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Primary osteosarcomas involving the base of the skull in middle-aged patients are rare. We describe the case of a 59-year-old Asian woman presenting with lethargy, epistaxis, left maxillary and mandibular pain, and headache. Computed tomography (CT) and magnetic resonance imaging (MRI) showed a large left greater sphenoid wing tumour with extensive local infiltration and intracranial extension. The tumour was diagnosed as osteosarcoma based on histological examination. In this report, we discuss the clinical presentations, radiological features, and imaging differential diagnoses of this case.

Osteosarcoma (OS) is a highly aggressive primary bone tumor. The mainstay for its treatment is multiagent chemotherapy and surgical resection, with a 50-70% 5-year survival rate. Despite the huge effort made by clinicians and researchers in the past 30 years, limited progress has been made to improve patient outcomes. As novel therapeutic approaches for OS become available, such as monoclonal antibodies, small molecules, and immunotherapies, the need for OS preclinical model development becomes equally pressing. Three-dimensional (3D) OS models represent an alternative system to study this tumor: In contrast to two-dimensional monolayers, 3D matrices can recapitulate key elements of the tumor microenvironment (TME), such as the cellular interaction with the bone mineralized matrix. The advancement of tissue engineering and biofabrication techniques enables the incorporation of specific TME aspects into 3D models, to investigate the contribution of individual components to tumor progression and enhance understanding of basic OS biology. The use of biomaterials that mimic the extracellular matrix could also facilitate the testing of drugs targeting the TME itself, allowing a larger range of therapeutics to be tested, while averting the ethical implications and high cost associated with in vivo preclinical models. This review aims at serving as a practical guide by delineating the OS TME ("what it is like") and, in turn, propose various biofabrication strategies to create a 3D model ("how to recreate it"), to improve the in vitro representation of the OS tumor and ultimately generate more accurate drug response profiles.

Guangxian Zhong,1,&ast; Yunqing Wang,1,&ast; Hongxiang Wei,1 Meifang Chen,2 Huangfeng Lin,1 Zhen Huang,1 Jinlong Huang,3 Shenglin Wang,1 Jianhua Lin1 1Department of Orthopaedics, The First Affiliated Hospital of Fujian Medical University, Fuzhou, 350004, People’s Republic of China; 2The Health Management Center, The First Affiliated Hospital of Fujian Medical University, Fuzhou, 350004, People’s Republic of China; 3Department of Hematology, The First Affiliated Hospital of Fujian Medical University, Fuzhou, 350004, People’s Republic of China&ast;These authors contributed equally to this work.Correspondence: Jianhua Lin; Shenglin Wang Tel/Fax +86 591 87981029Email jianhual@126.com; Shenglinwang0216@163.comPurpose: The aim of this research was to investigate the clinical significance of the expression of flap structure-specific endonuclease 1 (FEN1) in primary osteosarcoma.Methods: The expression of FEN1 was detected by immunohistochemistry analysis. The association of the expression of FEN1 in osteosarcoma with clinicopathological parameters was analyzed by using χ2 test or Fisher’s exact test. Survival analyses were performed by Kaplan–Meier method and Cox proportional hazards regression model.Results: Of the 40 osteosarcoma patients, 19 (47.5%) patients presented with FEN1 high expression, while in the non-neoplastic bone specimens, the FEN1 high expression was observed in 10% (3/30), the positive expression rate in osteosarcoma patients was significantly higher than that of non-neoplastic bone specimens (P< 0.01). Univariate analysis indicated that the progression-free survival (PFS) and overall survival (OS) were correlated with the expression level of FEN1 (PFS, P < 0.001; OS, P = 0.002), Enneking staging (PFS, P = 0.026; OS, P = 0.044) and chemotherapy response (PFS, P = 0.019; OS, P = 0.031). Multivariate analysis demonstrated that FEN1 expression was an independent prognostic factor for the PFS (HR = 4.73, P = 0.002) and OS (HR = 4.01, P = 0.038) of osteosarcoma patients.Conclusion: This study showed that FEN1 was overexpressed in osteosarcoma patients and positively associated with poor prognosis of osteosarcoma patients. Further studies should focus on the relative mechanisms and the targeted FEN1 therapies for osteosarcoma.Keywords: osteosarcoma, flap endonuclease 1, immunohistochemistry, prognosis

