Conventional Osteosarcoma Research Articles

A Novel System for the Surgical Staging of Primary High-grade Osteosarcoma: The Birmingham Classification.

Chemotherapy response and surgical margins have been shown to be associated with the risk of local recurrence in patients with osteosarcoma. However, existing surgical staging systems fail to reflect the response to chemotherapy or define an appropriate safe metric distance from the tumor that will allow complete excision and closely predict the chance of disease recurrence. We therefore sought to review a group of patients with primary high-grade osteosarcoma treated with neoadjuvant chemotherapy and surgical resection and analyzed margins and chemotherapy response in terms of local recurrence. (1) What predictor or combination of predictors available to the clinician can be assessed that more reliably predict the likelihood of local recurrence? (2) Can we determine a better predictor of local recurrence-free survival than the currently applied system of surgical margins? (3) Can we determine a better predictor of overall survival than the currently applied system of surgical margins? This retrospective study included all patients with high-grade conventional osteosarcomas without metastasis at diagnosis treated at one center between 1997 and 2012 with preoperative chemotherapy followed by resection or amputation of the primary tumor who were younger than age 50 years with minimum 24-month followup for those still alive. A total of 389 participants matched the inclusion criteria. Univariate log-rank test and multivariate Cox analyses were undertaken to identify predictors of local recurrence-free survival (LRFS). The Birmingham classification was devised on the basis of two stems: the response to chemotherapy (good response = ≥ 90% necrosis; poor response = < 90% necrosis) and margins (< 2 mm or ≥ 2 mm). The 5-year overall survival rate was 67% (95% confidence interval [CI], 61%-71%) and 47 patients developed local recurrence (12%). Intralesional margins (hazard ratio [HR], 9.9; 95% CI, 1.2-82; p = 0.03 versus radical margin HR, 1) and a poor response to neoadjuvant chemotherapy (HR, 3.8; 95% CI, 1.7-8.4; p = 0.001 versus good response HR, 1) were independent risk factors for local recurrence (LR). The best predictor of LR, however, was a combination of margins ≤ 2 mm and a less than 90% necrosis response to chemotherapy (Birmingham 2b HR, 19.6; 95% CI, 2.6-144; p = 0.003 versus Birmingham 1a; margin >2 mm and more than 90% necrosis HR, 1). Two-stage Cox regression model and higher Harrell's C statistic demonstrate that the Birmingham classification was superior to the Musculoskeletal Tumor Society (MSTS) margin classification for predicting LR (Harrell's C statistic Birmingham classification 0.68, MSTS criteria 0.59). A difference in overall survival was seen between groups of the Birmingham classification (log-rank test p < 0.0001), whereas the MSTS margin system was not discriminatory (log-rank test p = 0.14). Based on these observations, we believe that a combination of the recording of surgical margins in millimeters and the response to neoadjuvant chemotherapy can more accurately predict the risk of local recurrence than the current MSTS system. A multicenter collaboration study initiated by the International Society of Limb Salvage is recommended to test the validity of the proposed classification and if these findings are confirmed, this classification system might be considered the standard practice in oncology centers treating patients with osteosarcomas and allow more effective communication of margin status for research. Level IV, prognostic study.

Over-expression of Sox4 and β-catenin is associated with a less favorable prognosis of osteosarcoma.

The purpose of this study was to examine the association of the expression of Sox4 and β-catenin with the prognosis of osteosarcoma. A total of 108 cases of conventional osteosarcoma were involved in this study and 28 cases of osteochondroma served as controls. The expression of Sox4 and β-catenin was detected by using immunohistochemical staining and Western blotting. The results showed that Sox4 and β-catenin were over-expressed in 67 (62.03%) and 62 (57.41%) of 108 osteosarcoma cases, while in only 3 (10.71%) and 5 (17.86%) of 28 controls, respectively (P<0.05 for all). The expression of Sox4 and β-catenin was associated with the distant metastasis, pathological grade and Enneking stage of patients with osteosarcoma (P<0.05 for all). The mean overall survival time and the 5-year-survival rate in osteosarcoma patients with Sox4 and β-catenin over-expressed were significantly reduced as compared with those in Sox4 and β-catenin low-expression group (P<0.05 for all). Cox multifactor regression analysis revealed that the distant metastasis, Enneking stage, and the expression of Sox4 and β-catenin were independent risk factors of patients with osteosarcoma (P<0.05 for all). The findings indicated that overexpression of Sox4 and β-catenin is associated with a poor prognosis of osteosarcoma.

