Abstract
Background/Aim: Yolk sac tumour (YST) is a rare malignant ovarian germ cell tumour that often occurs in young women or adolescents and exhibits an unfavourable outcome. To evaluate the biological behavior of carcinomas in vitro, permanent tumour cell lines are required. However, previously, only a few human YST cell lines have been established. Therefore, we aimed to establish a novel YST cell line. Materials and Methods: We established a novel YST cell line, TC587, from an adolescent patient with ovarian YST. Results: The cell line expressed AFP and SALL4, the characteristics of YST. In addition, we evaluated somatic mutations using next-generation sequencing and revealed some pathogenic variants, including mutations in the NRAS, KIT, KMT2C, RSF1, and TP53 genes. Conclusion: The newly established TC587 cell line may represent an effective tool for developing treatments and conducting molecular analyses for YST.
Ovarian germ cell tumour is an extremely rare tumour comprising 2%-5% of all ovarian malignant tumours (1). Yolk sac tumour (YST) is the second most frequent histological subtype after ovarian dysgerminoma, it accounts for 20% of all ovarian germ cell tumours and predominantly occurs in women aged <35 years (1). Among all subtypes of malignant ovarian germ cell tumour, YST is a highly malignant germ cell tumour characterized by the expression of α-fetoprotein (AFP) and unfavourable outcomes, with a 5-year survival rate of 66.6% (2). Additionally, YST has 5-year survival rates of 92.5%, 75%, 30%, and 25% in patients with stage I, II, III, and IV disease, respectively (3). Therefore, to improve the aggressive course and unfavourable prognosis of YST long-term, new treatments, including molecular-targeted drugs based on molecular and genetic characteristics of YST are required. To evaluate the biological behavior of carcinomas in vitro, permanent tumour cell lines are required. However, only a few human YST cell lines have been established. Molecular, genetic, and biological characterization of YST in vitro and in vivo models is desired. In this study, we established and characterized a new YST cell line, TC587, derived from a recurrent tumour in a patient with YST.
Materials and Methods
Patient's clinical history. A 12-year-old girl was admitted to the Department of Pediatric Surgery, Kyushu University Hospital for increasing abdominal girth and vomiting. Her serum AFP and CA 125 levels were 32,421 ng/ml and 286 U/ml, respectively, whereas her human chorionic gonadotropin (HCG) level (<0.5 mIU/ml) was within the normal range. CT revealed a 10×8 cm2-sized pelvic tumour (Figure 1A). After a series of clinical and laboratory examinations, YST in the ovaries was diagnosed, and preoperative chemotherapy (ifosfamide, carboplatin, and etoposide) was initiated. Next, surgery (left oophorectomy and omentectomy) was performed. The primary resected sections were typified by the proliferation of oval to polygonal cells having prominent nucleoli and eosinophilic cytoplasm arranged in endodermal sinus, hepatoid, and glandular patterns (Figure 1B, C). Immunohistochemically, the tumour cells were positive for AFP, Cytokeratin AE1/AE3, and CAM5.2 but negative for EMA and HCG (Figure 1D-F). Despite postoperative adjuvant chemotherapy, repeat surgery (simple hysterectomy, bilateral salpingo-oophorectomy, and colostomy) was required because of local recurrence. The 20 specimens from the recurrent tumour exhibited similar histological and immunohistochemical features (data not shown) and were diagnosed as pure YST.
Chemosensitivity to 20 chemotherapeutic agents was evaluated using the succinate dehydrogenase inhibition (SDI) method as described previously (4). We performed chemotherapy using Actinomycin D, Vincristine and Adriamycin (regimen DD-4A) based on the results of the chemosensitivity test (Table I). Further, the tumour exhibited an aggressive clinical course, and the patient died 1 year after the initiation of therapy. No autopsy was performed. This study was conducted in accordance with the principles embodied in the Declaration of Helsinki.
Immunohistochemical analysis. Immunohistochemical analysis was performed for original tumours (first and second surgeries). The formalin fixed, paraffin-embedded sections were cut into 4 μm, dewaxed in xylen and rehydrated in a grade series of ethanol. Endogenous peroxidase was blocked with 3% hydrogen peroxide in methanol for 30 min, and washed twice with PBS. These sections were treated with trypsin for 30 min at room temperature for CAM5.2, and heated with microwave in 0.01 M citrate buffer (pH6) for 20 min at 99°C for Cytokeratin AE1/AE3. The endogenous biotin-avidin was blocked using an endogenous biotin-avidin blocking kit (Nichirei Corp., Tokyo, Japan), and washed twice with PBS. The primary monoclonal antibodies and antigen retrieval methods used in this study are summarized in Table II. Overnight primary antibody incubation at 4°C was performed, and then the immune complex was detected with Nichirei Histofine SAB-PO kit following the manufacturer's protocols (Nichirei Corp, Tokyo, Japan). The sections were developed with diaminobenzidine (DAB) and counterstained with Mayer's hematoxylin and mounted.
