Establishment and Characterization of a Recurrent Osteosarcoma Cell Line: OSA 1777.

Osteosarcoma (OSA) is the most common primary bone malignancy overall and is responsible for considerable adolescent mortality. Approximately 850 patients are newly diagnosed with OSA in the United States each year. While the 5-year survival rate for localized OSA has improved from <20% over 40 years ago to over 65% today, progress has dwindled over the past three decades. Therapeutic stagnation has occurred, in part, as a result of limited preclinical models and the overall heterogeneity of OSA among patients. In this study, we report the establishment and characterization of a novel OSA cell line: OSA 1777. This cell line was isolated from the recurrent tumor specimen of a 19-year-old female who initially experienced 99% tumor necrosis after neoadjuvant chemotherapy and eventually had local recurrence and metastases. We present OSA 1777 growth characteristics, tumor markers, chemotherapeutic sensitivities, and oncogenic spheroid formation. In a two-dimensional (2D) monolayer culture, OSA 1777 exhibited a spindle shape and 60 h doubling time. STR DNA profiling revealed a unique genomic identity not matching any existing human cancer cell lines from the ATCC or DSMZ databases. Consistent with the mesenchymal origin, western blot was positive for vimentin and negative for the carcinoma marker cytokeratin. Within three-dimensional (3D) culture, the cells formed spheroids of similar patterning and smaller size compared with MNNG-HOS and U2OS cell lines. The chemotherapeutic drug sensitivity of OSA 1777 was evaluated in both 2D and 3D culture systems. In summary, we report OSA 1777 as a novel biological model of OSA amenable to future studies focused on OSA that recurs despite an initially strong chemotherapeutic response. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:902-910, 2020.

Negative regulation of REST on NR2B in spinal cord contributes to the development of bone cancer pain in mice.

In this study, C3H/HeNCrlVr mice are implanted with sarcoma NCTC 2472 cells into the intramedullary space of the femur to induce ongoing bone cancer-related pain behaviors. During the progress of the bone cancer pain, the down-regulation in spinal REST (Neuron-restrictive silencer factor, NRSF/REST) with concomitant up-regulation in spinal NR2B (2B subunit of N-methyl-D-aspartate receptor, NR2B) protein expression are observed at days 5, 7, 10 and 14 post-inoculation. Immunofluorescence assay shows that almost all of REST and NR2B-positive signals encompass NeuN (neuron-specific nuclear protein, a neuronal marker)-positive signals in spinal cord of sham and tumor-bearing mice. Different from previous researches involved in the main distribution of REST in neural progenitors, the expression of REST in mature neurons in spinal cord of adult mice is observed. Intrathecal administration of AS-ODN of REST at days 0, 2, 4 and 6 post-inoculation further enhances expression of spinal NR2B at day 7 post-inoculation, which suggests the reduced suppression of spinal REST on NR2B during the development of bone cancer pain. In summary, our study provides the evidence that the negative regulation of REST on NR2B in spinal cord takes part in the exacerbation of bone cancer pain.

[Non-ossifying fibroma (metaphyseal fibrous defect)].

Non-ossifying fibroma (NOF) or metaphyseal fibrous defect (MFD) is benign fibroblast proliferation with the presence of osteoclast-like multinucleated giant cells. The most cases of NOF/MFD occur in the metaphysis of the long tubular bones of the lower extremities, more commonly in the metaphysis of the femur and in the proximal metaphysis of the tibia. This lesion has a characteristic X-ray pattern and requires no surgical intervention, except for cases of a pathologic fracture or a risk for the latter. The paper analyzes 35 NOF/MFD cases in children and adolescents. It has been found that one and all patients have undergone surgery, suggesting the low awareness of this abnormality among radiodiagnosticians, pathologists, and surgeons.

[A rare case of concurrent follicular and columnar cell variants of papillary thyroid carcinoma].

The paper describes a rare case of concurrent two different histological (follicular and columnar cell) variants of papillary carcinoma in one thyroid with columnar cell metastases to the lymph nodes and femoral bone. There are morphological features of and differences in BRAF status in the cells of two variants of papillary thyroid carcinoma.