Giant cell rich osteosarcoma revisited-diagnostic criteria and histopathologic patterns, Ki67, CDK4, and MDM2 expression, changes in response to bisphosphonate and denosumab treatment.

Defining giant cell-rich osteosarcoma (GCRO) as "an osteosarcoma in which more than 50% of the tumor consists of numerous uniformly distributed osteoclastic giant cells amidst oval or spindle mononuclear cells embedded in a fibrovascular stroma," eight such cases identified among 265 cases of osteosarcoma were analysed. Their age ranges from 11 to 33years, with peak incidence in the second decade and equal sex distribution. Seventy-five percent presented with pain, commonest in the knee, affecting the metaphysis. Most appeared radiologically as well-circumscribed expansile multiloculated osteolytic lesions, and many are displayed periosteal reaction. They showed several distinct histologic patterns: the stromal and giant cell, fibrohistiocytic, aneurysmal-cystic, osteoblastoma-like, and parosteal and fibrous dysplasia-like patterns. Focal subtle lacelike osteoid deposition, permeative infiltration into adjacent native bony trabeculae and over 30% Ki67 proliferative index were characteristic. There was no CDK4 and MDM2 amplification. In those having bisphosphonate and denosumab treatment, there was limited focal necrosis with reduction in the number of giant cells and broad trabecular woven bone formation but no giant osteoclast was seen. Two patients with initial diagnosis of giant cell tumor treated by curettage and local resection pursued aggressive clinical courses, died after 14 and 21months. The others survived 12 to 110months. GCRO accounts for about 3% of all osteosarcomas and apart from its more frequent diaphyseal location and associated normal bone-specific alkaline phosphate levels; it shares with conventional high-grade osteosarcoma the same patient demographics, sites of occurrence, absence of CDK4 and MDM2 amplification, and probably clinical course.

Chondroblastic variant of extraskeletal osteosarcoma of thigh: A rare case report

Extraskeletal osteosarcoma (ESOS) is a rare, malignant neoplasm comprising 1% of mesenchymal tumors. We hereby report a case of ESOS of the thigh which was diagnosed on histopathology. Magnetic resonance imaging showed two lobulated markedly heterogeneous masses in the subcutaneous fat of the lateral aspect of thigh measuring 5 cm × 4.2 cm × 4.2 cm and 7.1 cm × 5.7 cm × 6.0 cm. Femur and major neurovascular bundles were unremarkable. Excision biopsy revealed a circumscribed tumor comprising sheets and fascicles of spindle cells and abundant osteoid produced directly by malignant cells. Malignant cartilage and extensive areas of calcification were also seen. Hence, a diagnosis of ESOS was given. Prompt diagnosis and surgical resection are mandatory, because unlike conventional osteosarcoma, ESOSs are more aggressive and do not respond to chemotherapy.

Epithelioid Osteosarcoma with Pseudo-Glandular Structures: A Case Report and Review of the Literature

Epithelioid osteosarcoma is an uncommon subtype of conventional osteosarcoma that comprised of both typical osteosarcoma component and epithelioid cells. It often occurs in adolescent and children. In here, we report a case of epithelioid osteosarcoma of a 12-year-old Chinese boy. Microscopically, the tumor contains large epithelioid cell with abundant cytoplasm and osteoid forming malignant cells, and partially, the tumor cells arranged in pseudoglandular structures. To our knowledge, it is the first report of epithelioid osteosarcoma with pseudo-glandular structure. The epithelioid cells expressed vimentin and epithelial membrane antigen but did not expressed cytokeratin.

MEK inhibition induces apoptosis in osteosarcoma cells with constitutive ERK1/2 phosphorylation.