Background/aim Primary osteosarcoma of the breast is an exceedingly-rare malignant tumor that shares histological characteristics with osteosarcoma of the bone. Since effective therapies have not yet been established, standard therapy for osteosarcoma of the bone was examined in the present study for efficacy against primary osteosarcoma of the breast in a patient-derived orthotopic xenograft (PDOX) nude-mouse model. Materials and methods The PDOX mouse models were established by surgical implantation of the primary osteosarcoma of the breast specimen into the mammary gland of nude mice. Mice with tumors were randomized into four groups, each n=4: control group; cisplatinum (CDDP)-treatment group; doxorubicin (DOX)-treatment group; and CDDP/DOX-combination-treatment group. Mice were treated for twenty-one days, three weeks after implantation. Tumor size and body weight were measured during three weeks of treatment. Results Significant tumor growth inhibition was observed, compared to the control, in the CDDP-treatment group, the DOX-treatment group, and the combination-treatment-group. Only the combination treatment regressed the tumor. Conclusion CDDP and DOX which are standard first-line therapies for osteosarcoma, may be clinically effective against primary osteosarcoma of the breast, and in particular, their combination.

Osteosarcoma is the most common primary bone tumor and usually involves the long bones. Osteosarcoma of the skull, on the other hand, is relatively rare. Here, we present a 29-year-old man with a growing mass in the skull he first noticed after a fall while skateboarding. The initial clinical diagnosis was hematoma. While undergoing an evacuation surgery for a hematoma, a suspicious mass was detected which was biopsied. Histopathological evaluation showed high-grade osteosarcoma. The patient was referred to our hospital where he underwent definitive resection followed by adjuvant chemotherapy. His course was complicated by wound infection. Even though osteosarcoma of the skull is a rare finding, it should be suspected in a patient with a skull mass, and the history of prior head trauma does not exclude the diagnosis.

A 58-year-old female with a primary osteosarcoma of the breast (POB) is presented here. The diagnosis was based on the characteristic histological picture and on the immunohistochemical results. The possibility of the secondary lesion from a primary skeletal osteosarcoma or direct extension of an osteogenic sarcoma arising from nearby ribs or the sternum was excluded. Approximately, 150 cases of POB had been reported in the literature with tumor size affecting survival. The diagnosis needs to be made as early as possible to improve the survival of this disease.

Osteosarcoma is a common primary malignant tumor of long bones in which mesenchymal cells produce osteoid, affecting mainly young adults. Head and neck localization is rare, moreover at the level of the maxillary sinus. The certainty diagnosis is based on the clinical CT and histopathological findings. This histological type is associated with a low risk of metastasis. Early diagnosis and wide surgical resection of the tumor are the most important determinant factors of prognosis. In this article, we report a case where radiotherapy was used.

Primary osteosarcoma (OS) of the uterus is a distinctly rare and aggressive disease with fewer than 20 cases reported worldwide. We describe a case of primary uterine OS with rapid development of pulmonary and brain metastasis in a 50-year-old woman. Histopathologic examination of the uterine tumor showed atypical spindle cells producing an osteoid matrix with calcification in keeping with OS. Despite initial response to doxorubicin and ifosfamide, the patient succumbed to brain metastases just 8 months from diagnosis. Whole genome sequencing was performed on tumor and blood samples to analyze genetic alterations in this highly aggressive tumor. A pathogenic somatic missense mutation resulting in substitution of glutamate for lysine at position 653 within the protein kinase domain of the platelet-derived growth factor receptor beta (PDGFRB) was found. The PDGF pathway is involved in cell proliferation and angiogenesis, and it has been implicated in malignancy. Crucially, this pathogenic mutation may be amenable to PDGFR tyrosine kinase inhibition, representing a possible treatment approach in this rare sarcoma.

