actionable variants and no copy number alterations were identified in the known oncogenes. The copy number plot was normal and therefore, the tumor was considered homologous recombination proficient (HRP) (Figure 4). Moreover, it was determined to be microsatellitestable given that the microsatellite instability was 3.82%. The tumor mutation rate was 1.73 SNVs/Mbp, indicating no tumor mutation burden. We also examined the mutation signatures of the malignant struma ovarii. The most prevalent mutational signature was signature 1, which has been found in all cancer types and in most cancer samples ( Figure 5).    The DNA methylation analysis detected the methylation of FRMD6-AS2, SESN CYTL1, MIR4429, HIF3A, and ATP1B2 in malignant struma ovarii ( Table 2). All thes genes were reportedly associated with tumor growth suppression; thus, it is possible tha the methylation of these genes was involved in the carcinogenesis of malignant strum ovarii [8][9][10][11][12][13][14].

Discussion
Malignant struma ovarii is a very rare disease, with only approximately 200 case re ports in the published literature. Only 8% of patients with malignant struma ovarii hav hyperthyroidism; thus, malignant struma ovarii is rarely diagnosed based on the symp toms of hyperthyroidism [7]. In addition, preoperative diagnosis is very difficult becaus the MRI findings of malignant struma ovarii are nonspecific. Therefore, malignant strum ovarii is usually diagnosed after surgery. Since malignant struma ovarii is a rare diseas its carcinogenic mechanism is unknown, and no standard treatment has been established The DNA methylation analysis detected the methylation of FRMD6-AS2, SESN3, CYTL1, MIR4429, HIF3A, and ATP1B2 in malignant struma ovarii ( Table 2). All these genes were reportedly associated with tumor growth suppression; thus, it is possible that the methylation of these genes was involved in the carcinogenesis of malignant struma ovarii [8][9][10][11][12][13][14].

