Osteosarcoma in One of Identical Twins: Three Cases Report and a Literature Review

Background Osteosarcoma (OS) is the most common primary malignant bone tumor occurring mainly in children and young adults. OS is usually seen in sporadic cases, and it is an extremely rare phenomenon in blood relatives, particularly among identical twins. Case Presentation The present study reports three cases of OS occurring in only one of identical twins. The first case is a high‐grade OS in the left proximal tibia of a 16‐year‐old girl, treated with neo‐adjuvant chemotherapy, en bloc resection, and reconstruction with a modular knee tumor prosthesis. The second one is a high‐grade OS of the left proximal tibia of a 6‐year‐old girl. The patient was treated with neo‐adjuvant chemotherapy, en bloc resection, and reconstruction with inactived autograft. The third one is a conventional OS of the right proximal tibia of a 20‐year‐old woman. She was treated with neo‐adjuvant chemotherapy, en bloc resection, and reconstruction with a custom‐made prosthesis. Conclusions The occurrence of OS in one of identical twins is a relatively rare event but may present the best opportunity to understand the genetic mechanisms underlying the tumorigenesis and progression of this disease in humans. A longer follow‐up period and regular imaging evaluation are needed to confirm whether the identical twin of these patients will suffer OS in the future.


Introduction
O steosarcoma (OS) is one of the most common primary bone malignancies in children and adolescents 1 . OS accounts for 3.4% of pediatric tumors and 20% of primary bone cancers 2 . Nonetheless, it is a relatively rare neoplasia, with an incidence of 400-600 cases per year in the United States 3,4 . Most OS cases are usually sporadic in nature with no positive family history or identifiable predisposing factors. The risk of OS is increased in patients with various cancer predisposition syndromes, including hereditary retinoblastoma, Li-Fraumeni syndrome, Rothmund-Thomson syndrome, and Bloom syndrome [5][6][7] . OS is an even rarer phenomenon in siblings, occurring in fewer than 10 in 10,000 patients 8,9 .To the best of the authors' knowledge, there is only one report of OS affecting identical twins in the relevant English literature 4 .
We report three OS patients whose identical twin has not so far shown any evidence of malignant disease ( Table 1). The first case is a high-grade OS in the left proximal tibia of a 16-year-old girl, treated with neo-adjuvant chemotherapy, en bloc resection, and reconstruction with a modular knee tumor prosthesis. The second case is a highgrade OS of the left proximal tibia of a 6-year-old girl. The patient was treated with neoadjuvant chemotherapy, en bloc resection, and reconstruction with inactived autograft. The third case is a conventional OS of the right proximal tibia of a 20-year-old girl. She was treated with neoadjuvant chemotherapy, en bloc resection, and reconstruction with a custom-made prosthesis. We also review the clinical characteristics of 42 OS patients who have siblings described in detail in previous English literature ( Table 2).

Case 1
In June 2017, a 16-year-old Chinese girl, whose younger identical twin sister was healthy, was referred to our institute for complaints of left knee pain of approximately 3-months duration. Her past history was negative for trauma, infection, irradiation, or cancer. Anteroposterior (Fig. 1A) and lateral ( Fig. 1B) plain radiographs of the left lower extremity taken at an external hospital revealed mixed destruction of osteolytic and osteogenic focus in the metaphysis of the left proximal tibia, with periosteal reaction and soft tissue mass. The patient was admitted to our hospital for further examination and treatment. On physical examination, the findings were an obvious tender and slightly swollen area on the anterolateral aspect of the left proximal tibia with normal temperature and color, and both knees had full and symmetric range of motion (ROM).
Chest X-ray and computed tomography (CT) scans did not show any evidence of pulmonary metastases. Emission computerized tomography (ECT) was positive for the lesion in the left proximal tibia, but not elsewhere. The axial CT image (Fig. 1C) of the left lower extremity showed tumor bone formation in the medullary cavity, cortical penetration, and soft tissue mass. Percutaneous needle biopsy of the left proximal tibia was interpreted as an osteoblastic osteosarcoma (Fig. 1D).The immunohistochemical (IHC) analysis showed that the p16 protein was present in more than 50% of tumor cells and a strong positive vimentin expression was detected (Fig. 1E).The patient received neoadjuvant chemotherapy consisting of cisplatin (120 mg/m 2 ), ifosfamide (2 g/m 2 ), and doxorubicin (75 mg/m 2 ) for two cycles. After chemotherapy, there was apparent tumor calcification in the anteroposterior (Fig. 1F) plain radiograph of the left lower extremity. She was staged as IIB according to the Musculoskeletal Tumor Society (MSTS) staging system. On 4 August 2017, she underwent wide intra-articular resection of the left proximal tibia and reconstruction with a cemented, modular, rotating-hinge tumor knee prosthesis (Wego, Beijing, China) (Fig. 1G,H). The surgical margins of resected specimens were negative for tumor. The patient also completed six courses of postoperative chemotherapy without any complication. However, pulmonary metastases developed within 9 months after surgery. She was treated with three-dimensional conformal radiotherapy (3-DCRT) to a total dose of 20 Gy in six fractions. After radiotherapy, she was found to have new small pulmonary nodules. Then she received apatinib, a novel oral small-molecule tyrosine kinase inhibitor (TKI) targeting the intracellular domain of vascular endothelial growth factor receptor-2 (VEGFR-2). Unfortunately, the treatment failed. The patient died of spontaneous pneumothorax 3 years after initial diagnosis.

