Preliminary Application of Precision Genomic Medicine Detecting Gene Variation in Patients with Multifocal Osteosarcoma.

OBJECTIVES: The purpose of this study was to present our clinical experience of treating multifocal osteosarcoma (MFOS) in our center and gain more insight into the biology of this rare condition; in particular, to address with the help of precision genomic medicine the issue of whether the multiple osteosarcoma (OS) lesions in such patients are multi-centric or originate from one primary lesion and metastasize to other sites. Finally, we aimed to identify particular gene phenotypes and mutations that differentiate MFOS from OS with only one tumor. METHODS: Clinical data of patients with MFOS treated at our center between June 2007 and October 2014 were collected and analyzed retrospectively. High throughput sequencing of the whole exome of normal tissue and multiple lesions had been performed on samples from two patients (HJF and JZ) diagnosed in 2014. To explore the particular gene phenotype and clinical significance of MFOS, these sequencing results were analyzed and compared with those from patients with osteosarcoma in a single site. Seven patients with MFOS (three male and four female; average age 19.71 ± 3.35 years were enrolled in this study. Two of these patients declined treatment and died after 4 and 6 months, respectively. The remaining patients received standard treatment comprising neoadjuvant chemotherapy, surgery and chemotherapy. The chemotherapy regimen was lobaplatin (45 mg/m(2) ), doxorubicin (60 mg/m(2) ) and ifosfamide (12 g/m(2) ). Patients were followed up every 3 months after completing treatment and evaluated by the Enneking and Response Evaluation Criteria in Solid Tumors scoring systems. RESULTS: Up to the last follow-up on 1 December 2015, three patients were still alive. The event-free survival ranged from 4 to 144 weeks (median, 50.14 weeks), the mean (±SD) being 55.45 ± 45.47 weeks. Overall survival ranged from 16 to 388 weeks (median, 89 weeks; mean ± SD, 118.7 ± 147.7 weeks). The rates of mutation of the targeted drug-related genes were 133.5% ± 3.0% in the proximal tibia lesion and 113.1% ± 1.9% in the distal femur of patient HJF (P < 0.01) and 136.1% ± 10.8% in the proximal tibial lesion and 122.3% ± 5.5% in the proximal humerus of patient JZ (P = 0.0335). Furthermore, there were several anti-oncogenes in the somatic copy number variation lists analyzed from the two patients, especially TP53. However, no kataegis was found. CONCLUSIONS: Early and radical surgery accompanied by appropriate chemotherapy is the optimal means of treating MFOS. These patients may benefit from precision genomic medicine.

Imaging and Chemotherapeutic Comparisons of Iron Oxide Nanoparticles Chemically and Physically Coated with Poly(ethylene glycol)-b-Poly(ε-caprolactone)-g-Poly(acrylic acid).

We designed a new copolymer, poly(ethylene glycol)-block-poly(ε-caprolactone)-graft-poly(acrylic acid) (PAA-PEC), which could be chemically and physically coated onto iron oxide (Fe3O4) nanoparticles for theranostic applications. The chemically PAA-PEC-coated Fe3O4 nanoparticles (PAA-PEC-IO) were prepared using the carboxylic groups of PAA-PEC to bind the Fe3O4 nanoparticles during a co-precipitation reaction. Because of the amphiphilic properties of PAA-PEC, the compound self-assembled into a core-shell structure. The hydrophobic oleic acid-coated Fe3O4 nanoparticles could then be physically encapsulated inside the hydrophobic core of PAA-PEC (PAA-PEC-OA-IO) using an emulsion technique. A similar amount of iron content was controlled in both the PAA-PEC-IO and PAA-PEC-OA-IO (-23%). The particle diameters, morphologies, superparamagnetism, drug loading efficiency, and transversal relaxivity (r2) were studied and compared between the two magnetic nanoparticles. All results displayed the chemically-synthesized PAA-PEC-IO nanoparticles had higher potential than did the physically-synthesized PAA-PEC-OA-IO as an MRI contrast agent and a drug delivery carrier. Rodamine123-linked PAA-PEC-IO (PAA-PEC-IO-Rh123) was used as a molecular probe. Flow cytometric diagrams indicated that cellular internalization of PAA-PEC-IO occurred primarily through clathrin-mediated endocytosis.