Conventional high-grade osteosarcoma is the most common primary bone cancer with relatively high incidence in young people. Recurrent and metastatic tumors are difficult to treat. We performed a kinase inhibitor screen in two osteosarcoma cell lines, which identified MEK1/2 inhibitors. These inhibitors were further validated in a panel of six osteosarcoma cell lines. Western blot analysis was performed to assess ERK activity and efficacy of MEK inhibition. A 3D culture system was used to validate results from 2D monolayer cultures. Gene expression analysis was performed to identify differentially expressed gene signatures in sensitive and resistant cell lines. Activation of the AKT signaling network was explored using Western blot and pharmacological inhibition. In the screen, Trametinib, AZD8330 and TAK-733 decreased cell viability by more than 50%. Validation in six osteosarcoma cell lines identified three cell lines as resistant and three as sensitive to the inhibitors. Western blot analysis of ERK activity revealed that sensitive lines had high constitutive ERK activity. Treatment with the three MEK inhibitors in a 3D culture system validated efficacy in inhibition of osteosarcoma viability. MEK1/2 inhibition represents a candidate treatment strategy for osteosarcomas displaying high MEK activity as determined by ERK phosphorylation status.

P16 expression predicts neoadjuvant tumor necrosis in osteosarcomas: reappraisal with a larger series using whole sections

The presence of greater than or equal to 90% necrosis after neoadjuvant chemotherapy is a favorable prognostic factor in osteosarcomas. A recent study using tissue microarrays of 40 conventional osteosarcomas showed that p16 expression independently predicted the necrotic response to neoadjuvant chemotherapy. In this study, we investigated this finding using whole sections in a larger group of osteosarcomas. Cases of 83 patients who had pretreatment biopsies and received neoadjuvant chemotherapy and surgical resection were collected from 3 reference hospital archives. Age, sex, tumor size, tumor subtype, location, and percentage of tumor necrosis were recorded; 4-μm sections from pretreatment biopsies were stained for p16. More than 30% strong nuclear staining was regarded as positive. The median age was 17 years (5-68 years), and male/female ratio was 2.3. The mean tumor diameter was 9.9 cm (2-30 cm). Tumors were most commonly of the osteoblastic type (60%) and located at the femur (47%). p16 positivity was seen in 66% of the patients. The median pathologic necrosis was 65%, and 39% of the patients responded favorably (≥%90 necrosis) to neoadjuvant therapy. In univariate analysis, p16 expression significantly correlated with greater than or equal to 90% response (P = .022). On multivariate analysis, p16 expression (odds ratio [OR], 7.71; P = .008), female sex (OR, 8.62; P = .006), and smaller tumor size (OR, 0.86; P = .023) were independent predictors of favorable response to neoadjuvant chemotherapy. We confirmed the finding that p16 expression predicts postchemotherapy necrotic response in conventional osteosarcomas.

The prognostic and therapeutic factors which influence the oncological outcome of parosteal osteosarcoma.

The aim of this study was to evaluate the prognostic and therapeutic factors which influence the oncological outcome of parosteal osteosarcoma. A total of 80 patients with a primary parosteal osteosarcoma were included in this retrospective study. There were 51 females and 29 males with a mean age of 29.9 years (11 to 78). The mean follow-up was 11.2 years (1 to 40). Overall survival was 91.8% at five years and 87.8% at ten years. Local recurrence occurred in 14 (17.5%) patients and was associated with intralesional surgery and a large volume of tumour. On histological examination, 80% of the local recurrences were dedifferentiated high-grade tumours. A total of 12 (14.8%) patients developed pulmonary metastases, of whom half had either a dedifferentiated tumour or a local recurrence. Female gender and young age were good prognostic factors. Local recurrence was a poor prognostic factor for survival. Medullary involvement or the use of chemotherapy had no impact on survival. The main goal in treating a parosteal osteosarcoma must be to achieve a wide surgical margin, as inadequate margins are associated with local recurrence. Local recurrence has a significant negative effect on survival, as 80% of the local recurrences are high-grade dedifferentiated tumours, and half of these patients develop metastases. The role of chemotherapy in the treatment of parosteal osteosarcoma is not as obvious as it is in the treatment of conventional osteosarcoma. The mainstay of treatment is wide local excision.