Establishment of the TC587 cell line. Tumour tissue obtained during the second operation was minced with scissors and seeded in a 25-cm2 tissue culture flask (Corning Costar, Tokyo, Japan) at 37°C in a moist atmosphere containing 5% CO2. The culture medium was Ham's F12 containing 10% fetal bovine serum. When subconfluent layers were obtained, the cells were dispersed with phosphate-buffered saline (PBS) containing 0.1% trypsin and 1 mM EDTA and divided among several dishes. After 100 passages, we named this cell line “TC587” and then we carried out the assays described below.
Cells. TC587, Nec8, Hela and Hep3B were grown in Ham's F-12 (TC587), RPMI 1640 (NEC8), DMEM/F12 (Hep3B) and DMEM (Hela) supplemented with 10% fetal bovine serum.
Immunoblotting. Cells were washed twice with PBS, re-suspended in 2×SDS sample buffer, and denatured at 97°C for 3 min. The samples were separated via SDS-PAGE (Any kD™ Mini-PROTEAN® TGX™, Bio-Rad, Tokyo, JAPAN) and electrotransferred to a polyvinylidene fluoride membrane using the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Hercules, CA; 2.5 A, 25 V, 7 min). The membrane was blocked for 15 min in Blocking One-P (Nacalai Tesque) and incubated with primary antibodies in Hikari Solution A (Nacalai Tesque), followed by incubation with secondary antibodies and detection using Chemi-Lumi One Ultra (Nacalai Tesque). The primary antibodies used for immunoblotting are summarized in Table II.
Immunocytochemistry. Cells were plated on coverslips, washed twice with PBS, fixed in 4% paraformaldehyde in PBS for 10 min, permeabilized in 0.5% Triton X-100 in PBS for 5 min, and washed twice with PBS. A 10-min incubation in Blocking One-P was followed by overnight incubation at 4°C with anti-SALL4 and anti-AFP (Table II). The coverslips were then washed three times with TBS-T and incubated for 30 min at room temperature in Alexa 488 plus labelled goat anti-mouse or anti-rabbit antibody (1:1000, Thermo Fisher). Coverslips were again washed three times in PBS and mounted in ProLong Gold Diamond Antifade Reagent with DAPI (Life Technologies). Images were visualized using a fluorescence microscope (BZ-X700, Keyence, Osaka, Japan).
DNA extraction, Next-generation sequencing and bioinformatics. Total DNA from TC587 cells was isolated using a QIAmp DNA Micro kit (Qiagen, Hilden, Germany). Next, DNA quality was tested using the Genomic DNA ScreenTape system (Agilent). An amplicon library of the target exons was prepared using an Ion AmpliSeq Custom Panel (Applied Biosystems, Life Technologies) and designed using Ion AmpliSeq Designer (http://ampliseq.com) for 219 genes, including the Ion AmpliSeq Cancer Hotspot Panel v2 targeted genes and chromatin remodelling factor-related genes according to the manufacturer's instructions (Table III). The NGS library construction and analysis were performed by Cell Innovator (Fukuoka, Japan).
Cytogenetic analysis. A metaphase cytogenetic analysis was performed using the 20th passage of TC587 cells. The preparation was obtained using a standard trypsin-Giemsa banding technique. The karyotype was analyzed according to the rules of the International System for Human Cytogenetic Nomenclature (ISCN1995).
Results
Cell line establishment. We established a novel human yolk sac cell line (TC587) from the resected recurrent tumour (Figure 2A). Cultured cells comprised small, polygonal cells. The doubling time was approximately 36 h.
Immunocytochemical and immunoblotting results. Immunoblotting revealed that the cell line was positive for AFP and SALL4 and negative for Nanog, OCT3/4, and SOX2 (Figure 2B). Immunocytochemical staining also confirmed the expression of SALL4 and AFP (Figure 2C).