Image navigation assisted joint-saving surgery for treatment of bone sarcoma around knee in skeletally immature patients.

PURPOSE: It is challenge to perform a native joint preserving resection for skeletally immature patients with bone sarcomas around the knee. We determined whether tumor resection under image-guided navigation make joint-saving surgery possible for juxta-articular sarcomas around knee while adhering oncological principles. METHODS: We performed joint-saving limb salvage surgeries on eight patients with metaphyseal bone sarcomas (6 in proximal tibia, 2 in distal femur). Six tumors extend to and 2 beyond the epiphyseal line. Planned tumor resection under image-guided navigation was employed for obtaining clear surgical margin while maximizing host tissue preservation. All tumors were en bloc removed and intercalary defect were reconstructed by combination of allograft with vascularized fibula flap. All specimens were examined for resection margin. Patients were followed up at average of 40.5 months for evaluating of oncologic and functional outcomes. RESULT: Entire joints were preserved in 5 patients and partial joints were saved in 3 patients. Clear surgical margins were achieved in all patients. The minimum of surgical margin width in bone is 6 mm in this series. No patient experienced local recurrence. Bone union achieved in all cases during the study period. The average MSTS score was 27.1 at final follow-up. CONCLUSIONS: With careful patient selection, image navigation aided bone tumor resection was proved to be an effective way in joint-saving limb salvage procedures for treating skeletally immature patients with juxta-articular bone sarcomas around the knee.

Precise resection and biological reconstruction under navigation guidance for young patients with juxta-articular bone sarcoma in lower extremity: preliminary report.

BACKGROUND: It is a challenge to perform a joint-preserving resection for young patients with juxta-articular bone sarcomas. We determined whether osteotomy under image-guided navigation make joint-saving resection possible for juxta-articular lesions while adhering oncological principles. METHODS: Between June 2008 and July 2010, joint-preserving limb salvage surgeries were performed on 9 patients with juxta-articular bone sarcomas under navigation guidance. Computed tomography/magnetic resonance imaging fusion images were used for real-time navigation. Eight lesions located around the knee and 1 in hip. Six tumors extend to and 3 beyond the epiphyseal line. Planned osteotomy under image-guided navigation was employed for achieving clear surgical margin while maximizing host tissue preservation. All tumors were en bloc removed and intercalary defect were reconstructed by combination of allograft with vascularized fibula graft. All specimens were examined for resection margin. Patients were followed up for an average of 25.2 months for evaluating of functional and oncology outcomes. RESULTS: Entire joint were preserved in 6 patients and part of joint were saved in another 3 patients. The mean registration error for navigation was 0.40 mm (range, 0.31 to 0.62 mm). Clear surgical margin was obtained in all specimens. The average closest distance between the osteotomy line and tumor edge was 9.6 mm (range, 6 to 14 mm). Entire joint cartilage was preserved in 6 patients and portion of joint were saved in 3 patients (2 in proximal tibia, 1 in distal femur). No patient experienced local recurrence. Two patients developed lung metastasis. One died of disease and the other underwent metastasectomy and had no evidence of disease at the most recent follow-up. All reconstruction was in situ with the Musculoskeletal Tumor Society average score of 26.7 at final follow-up. CONCLUSIONS: With careful patient selection, image navigation-assisted surgery made it possible to resect the bone exactly as planned in length and orientation in the magnetic resonance imaging image, yielding a clear margin and preserving the entire or part of the articular cartilage in joint-sparing limb salvage procedures for treating skeletally immature patients with juxta-articular bone sarcomas. LEVEL OF EVIDENCE: Level IV--therapeutic study.

Angiopep-pluronic F127-conjugated superparamagnetic iron oxide nanoparticles as nanotheranostic agents for BBB targeting.

Pluronic® F127-modified water-dispersible poly(acrylic acid)-bound iron oxide (PF127-PAAIO) nanoparticles have been prepared as diagnostic agents. A blood-brain-barrier penetrating peptide, angiopep-2 (ANG), was further conjugated onto the surface of the PF127-PAAIO (ANG-PF127-PAAIO) for brain targeting. The ANG-PF127-PAAIO shows negligible cell cytotoxicity, better cellular uptake, and higher T2-weighted image enhancement than the PF127-PAAIO in U87 cells. Using an ex vivo blood-brain barrier (BBB) model, we showed that the ANG-PF127-PAAIO shows better permeability to bypass the BBB. This is because the ANG-PF127-PAAIO has a dual-targeting ability, recognition of the low-density lipoprotein receptor-related protein and clathrin-mediated receptor on the U87 surface. Thus, the ANG-PF127-PAAIO is a potential nanotheranostic agent for brain dysfunction.