Primary osteogenic sarcoma of the breast is a rare clinical entity with few cases described in the literature. Unfortunately, the prognosis for these patients is poor when compared to invasive carcinomas of the breast. We report a case of a 58-year-old female who developed a primary osteogenic sarcoma of the breast five years after being treated for invasive carcinoma of the ipsilateral breast without the use of radiotherapy.

Background: Although bone tumors are common pathologies in companion animals, limited reports describe nasalosteosarcoma (OSA) in cats.Case description: A case of nasal OSA in a local Libyan cat was admitted to the Veterinary Teaching Hospital at theFaculty of Veterinary Medicine, University of Tripoli–Libya, with nasal swelling and discharges and facial deformity. The radiological findings revealed nasal osteolysis with the absence of evidence of lung metastasis. In addition, fungal growth was not identified in microbiological culture. Furthermore, the pathological examination has grossly revealed a destructed nasal bone due to the presence of a tumor mass, with a mucohemorrhagic nasal discharge and absence of metastasis. OSA was confirmed histopathologically.Conclusion: This report presents the clinical, radiological, and pathological findings of a primary nasal OSA in aLibyan cat with no tumor metastasis to other body organs.Keywords: Cat, Nasal cavity, Primary osteosarcoma.

Primary extraskeletal osteosarcoma is an uncommon disease and has been reported to affect the uterus only rarely. Less than 20 cases have so far been reported in the English literature. The common clinical presentation is heavy bleeding per vaginam, and in virtually all cases, the diagnosis has been made at an advanced stage of the disease. Various authors have recommended adjuvant chemotherapy, but outcomes have so far been uniformly poor, with survival extended by months rather than years. We present two cases of this rare condition, which were diagnosed four months apart within our histopathology laboratory andconfirmed the very late presentation of the disease in one and the poor survival of both patients.

Background/Aim: Primary osteosarcoma of the mammary gland is a very rare disease, accounting for less than 1% of all mammary malignancies. There is no established first- line treatment and the prognosis is poor compared to normal breast cancer. We previously established the first patient tumor- derived animal model of this disease, grown subcutaneously in nude mice. In the present study, we established a patient derived orthotopic xenograft (PDOX) model of osteosarcoma of the breast and investigated the efficacy of cisplatinum (CDDP) and eribulin (ERB). Materials and Methods: PDOX models of primary osteosarcoma of the breast were divided into 3 groups (5-6 mice per group): untreated control; CDDP treatment; ERB treatment. The tumor volume in the 3 groups was compared after 2 weeks. Results: There were significant differences between control and CDDP, and control and ERB (p=0.036, 0.046, respectively). However, there was no significant difference between CDDP and ERB (p=0.964). Conclusion: CDDP and ERB are candidates for first-line clinical therapy of primary osteosarcoma of the breast.

Background Primary osteosarcoma of craniovertebral junction is extremely rare and reports in paediatric patients are limited. The symptoms are subtle and mostly underestimated. Case presentation. We present an unusual case of a 11-years old girl affected by an extensive and metastatic osteosarcoma of the atlas presented with cervicalgia and right torticollis refractory to medical treatment. The patient underwent open biopsy confirming the malignant histology. Conclusions Cervicalgia with or without a torticollis refractory to medical treatment, in absence of history of fell or trauma, is highly suspicious and should be considered as a warning sign for a severe pathology and it should not be neglected.

