Potential Specificity Between Mycorrhizal Fungi Isolated from Widespread Dendrobium spp. and Rare D. huoshanense Seeds.

In nature, orchid seed germination and seedling development depend on compatible mycorrhizal fungi. Mycorrhizal generalist and specificity affect the orchid distribution and rarity. Here, we investigated the specificity toward fungi in the rare D. huoshanense by mycorrhizal fungal isolation and symbiotic germination in vitro. Twenty mycorrhizal fungal strains were isolated from the roots of adult Dendrobium spp. (six and 12 strains from rare D. huoshanense and widespread D. officinale, respectively, and two strains from D. nobile and D. moniliforme, respectively) and 13 strains belong to Tulasnellaceae and seven strains belong to Serendipitaceae. Germination trials in vitro revealed that all 20 tested fungal strains can stimulate seed germination of D. huoshanense, but only nine strains (~ 50%) can support it up to the seedling stage. This finding indicates that generalistic fungi are important for early germination, but only a few can maintain a symbiosis with host in seedling stage. Thus, a shift of the microbial community from seedling to mature stage probably narrows the D. huoshanense distribution range. In addition, to further understand the relationship between the fungal capability to promote seed germination and fungal enzyme activity, we screened the laccase and pectase activity. The results showed that the two enzymes activities of fungi cannot be directly correlated with their germination-promoting activities. Understanding the host specificity degree toward fungi can help to better interpret the limited geographic distribution of D. huoshanense and provides opportunities for in situ and ex situ conservation and reintroduction programs.

Genome-wide analysis and prediction of genes involved in the biosynthesis of polysaccharides and bioactive secondary metabolites in high-temperature-tolerant wild Flammulina filiformis.

BACKGROUND: Flammulina filiformis (previously known as Asian F. velutipes) is a popular commercial edible mushroom. Many bioactive compounds with medicinal effects, such as polysaccharides and sesquiterpenoids, have been isolated and identified from F. filiformis, but their biosynthesis and regulation at the molecular level remains unclear. In this study, we sequenced the genome of the wild strain F. filiformis Liu355, predicted its biosynthetic gene clusters (BGCs) and profiled the expression of these genes in wild and cultivar strains and in different developmental stages of the wild F. filiformis strain by a comparative transcriptomic analysis. RESULTS: We found that the genome of the F. filiformis was 35.01 Mb in length and harbored 10,396 gene models. Thirteen putative terpenoid gene clusters were predicted and 12 sesquiterpene synthase genes belonging to four different groups and two type I polyketide synthase gene clusters were identified in the F. filiformis genome. The number of genes related to terpenoid biosynthesis was higher in the wild strain (119 genes) than in the cultivar strain (81 genes). Most terpenoid biosynthesis genes were upregulated in the primordium and fruiting body of the wild strain, while the polyketide synthase genes were generally upregulated in the mycelium of the wild strain. Moreover, genes encoding UDP-glucose pyrophosphorylase and UDP-glucose dehydrogenase, which are involved in polysaccharide biosynthesis, had relatively high transcript levels both in the mycelium and fruiting body of the wild F. filiformis strain. CONCLUSIONS: F. filiformis is enriched in a number of gene clusters involved in the biosynthesis of polysaccharides and terpenoid bioactive compounds and these genes usually display differential expression between wild and cultivar strains, even in different developmental stages. This study expands our knowledge of the biology of F. filiformis and provides valuable data for elucidating the regulation of secondary metabolites in this unique F. filiformis strain.

[Clinical effect of the SCMC APL-2010 regimen in treatment of acute promyelocytic leukemia in children: an analysis of 44 cases].