Surface osteosarcoma: Clinical features and therapeutic implications

IntroductionSurface osteosarcoma are rare variant of osteosarcoma that include parosteal osteosarcoma, periosteal osteosarcoma and high grade surface osteosarcoma. These lesions have different clinical presentation and biological behavior compared to conventional osteosarcoma, and hence need to be managed differently. GoalThe aim of this study is to analyze the clinico-pathological features and outcome of a series of surface osteosarcoma in an attempt to define the adequate treatment of this rare entity. Patient and methodIt is a retrospective and bicentric study of 18 surface osteosarcoma that were seen at the KASSAB’s Institute and SAHLOUL Hospital from 2006 to 2013. The authors reviewed the clinical and radiologic features, histologic sections, treatments, and outcomes in this group of patients. ResultsSeven patients were male (38.9%) and 11 were female (61.1%) with mean age of 25 years (range from 16 to 55 years). Eleven lesions were in the femur and 7 in the tibia. We identified 11 parosteal osteosarcoma (six of them were dedifferentiated), 3 periosteal osteosarcoma and 4 high grade surface osteosarcoma. Six patients had neoadjuvant chemotherapy and all lesions had surgical resection. Margins were wide in 15 cases and intra lesional in 3 cases. Histological response to chemotherapy was poor in all cases. The mean follow up was 34.5 months. Six patients (33.3%) presented local recurrence and 8 patients (44.4%) presented lung metastases. Six patients (33.3%) died from the disease after a mean follow up of 12 months (6–30 months); all of them had high grade lesions. ConclusionHistological grade of malignancy is the main point to assess in surface osteosarcoma since it determines treatment and prognosis. Low grade lesions should be treated by wide resection, while high grade lesions need more aggressive surgical approach associated to post operative chemotherapy.

Survival rate and prognostic factors of conventional osteosarcoma in Northern Thailand: A series from Chiang Mai University Hospital

BackgroundOsteosarcoma is a common and aggressive primary malignant bone tumor occurring in children and adolescents. It is one of the most aggressive human cancers and the most common cause of cancer-associated limb loss. As treatment in Thailand has produced a lower survival rate than in developed countries; therefore, this study identified survival rate and the poor prognostic factors of osteosarcoma in Northern Thailand. MethodsThe retrospective cases of osteosarcoma, diagnosis between 1 January 1996 and 31 December 2013, were evaluated. Five and ten year overall survival rates were analyzed using time-to-event analysis. Potential prognostic factors were identified by multivariate regression analysis. ResultsThere were 208 newly diagnosed osteosarcomas during that period, and 144 cases met the criteria for analysis. The majority of the osteosarcoma cases (78.5%) were aged 0–24 years. The overall 5- and 10-year survival rates were 37.9% and 33.6%, respectively. Presence of metastasis at initial examination, delayed and against treatment co-operation, and axial skeletal location were identified as independent prognostic factors for survival, with hazard ratios of 4.3, 2.5 and 3.8, and 3.1, respectively. ConclusionsThis osteosarcoma cohort had a relatively poor overall survival rate. The prognostic factors identified would play a critical role in modifying survival rates of osteosarcoma patients; as rapid disease recognition, a better treatment counselling, as well as improving of chemotherapeutic regimens were found to be important in improving the overall survival rate in Thailand.

The molecular landscape of extraskeletal osteosarcoma: A clinicopathological and molecular biomarker study.

Extraskeletal osteosarcoma (ESOSA) is a rare soft tissue neoplasm representing <5% of osteosarcomas and <1% of all soft‐tissue sarcomas. Herein, we investigate the clinicopathological and molecular features of ESOSA and explore potential parameters that may affect outcome. Thirty‐two cases were retrieved and histomorphology was reviewed. Clinical history and follow‐up were obtained through electronic record review. DNA from formalin‐fixed paraffin‐embedded (FFPE) tissue was extracted and processed from 27 cases. Genome‐wide DNA copy number (CN) alterations and allelic imbalances were analyzed by single nucleotide polymorphism array using Affymetrix OncoScan FFPE Assay. Massive high‐throughput deep parallel sequencing was performed using a customized panel targeting 410 cancer genes. Log rank, Fisher's exact test and Cox proportional hazards were used for statistical analysis. In this series of 32 patients (male n = 12, female n = 20), the average age was 66 years (19–93) and median follow up was 24 months (range 6–120 months). Frequent genomic alterations included CN losses in tumour suppressor genes including CDKN2A (70%), TP53 (56%) and RB1 (49%). Mutations affecting methylation/demethylation, chromatin remodeling and WNT/SHH pathways were identified in 40%, 27%, and 27%, respectively. PIK3CA and TERT promoter variant mutations were identified in 11% of the cases. Cases harbouring simultaneous TP53 and RB1 biallelic CN losses were associated with worse overall survival and local recurrence (p = 0.04, p = 0.02, respectively). CDKN2A losses and positive margins were also associated with worse overall survival (p = 0.002; p = 0.03, respectively). Our findings suggest that age above 60, positive margin status, simultaneous biallelic TP53 and RB1 losses and CDKN2A loss are associated with a worse outcome in ESOSA. Comparison between conventional paediatric osteosarcoma and ESOSA shows that, while both share genetic similarities, there are notable dissimilarities and mechanistic differences in the molecular pathways involved in ESOSA.