Highlights • The overall survival of osteosarcoma patients in the last 15 years has improved. • The proportion of patients with metastases at the time of diagnosis unchanged. • Consultation with specialists within 4 weeks can contribute to a better prognosis., Aims Examine: (1) If length of symptoms (LS) of children with osteosarcoma has improved in the last 15 years (2) Is delay in diagnosis related to the presence of metastases at presentation? (3) The impact of delay in diagnosis on prognosis. Methods 250 consecutive patients with a diagnosis of osteosarcoma of bone treated at a national bone tumor treatment center between 2004 and 2018 were studied retrospectively. Three groups comprising those diagnosed over a five-year period (Group 1: 2004–2008, Group 2: 2009–2013; Group 3: 2014–2018) were studied. Results There were 126 males and 124 females with a mean age 12.2 years. The median LS for all patients was eight weeks. The median LS for Group 3 was significantly shorter than that for other groups. Development of metastasis during follow-up period was significantly less in Group 3 compared to the other groups. Overall survival gradually improved over the whole study period. There was no difference in the proportion presenting with metastases at diagnosis between the three groups. The survival rates in patients with LS shorter than 4 weeks was better than those with LS longer than 4 weeks, irrespective of the study time period. Conclusions There has been an improvement in the LS in patients diagnosed with osteosarcoma over the last 15 years. The development of metastasis during follow-up has reduced and the overall survival in the last 15 years has improved. LS longer than 4 weeks is associated with a poorer prognosis.

Osteosarcoma is a malignant bone tumor with extremely high invasion, metastasis and mortality. The prognosis of patients with osteosarcoma remains poor. The ErbB receptor family was found to be overexpressed in human cancers and associated with poor prognosis. However, the role of ErbB receptor family in osteosarcoma has not been fully understood. The present study aimed to investigate the clinicopathological and prognostic significances of ErbB receptors in primary osteosarcoma. Western blot (WB), reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and fluorescence in situ hybridization (FISH) were used to detect the protein and gene expression of ErbB receptors in 60 primary osteosarcoma specimens and 30 non-neoplastic bone tissues. WB and RT-qPCR analyses showed that the protein and mRNA expression levels of EGFR, ErbB3 and ErbB4 in osteosarcoma specimens were significantly higher than those in non-neoplastic bone tissues. Seventeen (28.33%), 15 (25.00%) and 15 (25.00%) osteosarcoma specimens presented with amplification of EGFR, ErbB3 and ErbB4 gene, respectively, which were significantly higher compared with non-neoplastic bone tissues. The amplification of ErbB3 and ErbB4 in osteosarcoma was associated with advanced surgical stage. The amplification of EGFR, ErbB3, ErbB4 and the co-amplification of EGFR-ErbB3, EGFR-ErbB4, ErbB3-ErbB4 was linked with poor response to chemotherapy and distant metastasis. The amplification of EGFR, ErbB3 and ErbB4, as well as their co-amplification demonstrated independent prognostic values for reduced survival time of osteosarcoma patients and may serve as potential therapeutic targets for osteosarcoma patients in the future.

Editor's Note.-RadioGraphics continues to publish radiologic-pathologic case material selected from the "best case" presentations of the American Institute for Radiologic Pathology (AIRP), a program of the American College of Radiology. The AIRP conducts a 4-week Radiologic Pathology Correlation Course, which is offered five times per year. On the penultimate day of the course, the best case presentation is held at the American Film Institute Silver Theater and Cultural Center in Silver Spring, Md. The AIRP faculty identifies the best cases, from each organ system, brought by the resident attendees. One or more of the best cases from each of the five courses are then solicited for publication in RadioGraphics. These cases emphasize the importance of radiologic-pathologic correlation in the imaging evaluation and diagnosis of diseases encountered at the institute and its predecessor, the Armed Forces Institute of Pathology (AFIP).