Identification of CBX3 and ABCA5 as putative biomarkers for tumor stem cells in osteosarcoma.

Recently, there has been renewed interest in the role of tumor stem cells (TSCs) in tumorigenesis, chemoresistance, and relapse of malignant tumors including osteosarcoma. The potential exists to improve osteosarcoma treatment through characterization of TSCs and identification of therapeutic targets. Using transcriptome, proteome, immunophenotyping for cell-surface markers, and bioinformatic analyses, heterogeneous expression of previously reported TSC or osteosarcoma markers, such as CD133, nestin, POU5F1 (OCT3/4), NANOG, SOX2, and aldehyde dehydrogenase, among others, was observed in vitro. However, consistently significantly lower CD326, CD24, CD44, and higher ABCG2 expression in TSC-enriched as compared with un-enriched osteosarcoma cultures was observed. In addition, consistently higher CBX3 expression in TSC-enriched osteosarcoma cultures was identified. ABCA5 was identified as a putative biomarker of TSCs and/or osteosarcoma. Lastly, in a high-throughput screen we identified epigenetic (5-azacytidine), anti-microtubule (vincristine), and anti-telomerase (3,11-difluoro-6,8,13-trimethyl- 8H-quino [4,3,2-kl] acridinium methosulfate; RHPS4)-targeted therapeutic agents as candidates for TSC ablation in osteosarcoma.

A bone-seeking clone exhibits different biological properties from the ACHN parental human renal cell carcinoma in vivo and in vitro.

Metastatic bone disease caused by renal cell carcinoma (RCC) occurs frequently. Very little is currently known about the mechanism of preferential metastasis of RCC to bone. We hypothesize that RCCs that develop bone metastases have the capacity to facilitate their colonization in bone. To examine this hypothesis, we established bone-seeking (ACHN-BO) clones of the human RCC cell line ACHN by repeated four passages in nude mice and in vitro of metastatic cells obtained from bone. These clones were examined for distinguishing biological characteristics and compared with the ACHN parental cells (ACHN-P) in vivo and in vitro. Our results showed that the ACHN-BO cell line could be successfully obtained by in vivo selection through the lateral tail vein. This approach results in the development of multiple osteolytic lesions in the distal femora and proximal tibiae within four weeks after inoculation, with a success rate of 85-100% and no additional comorbidity. ACHN-P cells developed metastases in lung, bone, brain, ovary and adrenal glands. Conversely, ACHN-BO cells exclusively metastasized to bones with larger osteolytic lesions. Compared with the ACHN-P cell line, the proliferation ability in ACHN-BO6 was increased by 9.68 and 6.42%, respectively (P<0.05), while the apoptotic ratio decreased significantly (P<0.05) and cells were blocked in the S phase with suppressed migration and invasion capacities. The ACHN-BO6 cell line produced greater amounts of the pro-angiogenic factors VEGF and TGF-ß than ACHN-P. Our data suggest that these phenotypic changes allow RCC cells to promote osteoclastic bone resorption, survive and proliferate in bone, which consequently leads to the establishment of bone metastases. This model provides a reliable reproduction of the clinical situation and, therefore, is suitable for designing and evaluating more effective treatments for RCC bone metastasis.

MLL-ENL leukemia burden initiated in femoral diaphysis and preceded by mature B-cell depletion.