Second primary osteosarcomas in patients with retinoblastoma.

Second primary malignancies have become the leading cause of death in retinoblastoma survivors. Although osteosarcoma is the most common second malignancy, little is known about its clinical and therapeutic features. We retrospectively reviewed a database of patients with retinoblastoma and osteosarcoma occurring as a second malignancy between 1964 and 2010 at the National Cancer Center Hospital of Japan. Among 857 patients with retinoblastoma registered in the database, 10 (1.1%) developed osteosarcoma as a second malignancy. The median age at the onset of retinoblastoma was 3 months, being bilateral in nine patients and unilateral in one. Systemic chemoreduction was performed in three patients and intra-arterial chemotherapy in six; all patients received external beam radiotherapy. The median age at the onset of second primary osteosarcoma was 11.2 years; four were radiation-related and six were located in an extremity. Among five patients treated at our institute, four patients with tumors on an extremity were treated by wide resection with neoadjuvant and adjuvant chemotherapy. Three of these four patients (75%) were good responders to high-dose methotrexate-based multi-agent chemotherapy and survived with no evidence of disease (median follow-up period, 17.3 years). One patient whose temporal bone was affected underwent radiotherapy with chemotherapy but died after local recurrence. The clinical outcomes of second primary osteosarcoma in an extremity occurring in retinoblastoma survivors may be more favorable than those of conventional osteosarcoma. Early diagnosis of radiation-related osteosarcoma arising in the craniofacial region should be made at a stage where complete resection is possible.

Integrated analysis of gene expression and genomic aberration data in osteosarcoma (OS)

Cytogenetic analyses have revealed that complex karyotypes with numerous and highly variable genomic aberrations including single-nucleotide polymorphisms (SNPs) and copy number variants (CNVs), are observed in most of the conventional osteosarcomas (OSs). Several genome-wide studies have reported that the dysregulated expression of many genes is correlated with genomic aberrations in OS. We first compared OS gene expression in Gene Expression Omnibus (GEO) data sets and genomic aberrations in International Cancer Genome Consortium (ICGC) database to identify differentially expressed genes (DEGs) associated with SNPs or CNVs in OS. Then the function annotation of SNP- or CNV-associated DEGs was performed in terms of gene ontology analysis, pathway analysis and protein-protein interactions (PPIs). Finally, the expression of genes correlated with both SNPs and CNVs were confirmed by quantitative reverse-transcription PCR. Eight publicly available GEO data sets were obtained, and a set of 979 DEGs were identified (472 upregulated and 507 downregulated DEGs). Moreover, we obtained 1039 SNPs mapped in 938 genes, and 583 CNV sites mapped in 2915 genes. Comparing genomic aberrations and DGEs, we found 41 SNP-associated DEGs and 124 CNV-associated DEGs, in which 7 DGEs were associated with both SNPs and CNVs, including WWP1, EXT1, LDHB, C8orf59, PLEKHA5, CCT3 and VWF. The result of function annotation showed that ossification, bone development and skeletal system development were the significantly enriched terms of biological processes for DEGs. PPI network analysis showed that CCT3, COPS3 and WWP1 were the significant hub proteins. We conclude that these genes, including CCT3, COPS3 and WWP1 are candidate driver genes of importance in OS tumorigenesis.