OBJECTIVE: To study the clinical effect of the SCMC APL-2010 regimen in the treatment of acute promyelocytic leukemia (APL) in children. METHODS: A retrospective analysis was performed for the clinical data of 44 children with APL who received treatment with the SCMC APL-2010 regimen between April 2010 and July 2016. The Kaplan-Meier survival analysis was used to evaluate event-free survival (EFS) rate and overall survival (OS) rate. RESULTS: Of the 44 children with APL, 42 (95%) achieved a complete remission (CR) after one course of treatment and 1 achieved CR after two courses of treatment, with an overall CR rate of 98%. The 9-year EFS and OS rates were 96%±3% and 97.7%±2.2% respectively. As for adverse events, 41 (93%) had infection, 29 (66%) had granulocyte reduction, 12 (27%, 1 died) had differentiation syndrome, 16 (36%) had liver dysfunction, 12 (27%) had adverse gastrointestinal reactions, and 7 (16%) had QT prolongation, 1 (2%) had orchitis, and no secondary neoplasm was observed. CONCLUSIONS: Children with APL receiving the SCMC APL-2010 regimen have a good prognosis and can achieve a long-term survival, while treatment-related infection is commonly seen.

[Therapeutic efficacy of allogeneic hematopoietic stem cell transplantation in children with chronic myelogenous leukemia].

OBJECTIVE: To investigate the therapeutic efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in children with chronic myelogenous leukemia (CML), and to analyze the possible prognostic factors. METHODS: The clinical data of 20 children with CML who had received allo-HSCT was analyzed retrospectively to investigate possible prognostic factors, including age, sex, interval between diagnosis and transplantation, HLA matching between donors and recipients, illness status on transplantation and acute and chronic graft-versus-host disease (GVHD). RESULTS: At the end of follow-up, 13 of the 20 treated children had disease-free survival (DFS) and the rest (7 cases) died. Four died of severe acute GVHD, two of chronic GVHD and its complications, and one of relapse after transplantation. The three-year DFS was (64.6±1.1%). As shown by the univariate analysis, age was the most important prognostic factor in children with CML who had received allo-HSCT (P<0.05), and in children over 10 years, the prognosis was poor. No other of the above factors had a significant impact on prognosis (P>0.05). The multivariate logistic regression analysis also confirmed age as the only prognostic factor (P<0.01). Severe acute and/or chronic GVHD was the most important cause of patient death. 10/10 HLA-matched donors could improve the transplantation outcome. CONCLUSIONS: Allo-HSCT is an effective treatment for children with CML. To improve the prognosis and treatment outcome, children with CML aged over 10 years should receive allo-HSCT as early as possible. 10/10 HLA-matched donors are preferred in allo-HSCT and GVHD should be prevented.

The Clinical Characteristics of ARDS in Children With Hematological Neoplasms.

In order to explore the clinical characteristics of pediatric patients admitted to the pediatric intensive care unit (PICU) who suffered from hematological neoplasms complicated with acute respiratory distress syndrome (ARDS), we retrospectively analyzed 45 ARDS children with hematological neoplasms who were admitted to the PICU of Shanghai Children's Medical Center from January 1, 2014, to December 31, 2020. The 45 children were divided into a survival group and a non-survival group, a pulmonary ARDS group and an exogenous pulmonary ARDS group, and an agranulocytosis group and a non-agranulocytosis group, for statistical analysis. The main clinical manifestations were fever, cough, progressive dyspnea, and hypoxemia; 55.6% (25/45) of the children had multiple organ dysfunction syndrome (MODS). The overall mortality rate was 55.6% (25/45). The vasoactive inotropic score (VIS), pediatric critical illness scoring (PCIS), average fluid volume in the first 3 days and the first 7 days, and the incidence of MODS in the non-survival group were all significantly higher than those in the survival group (P < 0.05). However, total length of mechanical ventilation and length of hospital stay and PICU days in the non-survival group were significantly lower than those in the survival group (P < 0.05). The PCIS (OR = 0.832, P = 0.004) and the average fluid volume in the first 3 days (OR = 1.092, P = 0.025) were independent risk factors for predicting death. Children with exogenous pulmonary ARDS were more likely to have MODS than pulmonary ARDS (P < 0.05). The mean values of VIS, C-reactive protein (CRP), and procalcitonin (PCT) in children with exogenous pulmonary ARDS were also higher (P < 0.05). After multivariate analysis, PCT was independently related to exogenous pulmonary ARDS. The total length of hospital stay, peak inspiratory pressure (PIP), VIS, CRP, and PCT in the agranulocytosis group were significantly higher than those in the non-agranulocytosis group (P < 0.05). Last, CRP and PIP were independently related to agranulocytosis. In conclusion, children with hematological neoplasms complicated with ARDS had a high overall mortality and poor prognosis. Children complicated with MODS, positive fluid balance, and high VIS and PCIS scores were positively correlated with mortality. In particular, PCIS score and average fluid volume in the first 3 days were independent risk factors for predicting death. Children with exogenous pulmonary ARDS and children with agranulocytosis were in a severely infected status and more critically ill.