BACKGROUND: Molecular and cellular events that resulted in leukemia development are well characterized but initial engraftment and proliferation of leukemic cells in bone marrow and early modifications of the bone marrow microenvironment induced by engrafted leukemic cells remain to be clarified. DESIGN AND METHODS: After retro-orbital injection of 1,000 leukemic cells expressing Mixed Lineage Leukemia-Eleven Nineteen Leukemia fusion protein in non-conditioned syngenic mice, kinetics of leukemic burden and alterations of femoral hematopoietic populations were followed using an in vivo confocal imaging system and flow cytometry. RESULTS: Three days after injection, 5% of leukemic cells were found in femurs. Little proliferation of engrafted leukemic cells could then be detected for more than two weeks while the number of femoral leukemic cells remained stable. Twenty days after injection, leukemic cells preferentially proliferated in femoral diaphysis where they formed clusters on the surface of blood vessels and bone. B220(+) lymphoid cells were found near these leukemic cell clusters and this association is correlated with a decreased number of femoral B220(+)IgM(+) cells. Increasing the number of injected leukemic cells or conditioning recipient mice with γ-irradiation resulted in leukemic cell development in diaphysis and knee. Competition experiments indicate that proliferation but not engraftment is a rate-limiting factor of leukemic cells spreading in diaphysis. Finally, 30 days after injection leukemia developed. CONCLUSIONS: After retro-orbital injection of 1,000 leukemic cells expressing Mixed Lineage Leukemia-Eleven Nineteen Leukemia into syngenic mice, leukemic cell burden preferentially initiates in femoral diaphysis and is preceded by changes of femoral B-lymphoid populations.

[Clinicopathologic features of primary osteosarcoma in elderly patients].

OBJECTIVE: To study the clinical manifestations, radiologic findings, pathologic diagnosis and differential diagnosis of primary osteosarcoma in elderly patients. METHODS: Twelve cases of primary osteosarcoma occurring in patients older than 60 years were encountered during the period from 1985 to 2010. The clinical manifestations, radiologic features and pathologic findings were studied and the follow-up data were analyzed. RESULTS: The sites of involvement included long bones (number = 7), ilium (number = 1), craniofacial bones (number = 2) and soft tissue (number = 2). Radiologic examination showed a mixture of osteosclerotic and osteolytic lesions in 10 patients, soft tissue lesions with high-density areas in 2 patients and soft tissue lesions with periosteal reaction in 8 patients. Histologically, most cases showed features of conventional osteosarcoma. There were 2 cases of malignant fibrous histiocytoma-like osteosarcoma, 2 cases of chondroblastic osteosarcoma and 1 case of well-differentiated intraosseous osteosarcoma. Immunohistochemical study played little role in pathologic diagnosis. Ten patients had undergone amputation, including one patient who had received adjuvant chemotherapy beforehand. Nine patients had follow-up information available. Three of them died of lung metastasis and 1 died of cardiovascular disease. CONCLUSIONS: Primary osteosarcoma rarely occurs in elderly patients and can easily be missed. Correlation with clinical, radiologic and histologic features is important for arriving at a correct diagnosis.

Cytokine synthesis by chondroblastoma: relation to local inflammation.

PURPOSE: To investigate cytokine production by chondroblastoma in inducing local inflammation and adjacent-joint arthritis. METHODS: Immunohistochemical analyses of curetted tissues using anti-human interleukin (IL)-1 beta, IL- 6, IL-8, and tumour necrosis factor (TNF)-alpha were performed for 6 patients with chondroblastoma and 3 patients with giant cell tumour (GCT) of bone. In addition, prostaglandin E2, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, and TNF-alpha in the cyst fluid of one of the patients with chondroblastoma and 2 with GCT of bone were measured using immunoassay kits. RESULTS: More positive staining for IL-1 beta, IL-8, IL- 6, and TNF-alpha was shown in chondroblastoma than GCT of bone samples. Osteoclast-like giant cells in chondroblastomas showed positive staining for IL- 6 only. In addition, concentrations of IL-4, IL-6, and IL-8 in the cyst fluid were higher in the one patient with chondroblastoma than the 2 patients with GCT of bone. CONCLUSION: Cytokines such as IL-1 beta, IL-8, TNF-alpha, and particularly IL-6 play an important role in local inflammation in patients with chondroblastoma.

S100A6 expression and function in human osteosarcoma.