Pharmacological inhibition of Bcl-xL sensitizes osteosarcoma to doxorubicin.

High-grade conventional osteosarcoma is the most common primary bone tumor. Prognosis for osteosarcoma patients is poor and resistance to chemotherapy is common. We performed an siRNA screen targeting members of the Bcl-2 family in human osteosarcoma cell lines to identify critical regulators of osteosarcoma cell survival. Silencing the anti-apoptotic family member Bcl-xL but also the pro-apoptotic member Bak using a SMARTpool of siRNAs as well as 4/4 individual siRNAs caused loss of viability. Loss of Bak impaired cell cycle progression and triggered autophagy. Instead, silencing Bcl-xL induced apoptotic cell death. Bcl-xL was expressed in clinical osteosarcoma samples but mRNA or protein levels did not significantly correlate with therapy response or survival. Nevertheless, pharmacological inhibition of a range of Bcl-2 family members showed that inhibitors targeting Bcl-xL synergistically enhanced the response to the chemotherapeutic agent, doxorubicin. Indeed, in osteosarcoma cells strongly expressing Bcl-xL, the Bcl-xL-selective BH3 mimetic, WEHI-539 potently enhanced apoptosis in the presence of low doses of doxorubicin. Our results identify Bcl-xL as a candidate drug target for sensitization to chemotherapy in patients with osteosarcoma.

Pre-treatment serum lactate dehydrogenase and alkaline phosphatase as predictors of metastases in extremity osteosarcoma

BackgroundThe prognosis of patients with metastatic osteosarcoma remains poor. However, the chance of survival can be improved by surgical resection of all metastases. In this study we investigate the value of serum alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) in predicting the presence of metastatic disease at time of diagnosis. MethodsSixty-one patients with histologically confirmed conventional osteosarcoma of the extremity were included in the study. Only 19.7% of cases presented without evidence of systemic spread of the disease. Pre-treatment serum ALP and LDH were analysed in patients with and without skeletal or pulmonary metastases. ResultsSerum LDH and ALP levels were not significantly different in patients with or without pulmonary metastases (p=0.88 and p=0.47, respectively). The serum LDH and ALP levels did however differ significantly in patients with or without skeletal metastases (p<0.001 and p=0.02, respectively). The optimal breakpoint for serum LDH as a marker of skeletal metastases was 849 IU/L (AUC 0.839; Sensitivity=0.88; Specificity=0.73). LDH >454 IU/L equated to 100% sensitivity for detected bone metastases (positive diagnostic likelihood ratio (DLR)=1.32). With a cut-off of 76 IU/L a sensitivity of 100% was reached for serum ALP predicting the presence of skeletal metastases (positive DLR=1.1). In a multivariate analysis both LDH ≥850 IU/L (odds ratio [OR]=9; 95% confidence interval (CI) 1.8–44.3) and ALP ≥280 IU/L (OR=10.3; 95% CI 2.1–50.5) were predictive of skeletal metastases. LDH however lost its significance in a multivariate model which included pre-treatment tumour volume. ConclusionIn cases of osteosarcoma with LDH >850 IU/L and/or ALP >280 IU/L it may be prudent to consider more sensitive staging investigations for detection of skeletal metastases. Further research is required to determine the value and the most sensitive cut-off points of serum ALP and LDH in the prediction of skeletal metastases.

Abstract 78: Targeted next-generation resequencing in pediatric osteosarcoma patients