One-pot synthesis of PDMAEMA-bound iron oxide nanoparticles for magnetofection.

Superparamagnetic iron oxide nanoparticles (SPIONs) have been widely used for multiple biomedical applications. Magnetic-assisted transfection (magnetofection) using SPION is an attractive gene vector candidate. In this work, poly(2-dimethylamino)ethyl methacrylate-bound iron oxide nanoparticles (IO-PDMAEMA) were generated using a grafting-from approach via atom transfer radical polymerization (ATRP) for use as a gene vector. Preparing an iron oxide-initiator (IO-initiator) is a typical and important step. We designed a simple method to produce an IO-initiator containing bromide groups (IO-Br). The IO-Br was synthesized by reacting iron oxide nanoparticles with 2-bromoisobutyric acid using a one-pot solvothermal method at a high temperature. We optimized IO-PDMAEMA by controlling the PDMAEMA molecular weight, allowing higher gene expression with lower cytotoxicity. The hydrodynamic diameter of IO-Br was 76.7 nm, which increased to 361.7 nm after polymerization. Transversal relaxivity studies suggested that IO-PDMAEMA can be a contrast agent for magnetic resonance imaging. The magnetofection efficacy of IO-PDMAEMA/pDNA was measured in HEK 293T cells with or without fetal bovine serum (FBS). The IO-PDMAEMA/pDNA magnetoplexes exhibited remarkably improved gene expression in the presence of a magnetic field and 10% FBS compared with a commercial product, PolyMag/pDNA. No significant cytotoxicity of IO-PDMAEMA/pDNA was observed with different incubation time periods with or without the magnetic field. Confocal laser scanning microscopic images showed that the amount of internalized plasmid DNA increased in the assisted magnetic field.

Irregular osteotomy in limb salvage for juxta-articular osteosarcoma under computer-assisted navigation.

BACKGROUND: Joint-preserving limb salvage surgery has been expected to have good functional outcomes. However, it is still a unsolved problem to perform a joint preserving resection for patients with juxta-articular osteosarcoma invading epiphyseal line. We determined whether irregular osteotomy under image-guided navigation make joint-saving resection possible for juxta-articular osteosarcoma while adhering oncological principles. METHODS: We performed joint-preserving limb salvage surgeries on six patients with juxta-articular osteosarcoma of the long bone. Three lesions located in humerus, two in tibia and one in femur. Two tumors extend to and four beyond the epiphyseal line. CT and MRI data fusion images were applied for intraoperative navigation. Planned irregular osteotomy under image-guided navigation was employed for obtaining clear surgical margin while maximizing host tissue preservation. All tumors were en bloc removed and intercalary defect were reconstructed by allograft in one and combination of allograft with vascularized fibula graft in five patients. All specimens were examined for resection margin. Patients were followed up for average of 17.5 months for evaluating of functional and oncology outcomes. RESULT: Entire joint were preserved in three patients and part of joint were saved in another three patients. Clear surgical margin was obtained in all specimens with a minimum of 6-mm distance between tumor and osteotomy line. No patient experienced a local recurrence. One patient developed lung metastasis and had no evidence of disease at the most recent follow-up. All allografts but one healed during the study period. The MSTS average score was 88.8% at final follow-up. CONCLUSIONS: With careful patient selection, the irregular osteotomy under navigation guidance was proved to be an effective and safe technique for precise tumor resection in joint preserving limb salvage procedures for treating patients with juxta-articular osteosarcomas.

[Treatment of pelvic Ewing's sarcoma in children and the effect on the skeletal growth and development].