The genetics and clinical characteristics of children morphologically diagnosed as acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is characterized by t(15;17)(q22;q21), resulting in a PML-RARA fusion that is the master driver of APL. A few cases that cannot be identified with PML-RARA by using conventional methods (karyotype analysis, FISH, and RT-PCR) involve abnormal promyelocytes that are fully in accordance with APL in morphology, cytochemistry, and immunophenotype. To explore the mechanisms involved in pathogenesis and recurrence of morphologically diagnosed APL, we performed comprehensive variant analysis by next-generation sequencing in 111 pediatric patients morphologically diagnosed as APL. Structural variant (SV) analysis in 120 DNA samples from both diagnosis and relapse stage identified 95 samples with RARA rearrangement (including 94 with PML-RARA and one with NPM-RARA) and two samples with KMT2A rearrangement. In the eligible 13 RNA samples without any RARA rearrangement at diagnosis, one case each with CPSF6-RARG, NPM1-CCDC28A, and TBC1D15-RAB21 and two cases with a TBL1XR1-RARB fusion were discovered. These uncovered fusion genes strongly suggested their contributions to leukemogenesis as driver alternations and APL phenotype may arise by abnormalities of other members of the nuclear receptor superfamily involved in retinoid signaling (RARB or RARG) or even by mechanisms distinct from the formation of aberrant retinoid receptors. Single-nucleotide variant (SNV) analysis in 77 children (80 samples) with RARA rearrangement showed recurrent alternations of primary APL in FLT3, WT1, USP9X, NRAS, and ARID1A, with a strong potential for involvement in pathogenesis, and WT1 as the only recurrently mutated gene in relapsed APL. WT1, NPM1, NRAS, FLT3, and NSD1 were identified as recurrently mutated in 17 primary samples without RARA rearrangement and WT1, NPM1, TP53, and RARA as recurrently mutated in 9 relapsed samples. The survival of APL with RARA rearrangement is much better than without RARA rearrangement. Thus, patients morphologically diagnosed as APL that cannot be identified as having a RARA rearrangement are more reasonably classified as a subclass of AML other than APL, and individualized treatment should be considered according to the genetic abnormalities.

Chinese Black Truffle-Associated Bacterial Communities of Tuber indicum From Different Geographical Regions With Nitrogen Fixing Bioactivity.

It is well known that the microbes associated with truffle fruiting bodies play a very important role during the truffle lifecycle. Tuber indicum, commonly called Chinese black truffle, is a species endemic to Eastern Asia and in the genus of Tuber. Here, we reported the bacterial communities of T. indicum from different geographical regions and described the bacterial diversity from three compartments (soil, ectomycorrhizae and ascocarps) of T. indicum using high-throughput sequencing combined tissue culture. The results revealed that Bradyrhizobium was the dominant genus in fruiting bodies of T. indicum from nine geographical sites in China, and the microbes in T. indicum ascocarps were influenced by geological locations and soil characteristics. More specific bacterial taxa were enriched in the fruiting bodies than in the ectomycorrhizae and soil. In addition, 60 cultural bacteria were isolated from T. indicum fruiting bodies (4 families, 24 genera), and Pseudomonas, Alcaligenes faecalis, Microbacterium, and Arthrobacter were dominant. One of 13 strains that have potential nitrogen-fixation activities was further verified by an acetylene reduction assay (ARA). Together, this research provides new and important data for better understanding of the interaction between truffle and associated microbe and the biology of truffle itself.