Discussion
Malignant struma ovarii is a very rare disease, with only approximately 200 case reports in the published literature. Only 8% of patients with malignant struma ovarii have hyperthyroidism; thus, malignant struma ovarii is rarely diagnosed based on the symptoms of hyperthyroidism [7]. In addition, preoperative diagnosis is very difficult because the MRI findings of malignant struma ovarii are nonspecific. Therefore, malignant struma ovarii is usually diagnosed after surgery. Since malignant struma ovarii is a rare disease, its carcinogenic mechanism is unknown, and no standard treatment has been established. In case reports, surgery, thyroidectomy, and 131 I therapy are the most common treatments [4]. Here, we report a rare case of malignant struma ovarii with peritoneal dissemination. The patient was treated with surgery, 131 I therapy, and chemotherapy. However, peritoneal dissemination gradually increased. Therefore, we sought to identify the genetic lesions that may have led to the development of malignant struma ovarii, which can provide guidance for treatment. To that aim, we performed genetic and DNA methylation analyses. We detected the germline variants of RECQL4, CNTNAP2, and PRDM2, which are tumor suppressor genes. Furthermore, somatic UPD was observed in these three genes. In addition, the methylation of FRMD6-AS2, SESN3, CYTL1, MIR4429, HIF3A, and ATP1B2, which are associated with tumor growth suppression, was also detected. These findings suggest that somatic UPD and DNA methylation in tumor suppressor genes may be associated with the carcinogenesis of malignant struma ovarii.
Papillary carcinoma is the most common histological subtype of malignant struma ovarii, and follicular carcinoma, as seen in the present case, is rare [15]. Total thyroidectomy is recommended to confirm the diagnosis of malignant struma ovarii by excluding ovarian metastases from primary thyroid cancer to improve the effectiveness of 131 I therapy by allowing preferential uptake into residual struma or metastasis [16].
Previous reports have described different surgical procedures for malignant struma ovarii. In a population-level analysis of 68 patients, 33.0% of patients underwent unilateral oophorectomy, 28.6% underwent bilateral oophorectomy, 28.6% underwent oophorectomy and omentectomy, and 4.8% underwent debulking surgery [4]. The average age of the 68 patients was 43 years, and the overall survival rates at 5 and 10 years were 96.7% and 94.3%, respectively [4]. The 5-year and 10-year recurrent rates of the patients with MSO confined to the ovary were 27.1% and 35.2%, respectively [17]. Although the survival rate of malignant struma ovarii is relatively good, it is unclear whether conservative surgery is possible in young patients with peritoneal dissemination, as in the present case. The large tumor size (≥10 cm), adhesion to the surrounding organs, ascites (≥1 L), capsule rupture, stromal component higher than 80%, extensive papillary carcinoma, especially with solid components, necrosis, and more than 5 mitoses per 10 high-power fields have been reported as factors that predict malignancy [18]. Follicular cancer may be more susceptible to hematogenous metastasis than papillary cancer, as it is observed in primary thyroid carcinoma [19,20]. If more cases of malignant struma ovarii are accumulated, the prognosis can be predicted better, and treatment guidelines may be determined based on the prognosis prediction. Conservative surgery may be acceptable in cases without poor prognostic factors.
Very few studies have reported the treatment of malignant struma ovarii by means other than surgery and 131 I therapy. However, one case report described a patient with lung and bone metastases who survived more than 20 years after treatment with tegafur-uracil, paclitaxel/carboplatin, and oral etoposide [19]. In malignant tumors for which standard treatment has not been established or that are resistant to conventional treatments, genetic analysis using next-generation sequencing is now being used to guide treatment decision making. Associations between histological types and genetic variants were reported in primary thyroid carcinoma. BRAF V600E , RAS, and rearranged during transfection/papillary thyroid carcinoma (RET/PTC) variants are common in primary papillary thyroid carcinomas [21,22]. In primary PTCs, RET/PTC rearrangements correlate with young age, tumor morphology, and a high frequency of lymph node metastases [23]. BRAF variants have been reported to be associated with aging, advanced disease, classical papillary histology, and poor prognosis [24,25]. Conversely, in primary follicular thyroid carcinomas, RAS variants and PAX8/Peroxisome Proliferator-Activated receptor γ (PPARγ) fusions are found in approximately 80% of cases [26].
Primary PTC and malignant struma ovarii with papillary carcinoma have similar genetic variants and may share a mechanism of carcinogenesis [31]. BRAF variants in primary PTC were reported to overexpress cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and programmed cell death ligand-1 (PD-L1), which are responsible for immunosuppression [34,35]. These data may provide the rationale for immunotherapy in PTC originating from struma ovarii. However, the follicular carcinomas of malignant struma ovarii are rarer than papillary carcinomas; thus, there are only a few reports of the genetic analyses of these tumors. No major oncogenic gene variants, including KRAS, BRAF, P53, or PAX8/PPARγ gene fusions, have been found in the genetic analysis of follicular carcinoma originating from struma ovarii [36]. These cases suggest that the mechanism of carcinogenesis differs between the primary follicular thyroid carcinoma and follicular carcinoma originating from malignant struma ovarii [36]. To our knowledge, this is the first report of whole-exome sequencing and DNA methylation analysis in malignant struma ovarii with follicular carcinoma. RECQL4, CNTNAP2, and PR/SET domain 2 (PRDM2) variants (somatic UPD) were detected by whole-exome sequencing. RECQL4 is a DNA helicase that belongs to the RecQ helicase family. DNA helicases unwind double-stranded DNA into single-stranded DNA and regulate chromosome segregation. Therefore, RECQL4 is deeply involved in maintaining genome stability and is classified as a tumor suppressor gene [37]. CNTNAP2 encodes a member of the neurexin family that functions as a cell adhesion molecule and receptor. CNTNAP2 was reported to be an independent prognostic factor, with a high CNTNAP2 mRNA expression level associated with a good prognosis in glioma. Therefore, CNTNAP2 is also considered a tumor suppressor gene [38]. PRDM2 is a member of a nuclear histone/protein methyltransferase superfamily and a tumor suppressor gene. PRDM2 encodes a zinc finger protein that can bind to retinoblastoma protein, estrogen receptor. PRDM2 is involved in tumor suppression, and its expression is often silenced in many cancer types [39]. In this study, these three tumor suppressor genes exhibited somatic UPD, suggesting that they may be deeply involved in the development of malignant struma ovarii. DNA methylation analysis also revealed the methylation of multiple genes (FRMD6-AS2, SESN3, CYTL1, MIR4429, HIF3A, and ATP1B2) associated with tumor growth suppression. The methylation of these genes leads to tumor growth because DNA methylation inhibits transcription. Therefore, we believe that the methylation of these genes is related to the development of malignant struma ovarii.
Various analyses, such as transcriptome, methylome profiling, digital histopathology, and multiplexed immunofluorescence have been performed in high-grade serous ovarian carcinoma. The characteristics of high-grade serous carcinoma tumors have gradually become clear, leading to the discovery of new therapeutic strategies [40][41][42]. However, since malignant struma ovarii is a rare tumor, its carcinogenic mechanism remains unknown and no effective treatment has been established. Genetic and epigenomic analyses, such as those conducted in this study, will provide clues to clarify the carcinogenic mechanism of malignant struma ovarii.

Conclusions
This study is the first report of whole-exome sequencing and DNA methylation analysis in malignant struma ovarii. Genetic and DNA methylation analysis may help elucidate the mechanism of carcinogenesis in rare diseases and guide treatment decisions.
Author Contributions: H.Y. and K.N. drafted the manuscript. K.K., T.I., M.I., S.S., K.I. and S.R. performed the molecular genetic studies. K.N. participated in the study design. S.K. conceived the study, participated in its design and coordination, and helped in drafting the manuscript. All authors have read and agreed to the published version of the manuscript.