There is a critical need to identify markers that can accurately identify existing or predict future metastatic disease in patients with osteosarcoma since the majority of patients present with undetectable micrometastatic disease. We previously reported S100A6 is overexpressed in human osteosarcoma and increased expression of S100A6 by immunohistochemistry correlated with decreased clinical metastasis. We have established 11 primary cultures from biopsies of patients with osteosarcoma and ten of the 11 primary cultures have increased expression of S100A6 relative to normal human osteoblasts. To further explore possible mechanisms for metastasis suppression previously reported, we used in this report siRNA-mediated knockdown of S100A6 in four commonly used human osteosarcoma lines, then examined their cell adhesion, migration, and invasion properties. Knockdown of S100A6 expression inhibited cell adhesion and promoted cell migration and invasion in these lines. Conversely, S100A6 overexpression enhanced cell adhesion and inhibited cell invasion. Our data demonstrate S100A6 is commonly overexpressed in human osteosarcoma. S100A6 may inhibit osteosarcoma metastasis by promoting cell adhesion and inhibiting cell motility and invasion. Thus, S100A6 may be considered a potential marker for human osteosarcoma with prognostic value for identifying patients without metastases.

Hypoxia markers in human osteosarcoma: an exploratory study.

Neoplastic cells growing under hypoxic conditions exhibit a more aggressive phenotype by activating a cascade of molecular events partly mediated by hypoxia-inducible transcription factor (HIF-1alpha) and vascular endothelial growth factor (VEGF). The roles of these markers have been studied previously in several cancer lines. We ascertained the frequency of HIF-1alpha expression, VEGF expression, the degree of neovascularization, and cell proliferation in osteosarcoma samples. Samples from osteosarcoma patients were assessed for HIF-1alpha and VEGF protein expression using immunohistochemistry, neovascularization using antibodies for Factor VIII, and cell proliferation using the Ki-67 labeling index. Associations between these parameters and clinical features were examined. HIF-1alpha staining was positive in 35% of patients and metastases were present in 61% of these HIF-1alpha-positive patients. VEGF protein expression was detected in 69% of patients, 92% of whom were female. We observed an insignificant trend for a higher frequency of VEGF expression in the high-grade as compared to low-grade osteosarcoma. We observed no association between vascular density and proliferation index and any clinical parameters. We found an association between HIF-1alpha expression and metastatic disease and between VEGF expression and female gender.

Predictive value of bone marrow accumulation of Tc-99m tetrofosmin for subsequent development of distant metastases in breast cancer.

OBJECTIVE: We evaluated the predictive value of bone marrow accumulation of technetium (Tc)-99m tetrofosmin in patients with breast cancer for distant metastases in comparison with conventional prognostic factors such as clinical stage, tumor size, axillary lymph node (Node) status, and estrogen receptor (ER) status. METHODS: Bone marrow scans with Tc-99m tetrofosmin were performed on 64 patients with breast cancer who had no clinical evidence of distant metastases. Accumulation in the femoral marrow was classified into four patterns, no detectable, lower, higher, and intensively higher. Higher or intensively higher pattern was interpreted as abnormal. Thirty-six patients with abnormal accumulation (marrow-positive group) and 28 patients without abnormal accumulation (marrow-negative group) were enrolled in the follow-up study. The mean length of observation after scans was approximately 3 years. The predictive value of femoral marrow status and conventional prognostic factors for distant metastases was evaluated by statistical analysis. RESULTS: Univariate analysis showed a significantly higher incidence of subsequent bone metastases (36%>4%; P<0.005), and distant metastases (69%>18%; P<0.001) in the marrow-positive group when compared with the marrow-negative group. Conventional prognostic factors except tumor size were also significantly associated with the development of distant metastases; 77% in clinical stage 3>39% in clinical stages 1, 2, P<0.05; 64% in Node-positive>29% in Node-negative, P<0.01; and 70% in ER negative>27% in ER positive, P<0.005. These conventional factors were not significantly associated with bone metastases. The Cox proportional hazard ratio for bone metastases was markedly higher in femoral marrow status (hazard ratio=11.07). The distant metastases-free survival was significantly reduced in ER negative (P<0.0005), Node-positive (P=0.0215), and clinical stage 3 patients (P=0.0163). On the other hand, a more marked difference was observed in the femoral marrow status (P<0.0001). The hazard ratio for distant metastases was 2.44 in clinical stage, 2.74 in tumor size, 2.74 in Node, and 3.68 in ER, which were each independent prognostic factors associated with distant metastases. However, femoral marrow status was markedly associated with distant metastases (hazard ratio=5.27). CONCLUSIONS: Bone marrow accumulation of Tc-99m tetrofosmin can be a promising prognostic factor independent of conventional prognostic factors for predicting development of not only bone metastases but also distant metastases in breast cancer.