[Outcomes of 104 children with B-cell non-Hodgkin lymphoma].

OBJECTIVE: To analyze outcomes and prognostic factors of children with B-cell non-Hodgkin lymphoma (B-NHL). METHODS: One hundred and four newly diagnosed B-NHL children were enrolled in protocol of B-NHL 2001. The statistics were performed by SPSS 13.0. RESULTS: Of 104 children (79 males, the median age of 7.1 years), 60, 32 and 4 patients were diagnosed with Burkitt lymphoma, diffuse large B-cell lymphoma and unclassifiable B-cell lymphoma, respectively. Four patients were in stage Ⅰ, 27 stage Ⅱ, 55 stage Ⅲ and 18 stage Ⅳ; 1, 26 and 77 patients were allocated into R1, R2 and R3 risk groups, respectively. Three patients never got complete remission (CR), 9 patients relapsed after CR with the duration of relapse from 1 to 7 months after chemotherapy. The estimated 5-year EFS of 104 patients was (86.7 ± 3.5)%. Univariable analyses identified that risk factors for recurrence were of higher staging, elevated LDH, serum ferritin and poor early response. Age, sex, pathologic diagnosis, original tumor, bone or marrow involvement, C-MYC and risk group were not found to be associated with the risk of failure to treatment. Multivariable COX regression models confirmed serum ferritin as a significant independent prognostic marker. CONCLUSION: B-NHL 2001 protocol was reasonable for B-NHL children. Higher staging, elevated LDH, serum ferritin and poor early response increased risk for recurrence.

[Long-term follow-up of childhood low-risk ALL patients treated with SCMC-ALL-2005 protocol].

OBJECTIVE: To evaluate the long-term efficacy of SCMC-ALL-2005 protocol in treatment of low-risk childhood acute lymphoblastic leukemia (ALL). METHODS: From May 1, 2005 to April 30, 2009, 387 patients enrolled into SCMC-ALL-2005 protocol. Based on the characteristics of cell morphology, immunology, cytogenetics and molecular biology and treatment response, 158 patients were fit into the low-risk treatment group. All the cases were registered in pediatric oncology network database (POND). The clinical characteristics and outcome were analyzed. RESULTS: Until December 31, 2012, the 5-year event free survival (EFS) and overall survival (OS) is (77.76±3.37)% and (89.55±2.83)%, respectively. Median follow-up time is 5.33 y (3.75-7.70 y). Five patients (3.16%) died of complication, all of them were severe infections. Twenty-seven patients (17.09%) relapsed, including 13 bone marrow relapse (8.23%), 5 testis relapse (5.32% of boys, 2 of unilateral and 3 bilateral), 6 central nerve system relapse (CNS, 3.80%), 1 relapse in both bone marrow and CNS, 1 relapse in both bone marrow and testis, and 1 right ovary and fallopian tube relapse. Relapse is related to positive minimal residual disease. Two cases (1.27%) occurred second tumors, 4 patients (2.53%) gave up treatment in complete remission without special reasons. CONCLUSION: The EFS and life quality of SCMC-ALL-2005 protocol in the treatment of childhood low-risk ALL is satisfactory. The treatment-related mortality rate is lower, and the long-term EFS is higher than that of XH-99 protocol.

[Long-term outcomes of childhood lymphoblastic lymphoma: report of 70 cases].