Abstract Background: Osteosarcoma (OS) is the most common malignant bone tumor in children and adolescents. Recent genomic analysis demonstrated that OS are characterized as a high frequency of structural variations and chromosomal copy number changes. Meanwhile, specific genomic alterations such as RB1 or TP53 have been identified, although driver gene mutation for OS tumorigenesis has yet to be found. The aim of this study is to identify specific somatic mutations in OS patients by the method of comprehensive targeted resequencing. Patients and Methods: 13 pairs of tumor and non-tumor frozen tissues taken from the patients diagnosed as a primary conventional OS were subjected to this study. All tumor samples were collected at the time of preoperative biopsy and confirmed pathologically the tumor cell content more than 80%. We utilized a next generation sequencing technique with the Ion AmpliSeq Comprehensive Cancer Panel (CCP) containing 409 cancer-related genes. 40 ng of DNAs were used for multiplex PCR amplification and sequencing was run on the Ion Torrent Proton loaded with PIv2 chip. Data analysis including alignment to the hg19 human reference genome and variant calling was done using the Torrent Suite Software. Obtained genomic data was validated by visualizing in Integrative Genomics Viewer and candidate mutations were annotated by wANNOVAR database. Results: An adequate library was obtained from all samples for subsequent sequencing. Mean read length was 110 base pairs and an average coverage was approx.1500 for tumors and 400 for paired non-tumors. A total of 153 candidate non-synonymous somatic SNVs (median; 13.9 per tumor) were found with the threshold of over 5% frequency. After validation analysis and filtering process of variants, at least 12 SNVs were confirmed in 8 patients. Recurrent SNVs have not been observed among the 13 patients. There was a trend toward increased number of SNVs in dead patients comparing to alive patients (median 0 vs 1), although no significant difference was observed (P = .23, Wilcoxon test). Pathway analysis of the candidate SNVs showed significant correlations with three gene function categories including cell cycle, ATP binding and ligand-dependent nuclear receptors (p&amp;lt;0.02). We also calculated relative copy number changes in tumors using the coverage analysis data and found at least three gene amplification on chromosome 1p, 17p and 19p. Conclusion: We identified at least 12 non-synonymous somatic SNVs of cancer-related genes on the CCP panel in pediatric OS patients. Targeted sequencing is a useful and cost-effective tool to quickly survey known cancer-related mutations of pediatric OS. Citation Format: Shintaro Iwata, Yasutoshi Tatsumi, Tsukasa Yonemoto, Hiroki Nagase, Hiroto Kamoda, Takeshi Ishii, Miki Ohira. Targeted next-generation resequencing in pediatric osteosarcoma patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 78. doi:10.1158/1538-7445.AM2015-78

Oligo-Recurrence of Osteosarcoma Patients: Treatment Strategies for Pulmonary Metastases.

Distant metastases from osteosarcoma most commonly occur in the lungs. Osteosarcoma can be cured by complete surgical resection of all metastatic lesions if the number is limited (oligo-recurrence: ≤ 5 metastatic or recurrent lesions with controlled primary lesions). This study aimed to clarify the prognostic factors for osteosarcoma patients with pulmonary metastasis and determine their oligo-recurrence status. Patients with conventional osteosarcoma who underwent definitive surgery for the primary lesion and at least one thoracotomy for pulmonary metastases were recruited to this retrospective study. Clinicopathological information was collected on each thoracotomy from 1976 to 2011, and was then analyzed statistically. We counted the number of resected nodules that were pathologically confirmed as metastatic lesions from osteosarcoma. In total, 151 thoracotomies in 71 patients were analyzed. Forty-seven patients (66 %) underwent up to two thoracotomies, and the maximum number of thoracotomies was six. The median number of resected nodules on each thoracotomy was two, and the median total size of metastatic lesions was 20 mm. Incomplete surgical remission [relative risk (RR) 3.42], a less than 1-year interval from a previous thoracotomy (RR 1.97), more than three resected nodules (RR 2.42); and total size of more than 30 mm for pulmonary metastases (RR 2.19) were independent predictors of increased risk of tumor death by multivariate analysis. We propose that factors contributing to oligo-recurrence of patients with pulmonary metastatic osteosarcoma include complete surgical remission, an interval from a previous thoracotomy, number of resected nodules, and total size of pulmonary metastases.

Accuracy of different preoperative biopsy techniques in diagnosis of osteosarcomas and their value in prognostic evaluation