Disseminated metastatic disease of osteosarcoma of the femur in the abdomen: unusual metastatic pattern on Tc-99m MDP bone scan.

A 25-year-old patient with osteosarcoma of the right distal femur underwent a bone scintigraphy with Tc-99m methylene diphosphonate (MDP). Whole-body bone scan revealed extensive metastatic disease in the abdominal region. Abdominal computerized tomography confirmed the presence of ascites and calcified masses on the greater omentum and peritoneal surfaces. Here we describe a case of unusual metastatic pattern of an osteosarcoma showing extensive intraabdominal metastases without prominent lung involvement after intensive chemotherapy.

Rhemium-186-monoaminemonoamidedithiol-conjugated bisphosphonate derivatives for bone pain palliation.

To develop a radiopharmaceutical for the palliation of painful bone metastases based on the concept of bifunctional radiopharmaceuticals, we synthesized a bisphosphonate derivative labeled with rhenium-186 (186Re) that contains a hydroxyl group at the central carbon of its bisphosphonate structure, we attached a stable 186Re-MAMA chelate to the amino group of a 4-amino butylidene-bisphosphonate derivative [N-[2-[[4-[(4-hydroxy-4,4-diphosphonobutyl)amino]-4-oxobutyl]-2-thioethylamino]acetyl]-2-aminoethanethiolate] oxorhenium (V) (186Re-MAMA-HBP) and we investigated the effect of a hydroxyl group at the central carbon of its bisphosphonate structure on affinity for hydroxyapatite and on biodistribution by conducting a comparative study with [N-[2-[[3-(3,3-diphosphonopropylcarbamoyl)propyl]-2-thioethylamino]acetyl]-2-aminoethanethiolate] oxorhenium (V) (186Re-MAMA-BP). The precursor of 186Re-MAMA-HBP, trityl (Tr)-MAMA-HBP, was obtained by coupling a Tr-MAMA derivative to 4-amino-1-hydroxybutylidene-1,1-bisphosphonate. 186Re-MAMA-HBP was prepared by a reaction with 186ReO(4-) and SnCl2 in citrate buffer after the deprotection of the Tr groups of Tr-MAMA-HBP. After reversed-phase high-performance liquid chromatography, 186Re-MAMA-HBP had a radiochemical purity of over 95%. Compared with 186Re-MAMA-BP, 186Re-MAMA-HBP showed a greater affinity for hydroxyapatite beads in vitro and accumulated a significantly higher level in the femur in vivo. Thus, the introduction of a hydroxyl group into 186Re complex-conjugated bisphosphonates would be effective in enhancing accumulation in bones. These findings provide useful information on the design of bone-seeking therapeutic radiopharmaceuticals.

[Oncogenic osteomalacia in a 49-year-old female patient]. / Onkogene Osteomalazie bei einer 49-jährigen Patientin.

HISTORY AND CLINICAL FINDINGS: A 49-year-old woman presented with increased bone and muscle pain of the left thigh and also of the left ribs since 1 1/2 year. 2 osteolytic regions on the left proximal femur were punctured but no tumour-like changes were found. INVESTIGATIONS: Laboratory tests showed a severe hypophosphataemia (0.33 mmol/l), increased serum alkaline phosphatase activity and moderate elevated parathyroid hormone. Renal loss of phosphate was measured under phosphate substitution. The bone mineral density (DXA) was decreased. With octreotid scintigraphy a somatostatine receptor positive tumour was detected on the right proximal thigh and magnetic resonance scanning confirmed localization of the tumour. TREATMENT AND COURSE: Treatment with oral phosphate and calcitriol improved the complaints presently. After total removal of a mesenchymal tumour, normalization of serum phosphate occurred and the patient did not require any medicine and did not have complaints anymore. CONCLUSION: In a case of uncertain hypophosphataemic osteomalacia in adults it is essential to search for a tumour after exclusion of the rare differential diagnoses to enable a causal treatment of a potentially oncogenic osteomalacia.