Cytogenetic findings. Cytogenetic analysis of 10 samples of high-quality metaphase TC587 cells revealed an extremely complex karyotype, with chromosome numbers varying from 79 to 83. The cells featured the following composite chromosomal complements: 79~83<4n>, X, -X[10], add(X) (p11.2)[10], der(X)t(X;12)(p11.2;q12)[10], add(1)(p31)[10], del(1)(p34p36.1)[10], -2[10], add(2)(q11.2)[10], -3[9], add(3)(q11.2)[10], add(3)(q11.2)[3], -4[10], der(4)t(4;5) (q23;q13)ins(4;?)(q23;?)[10], -5[10], -5[10], add(5)(p13)[9], -6[10], -6[4], add(6)(p21)[2], add(7)(p13)[10], add(7)(p11.2) [9], del(7)(q11.2q21)[10], -8[10], -9[10], add(9)(p13)[10], -10[10], -10[10], add(10)(p11.2)[9], -12[10], -13[9], -14[10], -14[9], -15[10], -15[9], add(15)(p11.2)[9], -16[10], -17[10], add(17)(q21)[7], -18[10], -18[7], del(18)(q12)[9], +19[2], add(19)(q13.3)[10], -20[10], -20[3], add(20)(q13.1)[3], -21[10], -22[10], -22[3], +der(?)t(?;2)(?;q21)[7], +11~16 mar (Figure 2D).
Next-generation sequence analysis. Next-generation sequencing identified 42 somatic variants, including pathogenic variants of the NRAS, KIT, KMT2C, RSF1, and TP53 genes (Table IV). SNVs identified in these genes were missense variants, and all were associated with damaging effects on the coding product. One SNV identified in the SMARCA2 gene was a previously unknown stop-gained variant.
Discussion
The biology of YST is poorly understood. Permanent tumour cell lines serve as preclinical research tools for better understanding several tumour-related pathological aspects and exploring novel therapeutic targets. In this study, we established the novel YST cell line that exhibited the same protein expression patterns as YST. Only two YST cell lines had been previously reported (NOY1 and NOY2) (5). In addition, these two cell lines were established from the same patient, and it is difficult to investigate individual differences among patients with YST.
We also performed sequencing analysis using TC587 cells and identified various SNVs, including some pathogenic variants in the genes TP53, NRAS, KIT, KMT2C, and RSF1. The TP53 gene is a major tumor suppressor gene and the variants identified have also been reported in YST (COSIMC data) (6). TC587 cells carried the TP53 c.1040C>A variant, which has not been reported in YST. Variants in NRAS codons 12 and 61 have been reported in seminoma (16% and 59%, respectively) and non-seminoma (15% and 78%, respectively) (7, 8). In the COSMIC data, 1/27 patients with YST exhibited a c.34G>A mutation. TC587 cells carried the NRAS c.38G>A mutation, which has been reported in leukemia, malignant melanoma, and colorectal adenocarcinoma (8).
KIT mutations are common pathogenic mutations in gastrointestinal stromal tumour (GIST) that result in ligand-independent activation of tyrosine kinases. The most frequent mutations occur in KIT exon 11 (60%-70%), followed by a mutation in KIT exon 9. TC587 cells carried the KIT p.D816Y (exon 17) mutation, which encodes the second tyrosine kinase domain (9). In the COSMIC database, the KIT p.816Y mutation was found in GIST, acute myeloid leukemia (AML), and some germ cell tumours, including dysgerminoma, seminoma, and mixed germ cell tumour. AML featuring the D816Y mutation is highly resistant to imatinib mesylate (10).
The KMT2C gene encodes a histone methyltransferase that regulates gene transcription by modifying the chromatin structure. The KMT2C mutation identified in this study has been reported in various carcinomas, including endometrial carcinoma gastrointestinal carcinoma and urothelial carcinoma; however, this mutation has not been previously described in YST.
SMARCA2 (also called BRM) is a key component of the SWI/SNF chromatin-remodelling complex. TC587 cells carried the SMARCA2 p.Gln589 mutation, which created a stop codon. This mutation is not annotated in the ClinVar or COSMIC database, but is predicted to cause truncation of the SMARCA2 protein at the SNF ATPase and BRM domains.
The RSF1 gene encodes a chromatin-remodelling factor that has been linked to DNA damage response and DNA repair (10). No RSF1 mutations have been previously reported in germ cell tumours. TC587 cells featured the p.Pro1133Leu mutation, and the same variant has been reported in gastric cancer (COSMIC database). This pathogenic mutation might be a candidate clinical therapy target. Further investigation using the TC587 cell line is needed.
In summary, we have established and characterized a novel YST cell line. This cell line has some pathogenic mutations and expresses AFP and SALL4. Using this cell line, we hope to contribute to the individualization of YST treatment.
Acknowledgements
The Authors appreciate the technical assistance from Cell Innovator and the English language review from Enago (www.enago.jp).
Footnotes
Authors' Contributions
TI, KK and YO designed this study and wrote the manuscript. TI, KK and MO performed the experiments. TI, KK and YO performed histological re-evaluation of samples and confirmed the diagnosis. TT and YO supervised the experiments.
Conflicts of Interest
The Authors declare no conflicts of interest regarding this study.
- Received December 13, 2019.
- Revision received December 22, 2019.
- Accepted December 23, 2019.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved