The patient was a 49-year-old woman, Each Asian race, gravidity three, parity one, who had attained menarche at the age of 12 and had regular 28-day menstrual cycles lasting for five days before entering menopause in 2012. At the age of 42, she was diagnosed with invasive ductal carcinoma of the left breast (pT2N0M0, stage IIA). Then, she underwent modified radical mastectomy for the left breast and received tamoxifen for five years post-surgery. In January 2018, the patient at the age of 49, a pelvic mass was found during a routine physical examination. The patient had no uncomfortable symptoms. Computed tomography showed a round-like, well-defined, low density, mildly enhancing lesion measuring 5.6 cm×3.9 cm×5.0 cm directly behind the uterus. The detection of tumor markers revealed that the CA125 level was 43.3 IU/mL, while β-HCG and other tumor marker levels were within the normal range.
This patient underwent subsequent laparoscopic surgery, including total hysterectomy, bilateral salpingo-oophorectomy, pelvic lymphadenectomy, paraaortic lymphadenectomy, partial omentum resection, and appendicectomy. During laparoscopic surgery, the right ovary was observed with an abnormally enlarged morphology. This involved a solid mass contained in an intact capsule. Pathological analysis indicated that the tumor was a dysgerminoma involving the surface of the right ovary with a small amount of the gonadoblastoma component regionally. The patient was diagnosed with stage IC ovarian gonadoblastoma and dysgerminoma. After the operation, the patient received four cycles of combination chemotherapy containing bleomycin, etoposide, and cisplatin. No recurrence was found during 31 months of regular follow-up after chemotherapy. Written informed consent was obtained from the patient for publication of this case report and any accompanying images.
The dysgerminoma was comprised of large and identical tumor cells separated by collagen fiber bands, and tumor nuclei were similar in size. The nucleoli were obvious, even during the stage of karyomitosis (Fig. 1a). The gonadoblastoma was arranged in the form of nests, which were comprised of germ cells mixed with sexual cord cells similar to immature Sertoli cells and granulocytes. The germ cells were round with transparent cytoplasm and large, round nuclei. The sex cord cells were small and deeply stained, the nuclei were slender, and the nucleoli were not obvious (Fig. 1g). Immunohistochemical analysis indicated that the dysgerminoma component had positive staining for D2-40 (Fig. 1b), OCT3/4 (Fig. 1c), PLAP (Fig. 1d), SALL4 (Fig. 1e), and CD117 (Fig. 1f) and negative staining for α-inhibin, Calretinin and EMA. In the gonadoblastoma component, the sex cord cell component showed positive staining for α-inhibin (Fig. 1h) and Calretinin (Fig. 1i), while the germ cell component indicated positive staining for SALL4 (Fig. 1j), PLAP (Fig. 1k), and OCT3/4 (Fig. 1l). These features supported the diagnosis of gonadoblastoma.
Karyotype analysis of peripheral blood lymphocytes revealed that the woman had a 46, XX female karyotype (Fig. 3). Genomic DNA was extracted from both paraffin-embedded sections and peripheral blood according to standard procedures. The primary paraffin-embedded tissues were dewaxed in xylene and rehydrated in ethanol and then positioned in proteinase K buffer before DNA extraction. Then DNA was fragmented, exome-enriched, and sequenced. Whole-Exon Sequencing (WES) was conducted at BGI with ~ 170x sequencing depth for the peripheral blood sample and ~ 428x sequencing depth for the paraffin-embedded sample, respectively. After removing the adaptors and low-quality reads (Phred score < 15) produced by the sequencing process, clean reads were further mapped to the human reference genome assembly GRCh19 using the Burrows-Wheeler-Alignment (BWA) tool. Duplicate reads were removed by Picard. Small mutations were called and filtered respectively by GATK4 for both the sequenced samples. Germline mutations were obtained from the peripheral blood sample directly. Somatic mutations were obtained by filtering out the germline mutations from the mutations called in paraffin-embedded sections. Both germline mutations and somatic mutations were annotated by ANNOVAR. Public database, including the latest 1000 Genomes Project dataset (1KG), Exome Aggregation Consortium dataset (ExAC), gnomAD dataset (gnomAD), the COSMIC cancer database and the Clinvar database were used in the annovation. We filtered both somatic mutations and germline mutations by the following rules: 1) variants beside exonic or splicing region were filtered out; 2) variants annotated as synonymous SNV or unknown were filtered out; 3) variants that were reported in one of the 1KG, ExAC or gnomAD with allele frequency equal or greater than 1% were filtered out; 4) somatic variants with allele frequency smaller than 5% were filtered out. After filtering, 84 variants were left for somatic mutations, and 516 variants were left for germline mutations. We subsequently obtain pathogenic variants from germline mutations and somatic mutations separately. For the germline mutations: mutations with pathogenic clinical significance annotated by Clinvar were remained. For the somatic mutations: insertions, deletions and splicing variants were remained as they affect the amino acid sequences of the proteins. We predict the pathogenicity of the single nucleotide variants by SIFT, Polyphen2 (HDIV/HVAR), LRT, MutationTaster, MutationAssessor, MetaSVM/LR, M-CAP, PROVEAN, FATHMM and FATHMM-MKL. Variants annotated as pathogenic or like pathogenic by at least three software were remained for further analysis. Thirty-nine variants, with four germline mutations (i.e., GJB2, SPINK5, SMPD1, CEP290) and 34 somatic mutations (e.g., KIT, USP6, NANOG) left marked as deleterious variants as shown in Table 1. Figure 2 demonstrates the distribution of the variants on the human genome.
Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed for germline mutations and somatic mutations separately (Fig. 4). The top 10 enriched cellular component, biological process and molecular function in GO and the top 30 KEGG pathways were displayed. Somatically altered genes showed significant enrichment for sex differentiation and development of the first sex character (Fig. 4a). This result may confirm the tissue origin of this tumor, which originates from germ cells. Notably, KIT was implicated in the differentiation of germ cells. In addition, somatic mutated genes were mainly enriched in the dysregulation of transcriptional activity and DNA replication, which might be the putative triggering factors. Results of the KEGG analysis showed that somatic mutated genes were enriched in signaling pathways related to cell proliferation and differentiation, such as the Forkhead box protein O (FoxO) signaling pathway, EGFR tyrosine kinase inhibitor resistance, the ERBB2 signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, homologous recombination deficiency and mRNA surveillance (Fig. 4b). KIT, encoding a receptor tyrosine kinase, is involved in mitogen-activated protein kinase (MAPK) and PI3K-Akt signaling pathways. No remarkable GO and KEGG enrichment were observed for the germline mutated genes (Fig. 4c and d).