To study the difference in pathologic diagnostic accuracy among different histologic subtypes of osteosarcoma and different methods of preoperative biopsy, and the influence of diagnostic accuracy on prognosis of osteosarcoma. The preoperative biopsies, complete clinical, radiological and pathological data of 347 pathologically confirmed osteosarcomas were evaluated. According to the Pathological Diagnostic and Technical Specifications, the accuracy of preoperative biopsies was divided into 6 grades. 1: definite diagnosis, 2: basically definite diagnosis, 3: significant diagnosis, 4: descriptive diagnosis, 5:inadequate sampling, 6:misdiagnosis. 1 to 3 were defined as successful diagnosis,while 4 to 6 were defined as unsuccessful diagnosis. Of the 347 biopsies, 252 were CT-guided needle biopsies by the radiologists, and 95 were core-needle biopsies by orthopedic surgeons without CT-guidance. The latter showed a higher overall biopsy success rate (97.9%) in all osteosarcomas. Biopsies by surgeons showed a higher biopsy success rate (95.4%) in conventional osteosarcoma, but lower success rate in telangiectatic (55.6%) and low-grade central osteosarcomas (63.7%). The accuracy of pathologic diagnosis of preoperative biopsy was related to patients' age, serum AKP level, imaging diagnosis, method of biopsy and the subtype of osteosarcoma. Comparing the groups with successful and unsuccessful diagnosis, there were significant differences in recurrence rate and mortality after operation (P<0.01). The accuracy of pathologic diagnosis of preoperative biopsy are related to recurrence rate and mortality after operation. Biopsy by orthopedic surgeons without CT-guidance is reliable and safe, followed by primary diagnosis at frozen section and final diagnosis by routine pathologic sections for osteosarcomas located in the long bones of the extremities. Close integration of the preoperative pathologic diagnosis with clinical and radiological data will improve the accuracy of diagnosis.

Aven-mediated checkpoint kinase control regulates proliferation and resistance to chemotherapy in conventional osteosarcoma.

Conventional high-grade osteosarcoma is the most common primary bone sarcoma, with relatively high incidence in young people. In this study we found that expression of Aven correlates inversely with metastasis-free survival in osteosarcoma patients and is increased in metastases compared to primary tumours. Aven is an adaptor protein that has been implicated in anti-apoptotic signalling and serves as an oncoprotein in acute lymphoblastic leukaemia. In osteosarcoma cells, silencing Aven triggered G2 cell-cycle arrest; Chk1 protein levels were attenuated and ATR-Chk1 DNA damage response signalling in response to chemotherapy was abolished in Aven-depleted osteosarcoma cells, while ATM, Chk2 and p53 activation remained intact. Osteosarcoma is notoriously difficult to treat with standard chemotherapy, and we examined whether pharmacological inhibition of the Aven-controlled ATR-Chk1 response could sensitize osteosarcoma cells to genotoxic compounds. Indeed, pharmacological inhibitors targeting Chk1/Chk2 or those selective for Chk1 synergized with standard chemotherapy in 2D cultures. Likewise, in 3D extracellular matrix-embedded cultures, Chk1 inhibition led to effective sensitization to chemotherapy. Together, these findings implicate Aven in ATR-Chk1 signalling and point towards Chk1 inhibition as a strategy to sensitize human osteosarcomas to chemotherapy.

The Time of the Insult/Triggering Event in Primary Osteosarcoma and Ewing’s Sarcoma of Bone as Determined by Incubation Period Modeling and Age Distribution of Such Malignancies

The time for the triggering event in neoplasms can be estimated using incubation period modeling techniques. We applied these techniques to primary osteosarcoma and Ewing’s sarcoma of bone using the Surveillance Epidemiology and End Results database for all cases of osteosarcoma or Ewing’s sarcoma of bone from 1993 through 2010. Secondary neoplasms were excluded. The age at diagnosis, gender, ethnicity, and anatomic location were collected. The time (xt) of the insult/triggering event was calculated using the best fit frequency distribution of age at diagnosis. There were 4,356 patients with osteosarcoma and 1,832 patients with Ewing’s sarcoma. The Pearson IV distribution was the best fit for both osteosarcoma (r2=0.987) and Ewing’s sarcoma (r2=0.99). For these distributions xt is −0.7 years of age (4 weeks after conception) for Ewing’s sarcoma, 0.45 years for long bone osteosarcoma, and 10.4 years for parosteal osteosarcoma. This confirms the genetic etiology of Ewing’s sarcoma since an xt is 4 weeks after conception. Long bone osteosarcoma is not entirely genetic, as xt was 0.4 years for conventional osteosarcoma and 10.4 years for parosteal osteosarcoma. The etiologies for those two different types of osteosarcoma are thus different.