OBJECTIVE: To explore the effect of neo-adjuvant chemotherapy and computer-assisted surgery on children and adolescents with primary pelvic Ewing's sarcoma, and assess the therapeutic effect on the pelvic skeletal growth and development. METHODS: This is a retrospective analysis of 10 children with primary pelvic Ewing's sarcoma treated between Jan 2001 and Oct 2010 at the Department of Oncologic Orthopaedics at Xijing Hospital. There were 3 girls and 7 boys in the age of 7 to 16 years (average 12.7 years). All patients were pathologically diagnosed as Ewing's sarcoma. There were two cases in the sacroiliac joint, one in the ilium, one in the pubic bone, and 6 cases in peri-acetabular area including 5 below the triradiate cartilage and one above the triradiate cartilage, without cartilage invasion. All patients underwent neo-adjuvant chemotherapy, resection and reconstruction surgery and postoperative chemotherapy. CDP, ADM and IFO regimen chemotherapy were given as the main treatment. Five cases were treated by traditional resection and reconstruction, and after 2008, five cases were treated by computer-assisted surgery. During the reconstruction, the hip rotation center was put at a depressed location. All of the 10 cases underwent postoperative radiotherapy in a dose of 45-55 Gy. RESULTS: All patients were followed-up for 12-72 months (mean: 37.8 months). One child had tumor recurrence and lung metastasis and 9 patients had no evidence of disease (NED). After neo-adjuvant chemotherapy, the oncologic statuses (RECIST) were: 1 CR, 8 PR and 1 SD. The functional recoveries after surgery (Enneking's) were: 4 cases excellent, 4 good, 1 fair and 1 poor. Five cases who underwent computer-assisted surgery achieved a good reconstruction without local recurrence. There were no effects on skeletal growth in 8 cases. An unbalanced hip rotational center occurred in one case, and a compemsatory scoliosis was found in another case. There were no serious complications in all patients. CONCLUSIONS: The comprehensive treatment including neo-adjuvant chemotherapy, resection-reconstruction surgery and postoperative chemoradiotherapy may give a good control to primary pelvic Ewing's sarcomas in children and adolescents. The computer-assisted surgery used for accurate tumor resection and pelvic reconstruction is a good alternative when treating young patients with malignant pelvic tumors. The triradiate cartilage in children's acetabulum could be a natural barrier resistant to the invasion of Ewing's sarcomas.

Design of magnetic nanoparticles-assisted drug delivery system.

Magnetic nanoparticles (MNPs) have been designed for multifaceted applications such as contrast agents in magnetic resonance imaging (MRI) diagnosis, drug/gene carriers for different kinds of therapeutic agents, tissue repair, hyperthermia, immunoassay, and cell separation/sensing. This review highlights synthesis methods, stabilizers used for surface coating on MNPs, and target ligands for ferrying payloads to an interested disease area. Some of the recent biomedical applications of MNPs in the field of drug and DNA targeting delivery are extensively reviewed.

The use of massive allograft with intramedullary fibular graft for intercalary reconstruction after resection of tibial malignancy.

Reconstruction after intercalary excision of tibia malignancy is challenging. The combined use of a vascularized fibular flap and allograft can provide a reliable reconstructive option. Eight patients underwent reconstruction with an allograft and vascularized fibula following tibia malignancy resection. Patients were examined clinically and radiographically. The average age of patients was 16.5 years. The mean follow-up time was 38.4 months. Contralateral free fibula flap was used in three patients and ipsilateral pedicle fibula in five. The average length of defect was 11.8 cm and of fibula flap was 15.9 cm. Primary union was achieved in seven patients. The average time for bone union was 5.8 months at fibula-tibia junction and 14.1 months at allograft-tibia junction. Five patients had 10 complications. The Musculoskeletal Tumor Society average score was 90.8% at final follow-up. Intramedullary fibular flap in combination with massive allografts provide an excellent option for reconstruction of large bony defects after tibial malignancy extirpation. Ipsilateral pedicle fibula transportation had the advantages of short operation time and avoidance of donor site complications compared with the contralateral free fibula transfer.

The use of allograft shell with intramedullary vascularized fibula graft for intercalary reconstruction after diaphyseal resection for lower extremity bony malignancy.

