The cultivation and identification of tumor stem cells from neuroblastoma derived tumor spheres

Background and Objective: Since the proposal of the tumor stem cell hypothesis, considerable interest has been devoted to the isolation and purification of tumor stem cells. Tumor stem cell enrichment from primary tumor derived cell spheres has been demonstrated in specific, serum-free media. This goal of this study is to establish a method of cultivating floating tumor spheres from neuroblastoma cells and to confirm that neuroblastoma spheres are rich in tumor stem cells. Methods: Bone marrow aspirates were obtained from pediatric patients diagnosed with stage IV neuroblastoma. Primary tumor cells were isolated and cultivated in serum-free, stem cell-selective medium. Single sphere-forming cells were cultivated under serum-free conditions; their cloning efficiency and monoclonal tumor sphere formation rates were calculated. The expression of stem cell marker genes Oct-4 and Bmi-1 was detected by RT-PCR in sphere-forming cells and parental neurolastoma cells. Sphere-forming cells were injected into the armpit of nude mice with subsequent assessment for tumor growth. Sphere-forming cells were cultivated in differentiation medium containing 5 μmol/L 13-cis retinoic acid; changes in cell morphology were observed. Results: Neuroblastoma cells formed non-adherent neurospheres under serum-free, stem cell-selective conditions after a period of 4 to 6 days. A single cell dissociated from a neurosphere could reform a monoclonal sphere; cloning efficiency and monoclonal sphere formation rates were 55.3% and 26.3%, respectively. RT-PCR results revealed heightened tumor sphere expression of Oct-4 and Bmi-1 as compared with parental tumor cells. Fourteen days after injection of 104 sphere-forming cells into nude mice, a neuroblastoma xenograft formed. Treatment of sphere-forming cells with 13-cis retinoic acid induced a gradual differentiation to neuronal cell morphology. Conclusions: Neuroblastoma derived tumor spheres enrich tumor stem cells and the cultivation of primary neuroblastoma cells in serum-free, stem cell-selective medium is an effective method to dissociate and purify tumor stem cells in vitro.

Cytogenetic Characteristics of Childhood Acute Lymphoblastic Leukemia:A Study of 1541 Chinese Patients Newly Diagnosed between 2001 and 2014

Objective:Cytogenetic abnormalities have been proven to be the most valuable parameter for risk stratification of childhood acute lymphoblastic leukemia(ALL).However,studies on the prevalence of cytogenetic abnormalities and their correlation to clinical features in Chinese pediatric patients are limited,especially large-scale studies.Methods:We collected the cytogenetics and clinical data of 1541 children newly diagnosed with ALL between 2001 and 2014 in four Chinese hospitals,and retrospectively analyzed their clinical features,prognosis and risk factors associated with pediatric ALL.

Association of image-defined risk factors,tumor resectability,and prognosis in children with localized neuroblastoma

Background Although localized neuroblastoma has a good prognosis,some cases have undergone treatment failure or recur-rence.Apart from biologic features such as MYCN status,we wondered whether some characteristics of growing tumors are prognostic,such as a well-encapsulated mass without infiltration of vital organs.We analyzed the diagnostic utility of image-defined risk factors(IDRFs)to predict successful treatment and prognosis.The overall goal was to achieve maximum cure rates for patients with localized neuroblastoma through a better understanding of clinical characteristics.Methods We retrospectively reviewed the images of patients with localized neuroblastoma who were enrolled between June 1998 and December 2012 at a single institution in Shanghai,China.Unequivocal categorization regarding IDRFs was available in 67 patients.IDRF was assessed at diagnosis and after four cycles of neoadjuvant chemotherapy,on average.The median follow-up period was 84 months(range: 48-132 months)after diagnosis.Results MRI and CT indicated a total of 177 IDRFs in these 67 patients.Logistic regression analysis revealed a highly significant negative correlation between the numbers of IDRFs and the possibility of complete removal of neuroblastoma.Intraspinal extension of the tumor,compression of the trachea,and encasement of the main artery in localized neuroblastoma were predictors for incomplete tumor resection.According to univariate analysis,≥4 IDRFs and intraspinal extension of the tumor were significant indicators of poor prognosis.Conclusions The number of IDRFs was useful in predicting surgical outcome and event-free survival.The number of IDRFs should be considered in protocol planning,instead of IDRF presence or absence.

Renal tumor in developing countries:142 cases from a single institution at Shanghai,China

Background:The clinical management of children with renal tumors including Wilms'tumor,clear cell sarcoma,rhabdoid tumor and other renal tumors in our center was designed according to the National Wilms'Tumor Study Group protocols.Methods:A total of 142 consecutive patients who had been diagnosed as having renal tumors at Shanghai Children's Medical Center were reviewed retrospectively in the period of December 1998 and September 2012.Diagnosis and treatment were decided by a multi-disciplinary team including oncologists,surgeons,pathologists and sub-specialized radiologists.Results:The median age of the patients at the time of diagnosis was 27 months.The tumor stages of the patients were as follows:stage I 24.6%,stage II 23.2%,stage III 32.3%,stage IV 14.1%,and stage V 5.6%.Favorable histology was diagnosed in 80.3%,anaplasia in 4.2%,clear cell sarcoma in 9.8%,rhabdoid tumor in 4.9%,and other renal tumors in 0.7%of the patients.The event-free and overall 5-year survival rates were 80%and 83%,respectively.Tumor relapse and progress was seen in 25 patients(17.6%).The median relapse time was 6 months(range:2-37 months).Seven relapsing patients were re-treated and four of them got second complete remission(three in stage II,one in stage I).Conclusion:A multi-disciplinary team work model is feasible in developing countries,and the renal tumors protocols basically from developed countries are safe in developing countries.

Using sound Clinical Paths and Diagnosis-related Groups(DRGs)-based payment reform to bring benefits to patientcare: A case study of leukemia therapy

China has been undertaking a profound reform on health care.Although more than 1.16 billion people have been covered by rural and urban medical insurance to date,the level of reimbursement from insurance is very limited,especially for critical diseases such as leukemia.This places heavy economic burdens on patients.Under these circumstances,systems innovation is imperative for the efficient utilization of limited funding.In this respect,certain valuable experience from other countries may prove helpful.The prospective payment system of Diagnosis-related Groups(DRGs),Clinical Paths,and the Comparative Effectiveness Analysis adopted by the National Institute of Health and Clinical Excellence(NICE,UK),can befine tools to reduce medical costs and improve quality of services.Treatments of acute promyelocytic leukemia at Rui-Jin Hospital,and childhood acute lymphoblastic leukemia at Shanghai Children’s Medical Center,can be taken as suitable models to illustrate the crucial role of Clinical Paths in guaranteeing clinical and cost effectiveness of medical services for critical diseases,and to satisfactorily justify the feasibility of DRGs in China.