Purpose: Older osteosarcoma patients have a very poor prognosis and treatment for them remains a challenge. The outcomes and potential prognostic factors of primary or secondary older osteosarcoma patients are rarely documented. Therefore, we examined the prognosis of the two special cohorts to identify possible prognostic factors, and provide optimal treatment strategy for them. Methods: The Surveillance, Epidemiology, and End Results (SEER) program database was used to identify osteosarcoma patients aged over 40 years from 1973 to 2015. The prognostic analysis was performed using the Kaplan-Meier method and a Cox proportional hazards regression model. Results: In total, 1162 primary older osteosarcoma patients and 444 secondary older osteosarcoma patients were eligible for this study. The OS and CSS rates of the primary older osteosarcoma patients at 5-year were 38.5% and 37.1%, respectively. The 3- and 5-year OS rates of the secondary older osteosarcoma patients were 22.8% and 14.6%, respectively. On multivariate analysis of the primary older osteosarcoma patients, age > 60, male, axial site, high grade, metastasis, tumor size>10 cm, no surgery, and radiation treatment were negatively associated with OS. In terms of CSS, age, gender, decade of diagnosis, tumor site, tumor grade, tumor stage, tumor size, and surgery were independent prognostic factors. A multivariate Cox regression model showed that secondary older osteosarcoma patients of high grade, metastasis, tumor size > 10 cm, no surgery, and no chemotherapy were independent predictors of decreased OS. Conclusions: Surgery in combination with chemotherapy should be recommended for the treatment of the secondary older osteosarcoma patients, while for the primary older osteosarcoma patients, only surgery should be recommended.

Background/aim Sarcomas of the breast are extremely rare malignant tumors and comprise only 5% of all sarcomas and fewer than 1% of breast cancers. Primary osteosarcoma of the breast is histologically indistinguishable from osteosarcoma of the bone. Effective therapies of this recalcitrant disease have not yet been developed. Materials and methods A patient-derived xenograft (PDX) mouse model of primary osteosarcoma of the breast was established by subcutaneous implantation of the surgical specimen, along with surrounding normal tissue. Hematoxylin and eosin (H&E) staining was performed on paraffin-embedded histological sections of the original tumor resected from the patient and from implanted tumors that grew in nude mice. Results Tumors grew in 46 of 51 mice implanted with the original surgical specimen. The H&E-stained slides of the mouse-grown tumor and the original patient tumor matched, both showing large areas of spindle-shaped cells, characteristic of osteosarcoma. Conclusion The first PDX mouse model of primary breast osteosarcoma was established which will enable testing of novel therapeutics as well as basic research of osteosarcoma of the breast.

Osteosarcoma is the most common primary malignant bone tumor, and its standard treatment is a combination of surgery and chemotherapy. A poor response to chemotherapy causes unfavorable oncological outcomes. We investigated the correlation between osteoclast differentiation in biopsy specimens and the efficacy of neoadjuvant chemotherapy in resected specimens. Forty-nine patients who underwent neoadjuvant chemotherapy and subsequent surgical treatment at our institution between 1999 and 2018 were enrolled. Using medical records, we investigated the age, sex, tumor size, location, subtype, staging, chemotherapy agents (doxorubicin, cisplatin, ifosfamide, and methotrexate), number of neoadjuvant chemotherapy courses, number of osteoclasts in biopsy specimens, and efficacy of neoadjuvant chemotherapy according to the Rosen and Huvos classification (Grade I-IV) in resected specimens. Univariate and multivariate analyses were performed to identify factors predictive of a good response in resected specimens after neoadjuvant chemotherapy. A good response (Grade III/IV) was detected in 25, while a poor response (Grade I/II) was detected in 24. According to the multivariate analysis, ≥ 46 years old (odds ratio [OR], 0.05; 95% confidence interval [CI], 0.01-0.45; p 0.01) and ≥ 5 mature osteoclasts in a biopsy specimen (OR, 36.9; 95% CI, 6.03-225; p 0.01) were significantly associated with the neoadjuvant chemotherapy efficacy. The accuracy for predicting a good response to chemotherapy based on ≥ 5 osteoclasts in a biopsy specimen in patients 46 years old was 85%. The number of mature osteoclasts in biopsy specimens is a simple factor for predicting the efficacy of chemotherapy before treatment, although further studies will be required to determine the underlying mechanism.