[Clinicopathologic study of 10 cases of osteomalacia or rickets-associated mesenchymal tumors].

OBJECTIVE: To study the clinicopathologic features of osteomalacia or rickets-associated mesenchymal tumors. METHODS: The clinical and pathologic findings of 10 cases of osteomalacia or rickets-associated mesenchymal tumors were evaluated. Hematoxylin and eosin stain, immunohistochemistry and histochemistry were performed on the archival paraffin sections. RESULTS: Amongst the 10 patients studied, 6 were males and 4 were females. Their age at the time of operation ranged from 28 to 69 years ( mean = 45.6 years). A history of long-standing bone pain, arthralgia, limitation in movement, hypophosphatemia and hyperphosphaturia was present in all cases. The duration of symptoms ranged from 2 to 27 years (mean = 9.6 years). The tumor size ranged from 1 to 7 cm (mean size = 3.52 cm). Microscopically, the tumors were composed of various mesenchymal cells, including spindled fibroblast-like cells, adipocytes, chondroid cells and mucinous cells. The background was rich in blood vessels. In 8 of the 10 cases, there was also dystrophic calcification in an unusual flocculent or "grungy" pattern. Peripheral woven bone shell formation was noted in 2 cases and non-urate crystal deposition in 2 cases. Mitotic figures were rare in 9 cases. In 1 of the 10 cases however, mitotic figures and bizarre cells were commonly encountered. On immunohistochemical study, the tumor cells were all positive for vimentin. There was focal positivity for smooth muscle actin and CD34 in 5 and 3 cases respectively. The staining for desmin, S-100 and AE1/AE3 was negative. Ki-67 proliferation index was less than 4% in 8 cases and 30% in 1 case. Alcian blue-positive mucinous matrix and mucinous degeneration around vessels were noted in 8 cases. CONCLUSIONS: Most of the osteomalacia or rickets-associated tumors are either benign or low-grade malignant mesenchymal tumors. They can be mistaken as other neoplasms due to the morphologic heterogeneity present. Thorough understanding of the associated clinical features and laboratory investigation results is helpful in arriving at the correct diagnosis.

VEGF expression in osteosarcoma correlates with vascular permeability by dynamic MRI.

Dynamic enhanced magnetic resonance imaging has been used to assess tumor angiogenesis in osteosarcoma. Vascular endothelial growth factor has been shown to correlate with pulmonary metastasis and a poor prognosis in osteosarcoma. The purpose of this investigation was to determine whether vascular endothelial growth factor expression in osteosarcoma correlates with vascular permeability detected by dynamic enhanced magnetic resonance imaging and to explore the role of dynamic enhanced magnetic resonance imaging as a noninvasive means of assessing tumor angiogenic activity. Fifty-five osteosarcoma patients with osteosarcoma enrolled in a treatment protocol that included dynamic enhanced magnetic resonance imaging. In 15 patients, tumor tissues were available for vascular endothelial growth factor immunohistochemical studies. A two-compartment model used the exchange rate constants (kep) between the plasma and tumor compartments to quantify vascular permeability during dynamic magnetic resonance imaging studies. Immunohistochemical staining for vascular endothelial growth factor was graded according to the intensity and number of positively stained cells. Vascular endothelial growth factor-positive tumors showed higher mean vascular permeability when compared with vascular endothelial growth factor-negative tumors. Vascular permeability also correlated with increasing vascular endothelial growth factor expression. The preliminary results in this study show an association between vascular endothelial growth factor and dynamic MR signal enhancement in osteosarcoma. Dynamic enhanced magnetic resonance imaging should be investigated as a means to prognosticate osteosarcoma patients with osteosarcoma according to their tumor angiogenic activity.