Case 2
In June 2016, a 6-year-old Chinese girl was admitted to our hospital with 2-months history of left knee pain. No history of trauma, infection, irradiation, or cancer was found. The anteroposterior and lateral radiographs ( Fig. 2A) before chemotherapy showed osteolytic lesion in the metaphysis of left proximal tibia. Bone scans were negative for bone metastasis. No definite metastatic nodules were found in CT images of the lungs. A core needle biopsy was performed with diagnosis of high-grade chondroblastic OS (Fig. 2B). p53 protein accumulation was seen in 40% of tumor cells and S-100 expression was seen in 25% of tumor cells (Fig. 2C). After two cycles of neoadjuvant chemotherapy, the magnetic resonance imaging (MRI) (Fig. 2D) showed a destructive lesion of the proximal tibia extending from the metaphysis to the epiphysis beyond the epiphyseal line or plate. Then she underwent wide tumor resection and reconstruction of the intercalary bone defect with alcohol-induced devitalized bone segment and plate. Then she completed 10 cycles of postoperative adjuvant chemotherapy. Radiographs (Fig. 2E) taken 1 year after operation demonstrated good bone union at the graft-host junction. However, surgical-related complications including posterior knee dislocation (Fig. 2E) and lower limb discrepancy (Fig. 2F) were identified. At last follow-up in September 2020, the patient was continuously disease-free and without functional deficits. Her identical twin sister remains healthy through the follow-up period.

Case 3
A 20-year-old Chinese female, with no family history of malignant tumors or irradiation, fell and hurt her right knee in physical education class in school in January 2018. Since that time, she experienced constant pain in the proximal leg for 3 months. X-ray films of her right knee (Fig. 3A) revealed a destructive lesion at the proximal tibia with soft tissue extension. Axial CT image (Fig. 3B) showed tumor new bone formation and cortical discontinuity on medial aspect. MRI (Fig. 3C) showed a tumor with low signal intensity on sagittal T1-weighted imaging (T1WI) and high signal intensity on coronal T2-weighted imaging (T2WI) with evidence of a posterior soft tissue mass. No metastatic lesions were seen on CT scans of the chest and abdomen. A biopsy of the lesion was done on 29 April 2018 and a diagnosis of primary conventional OS was made. The patient was started on two cycles of DIA neoadjuvant chemotherapy. Then she underwent wide resection of bone sarcoma and modular knee tumor prosthetic replacement (Wego, Beijing, China) (Fig. 3D). The postoperative pathological analysis confirmed the diagnosis of high-grade fibroblastic OS (Fig. 3E). Strong positivity for SATB-2 protein was seen in the majority of tumor cells by IHC (Fig. 3F). This was followed by postoperative adjuvant chemotherapy. However, the patient died of respiratory failure due to advanced pulmonary metastasis in October 2019.
Pediatric OS is characterized by multiple somatic chromosomal lesions, including structural variations and copy number alterations (CNAs) 30 . The OS genome has long been known to be complex and heterogeneous, with few common features between tumors. Previously, various somatic mutations and copy number changes involved in the pathogenesis and development of OS have been detected by NGS approaches 31 . Recently, we reviewed the top 10 frequently mutated genes (e.g., TP53, RB1, PTEN, DLG2, MYC, ATRX, NF1, CCNE1, CDKN2A, and PIK3CA) and some tumorspecific CNAs (e.g., MYC, CCNE1, VEGFA, BRCA1/2, TP53, RB1, CDKN2A/2B) in OS tissues identified by NGS technology 32 . More recently, Mirabello and her colleagues found that a higher-than-expected frequency of pathogenic or likely pathogenic germline variants existed in genes not previously linked to OS (e.g., CDKN2A, MEN1, VHL, POT1, APC, MSH2, and ATRX) 33 . Furthermore, some studies have indicated that familial occurrence of OS may present an inherited genetic predisposition to this tumor 19,34 . Several genetic variants or molecular abnormalities have been identified to be associated with the cooccurrence of OS in siblings, such as germline mutation of RB1, TP53, or ATRX genes 8,24 , loss of heterozygosity of RB1 and TP53 24 , 13;14 chromosomal rearrangement 19 , HLA phenotypes 15,21 , and RECQL4 mutation 25 . The occurrence of OS in identical twins is a relatively rare event but may present the best opportunity to understand the genetic factors and molecular mechanisms underlying the tumorigenesis and progression of this disease in humans 4 . Therefore, it is necessary to perform NGS for identical twins in the following study.
However, the duration of follow-up was relatively short in the present study. A longer time follow-up and regular imaging evaluation are needed to confirm whether the identical twin of these patients will suffer OS in the future.

Acknowledgments
T he authors sincerely thank Doctor Minmin Yu and Zhiyong Wei for their help in processing and analyzing tumor samples.

Authorship Declaration
A ll authors listed meet the authorship criteria according to the latest guidelines of the International Committee of Medical Journal Editors, and they are all in agreement with the manuscript.

Availability of Data and Materials
A ll data generated or analyzed during this study are included in this published article.

Ethics Approval and Consent to Participate
O ur study was approved by the Ethics Committee of the PLA 960th hospital. All adult patients and parents for children who participate in the study provided written informed consent. A copy of the consent form is available for review.

Patient Consent for Publication
T he patient provided written informed consent for the publication of associated data and accompanying images.