Purpose: Primary osteosarcoma of the spine is a rare osseous tumour. En bloc resection, in contrast to intralesional resection, is the only procedure able to provide Enneking appropriate (EA) margins, which has improved local control and survival of patients with primary osteosarcoma of the spine. The objective of this study is to compare the risk of local recurrence, metastases development and survival in patients with primary osteosarcoma of the spine submitted to Enneking appropriate (EA) and Enneking inappropriate (EI) procedures. Methods: A systematic search was performed on EBSCO, PubMed and Web of Science, between 1966 and 2018, to identify studies evaluating patients submitted to resection of primary osteosarcoma of the spine. Two reviewers independently assessed all reports. The outcomes were local recurrence, metastases development and survival at 12, 24 and 60 months. Results: Five studies (108 patients) were included for systematic review. These studies support the conclusion that EA procedure has a lower local recurrence rate (RR 0.33, 95% CI 0.17-0.66), a lower metastases development rate (RR 0.39, 95% CI 0.17-0.89) and a higher survival rate at 24 months (RR 1.78, 95% CI 1.24-2.55) and 60 months (RR 1.97, 95% CI 1.14-3.42) of follow-up; however, at 12 months, there is a non-significant difference. Conclusions: EA procedure increases the ratio of remission and survival after 24 months of follow-up. Multidisciplinary oncologic groups should weigh the morbidity of an en bloc resection, knowing that in the first year the probability of survival is the same for EA and EI procedures. These slides can be retrieved under Electronic Supplementary Material. info:eu-repo/semantics/publishedVersion

BACKGROUND: Osteosarcomas are a rare, heterogeneous and malignant group of bone tumors that have a high potential for metastasis and aggressive growth patterns. Treatment of metastasized osteosarcoma is often insufficient and research is compromised by problems encountered when culturing cells or analyzing genetic alterations due to the high level of intratumoral and intertumoral heterogeneity. The chick chorioallantoic membrane (CAM) model, a 3D-in-vivo-tumor-model, could potentially facilitate the investigation of osteosarcoma heterogeneity at an individual and highly specified level. OBJECTIVE: Objective was to establish the grafting and transplantation of different primary osteosarcoma tissue parts onto several consecutive CAMs for tumor profiling and investigation of osteosarcoma heterogeneity. METHODS: Various parts of primary osteosarcoma tissue were grafted onto CAMs and were transplanted onto another CAM for five to seven consecutive times, enabling further experimental analyzes. RESULTS: Primary osteosarcoma tissue parts exhibited satisfactory growth patterns and displayed angiogenic development on the CAM. It was possible to graft and transplant different tumor parts several times while the tissue viability was still high and tumor profiling was performed. CONCLUSIONS: Primary osteosarcoma tissue grew on several different CAMs for an extended time period and neovascularization of serial transplanted tumor parts was observed, improving the versatility of the 3D-in-vivo-tumor-model.

Osteosarcoma (OSA) arising from the digits, metatarsal and metacarpal bones is rare and may carry a better prognosis compared with other locations. The aim of this study was to retrospectively evaluate the biological behaviour, the progression free interval (PFI), the survival time (ST) and evaluate the effect of adjuvant chemotherapy for OSA affecting these bones. Medical records from two academic institutions were reviewed and 15 cases were included. Descriptive statistics were used for signalment and history. For estimation of median PFI and median ST, the Kaplan-Meier method was utilized. The prognostic effect of chemotherapy, lymphocyte and monocyte count was investigated. Log-rank analysis was used to compare PFI and ST between groups. The overall median PFI and median ST were 377 and 687 days, respectively. No significant differences were noted for any of the variables evaluated. In this study, dogs affected by OSA of digits, metacarpal and metatarsal bones appear to have a longer ST compared with dogs with OSA of other appendicular locations. A study with a larger number of patients is needed to confirm these results and investigate the potential benefit of adjuvant chemotherapy.