BACKGROUND: Reconstruction after excision of the femur and tibia malignancy is a challenging issue for the reconstructive surgeon. The combined use of a vascularized fibular flap and allograft can provide a reliable reconstructive option. This article describes the authors' experience with this technique for the treatment of large-segmental bone defects after intercalary resection of lower extremity malignancy. METHODS: From 2003 to 2008, 11 patients that had intercalary resection of lower extremity malignancy underwent reconstruction with an allograft and vascularized fibular construct. Time to union was recorded through evaluation of plain radiographs. Patients were examined clinically and radiographically and were assessed functionally with MSTS score. RESULT: The average age at time of operation was 18.5 years. The mean follow-up time was 34.1 months. The oncology result was continuous disease free in 7 patients, no evidence of disease in 2, alive with disease in 1, and died of disease in 1. Free vascularized fibula flap was used in 7 patients and ispilateral pedicle vascularized fibula in 4. The average length of the resected segment was 12.1 cm and that of the fibula flap was 16.2 cm. The primary unions were achieved in all patients except one with tibia reconstruction. The average time for bone union was 5.4 months at fibula-host junction and 11.8 months at allograft-host junction. There were no allograft fractures and no infections. Five patients had 7 local complications. The MSTS average score was 91.8% at final follow-up. The mean time of weight-bearing was 12.4 months. CONCLUSIONS: Intramedullary fibular flap in combination with massive allografts provide an excellent option for reconstruction of large-bony defects after lower extremity malignancy extirpation. The viability of the fibula is a cornerstone in success of reconstruction that prevents allograft nonunion and result in decreased time to bone healing, leading to earlier patient recovery and return of function.

Limb salvage surgery for calcaneal malignancy.

BACKGROUND: Advances in oncologic treatment modalities and wide resection have made limb salvage procedures in calcaneal malignancy increasingly possible. However, reconstructions of the calcaneal remain a major surgical challenge because of the rarity and specific anatomy of this condition. METHODS: we retrospectively reviewed five patients who had primary calcaneal malignancy and underwent total calcanectomy and reconstructions with the distally pedicled osteocutaneous folded fibular flap between 2001 and 2007. The diagnoses included chondrosarcoma in 2, Ewing's sarcoma in 2 and osteosarcoma in 1. Three tumors were classified as stage-II B lesions, one as I A and one as II A. Wide resection margins were achieved in all patients. The patients were followed up for a mean of 50.4 months (range, 32-76 months). Patients were examined clinically and radiographically and were assessed functionally with Musculoskeletal Tumor Society (MSTS) score and American Orthopaedic Foot and Ankle Society (AOFAS) score. RESULTS: Four patients had no evidence of disease, and lung metastasis was found in one patient who was alive with disease. No local recurrence occurred in this series. At latest evaluation four patients had no evident limp or limitation of daily activities and one patient had mild limp. The average MSTS 93 score and AOFAS score were 83.2% and 86.4% at the last follow-up, respectively. All fibular flaps survived and bone unions achieved successfully. Fibula hypertrophies were seen in three patients. The overall mean time for bone union was 6.2 months. The mean time to full weight-bearing was 8.6 months. The average two-point discrimination was 2.3 cm at the time of final follow-up. Two patients had surgery-related complications including hematoma in 1 and skin margin necrosis in 1. CONCLUSIONS: Following the wide resection of calcaneal malignancy, biological reconstruction using distally pedicled osteocutaneous fibular has proven to be a successful limb salvage procedure, offering satisfactory local tumor control and functional restoration of the lower extremity function.

Layered inorganic/enzyme nanohybrids with selectivity and structural stability upon interacting with biomolecules.

Effective intercalation of protein molecules within the galleries of montmorillonites can be achieved via simple space enlarging and exchange processes while retaining the native conformation of the guest protein and the multilayered structure of the bioinert host plates. The capacity of accommodating protein molecules in the galleries can be markedly larger than that governed by the Langmuir-type adsorption of protein molecules on the external surfaces of particles. The basal spacing in the multilayered structure of clay is abruptly enlarged when the extent of protein intercalation increases to a critical point. Beyond this critical point, the nanohybrids show well-preserved catalytic activity in hydrolyzing small substrates while establishing a barrier to interactions with large biomacromolecules. Furthermore, the structural stability of the inorganic/organic nanohybrids is enhanced such that neither exchange of biomolecules nor exfoliation of layered clay particles occurs when exposed to other proteins. The results indicate that, through the benign accommodation of protein species between the inorganic platelets, this nanoscaled manipulation of protein functions can be highly useful in developing new inorganic/enzyme nanohybrids for protein therapeutics and tissue engineering.