Uterine osteosarcomas are very rare and aggressive neoplasms. We present here a case of primary osteosarcoma of the uterus with peritoneal, omental and mesenteric metastasis. Treatment consisted of total abdominal hysterectomy with bilateral salpingo-oophorectomy followed by chemotherapy. However, she succumbed to her illness after 4 months of the surgery. Treatment options of this rare entity are unclear, and the prognosis is dismal. This case report is being presented to highlight the importance and rarity of primary uterine osteosarcoma and the need to differentiate it from the more common differentials, i.e. malignant mixed Mullerian tumour and uterine leiomyosarcoma.

Introduction: Osteosarcoma is a common primary sarcoma of the bone, constituting approximately 0.07% of all neoplasms. Rarely, some patients with osteosarcoma have multiple skeletal sites involvement, either synchronous or metachronous. Metachronous osteosarcoma is a rare form of osteosarcoma in which osteosarcomatous lesions occur distant from the primary osteosarcoma site more than 6 months after the initial treatment without pulmonary manifestation. Case Report: We present a case of metachronous osteosarcoma in a 16-year-old female who was initially treated for non-metastatic osteosarcoma of the left distal femur with neoadjuvant chemotherapy, wide excision, endoprosthesis and followed by adjuvant chemotherapy. Serial follow-up did not show evidence of distant metastasis. Nevertheless, she presented 3 years later with a progressively enlarging left proximal forearm mass and re-staging revealed an osteosarcoma of the same histological type. She underwent combination of chemo- and radio- therapy but subsequently succumbed to the disease due to lung metastasis. Conclusion: With the advent of diagnostic and therapeutic techniques, the 5-year survival rate of nonmetastatic osteosarcoma ranges around 60-70%. Albeit it is rare, a metachronous osteosarcoma can present difficult challenges to the treating physician.

Background:Osteosarcoma cancer stem cells (CSCs) are defined as a subpopulation of osteosarcoma cells, which have the ability of self-renewal, proliferation and differentiation. This study aimed to identify CSCs from human osteosarcomain vitro.Methods:Osteosarcoma CSCs were isolated and cultured with sphere-forming assay technique on an ultra-low well attachment surface plate. After sarcosphere colonies were formed, we conducted reverse transcriptase-polymerase chain reaction (RT-PCR) to detect the expression of genes of embryonic stem cells such asNANOG, Oct3/4, STAT3 and gene of MSC CD133. Immunofluorescence analysis (IFA) of alkaline phosphatase (ALP), osteocalcin, and CD 133 was also performed to see the expression of osteosarcoma CSC surface protein with immuno-enzymatic staining principle. We also performed alizarin red staining to evaluate calcification in osteosarcoma CSCs.Results:The culture sphere-of the osteosarcoma cells showed three dimension round shaped colonies (sarcospheres) in slightly hypoxicand serum free condition which was not attached to the substrate with tight density. RT-PCR demonstrated that sarcospheres expressed genes which encodeNANOG, Oct3/4 STAT 3, but not for CD 133. IFA showed positive protein expression of ALP, osteocalcin and CD 133 which was moderate, strong, and weak positive respectively. Sarcospheres also had a positive reaction toward alizarin red staining.Conclusion:Osteosarcoma CSCs could be isolated from human osteosarcoma by sphere-forming assay technique and characterized by the expression of genes of embryonic stem cells,such asNANOG, Oct3/4, STAT3 and IFA of ALP, osteocalcin, and CD 133.

Primary calvarial (excluding jaw) osteosarcoma is rare. We report two cases highlighting their unusual presentation and histopathologic variability – a 30-year-old female who presented with progressively increasing headache and a rapidly growing frontal swelling and a 16-year-old girl who had a rapidly growing mass in the left frontoparietal region which had recurred thrice but was otherwise asymptomatic. The lesions were osteolytic on neuroimaging and histopathological examination confirmed osteosarcoma. These two unusual cases are discussed along with a brief review of literature.