OBJECTIVE: To summarize long-term outcomes of childhood lymphoblastic lymphoma (LBL) with protocol CCCG-97 and -2002. METHODS: From November 1998 to October 2010, 70 consecutive newly diagnosed childhood LBL (5 B-LBL and 65 T-LBL) were enrolled in this study, in which 22 received CCCG-97 and 48 CCCG-2002 protocols. St.Jude staging system was adopted. Patients were divided into three risk groups based on clinical stage and serum LDH, and received chemotherapy with different intensity. The factors, which were possibly associated with the prognosis, were analyzed. The survival rates were evaluated by Kaplan-Meier analysis. RESULTS: The patients were 1.5 to 14 years old with the median age of 8 years old. They were evaluated as stage I-II for 6 , stage III41, and stage IV23 (15 were BM positive and 8 multiple bone metastases). Until Dec.31th, 2011,the mean follow-up was 62.5 months (range, 14 to 161 months) with the median follow-up of 48 months. 1-year overall survival (OS) was 74.3%, and 5- year event-free survival (EFS) 64.1% (abundance as event). Thirteen patients were complicated with serious condition during chemotherapy and 1 died of complication. Univariate analysis indicated that delayed and/or non-completed response on days 33 and 63 of induction was the unfavorable prognostic factor. CONCLUSION: Primary LBL usually located in the mediastinum. 90% of the patients was at advanced stage III-IV at first presentation. The 5-year EFS was 64.1%. Patients not achieved CR at days 33 and 63 at the end of induction was a poor prognostic factor.

Minimal residual disease is a prognostic marker for neuroblastoma with bone marrow infiltration.

OBJECTIVE: This pilot study focused on whether flow cytometry (FCM) detection of minimal residual disease in bone marrow (BM) could predict the outcome of patients with advanced neuroblastoma (NB). PATIENTS AND METHODS: Fifty-seven stage 4 NB patients with BM infiltration were enrolled in this study. All of them received NB-2001 protocol. BM samples were examined for tumor cell contamination by both morphology and FCM with CD45-FITC/CD81-PE/CD56-PECy5 monoclonal antibodies cocktail at diagnosis and after 4 courses of chemotherapy. RESULTS: BM samples of all patients were positive at diagnosis by FCM, and samples from 30 patients became negative after 4 courses of chemotherapy, 10 patients relapsed (33.3%) in mean 45.5 months, range 7 to 69. Another 27 patients remained positive, and 20 of them relapsed (74.1%) in mean 24.2 months, range 8 to 48. There was a statistically significant difference in event-free survival between the 2 groups (P = 0.002). CONCLUSIONS: Persistence of minimal residual disease in BM may work as a chemotherapy response marker and predict the prognosis in advanced NB.

[ALL-2005 protocol experience in the therapy of children and adolescents over 10 years of age with acute lymphoblastic leukemia].

OBJECTIVE: To explore the incidence, clinical characteristics and prognosis of children and adolescents over 10 years of age with acute lymphoblastic leukemia (ALL). METHODS: From May 1, 2005 to April 30, 2009, 67 newly diagnosed ALL children and adolescents over 10 years of age were enrolled in protocol of ALL-2005. All of the clinical characteristics of the patients were analyzed. The statistics was done by SPSS 13.0. RESULTS: There were 40 males (59.7%) and 27 females (40.3%). The mean age at diagnosis was 12.3 ± 1.7 (10.0 to 17.8) years with median age of 12.2 years. Of 67 patients, 48 were in medium risk group, and 19 in high risk group. During induction therapy, 83.6% and 86.6% patients had good response to prednisone and bone marrow blasts ≤ 5% at day 19, respectively. The overall hematologic response rate in these 67 patients was 88.1% (59) in complete remission (CR) after induction therapy, 15 patients relapsed with mean continuous CR period of (14.9 ± 9.9) months. The five-year event-free survivals (EFS) and overall survivals (OS) were (64.4 ± 6.3)% and (74.1 ± 6.1)%, respectively. According to univariate analysis, elevated serum ferritin, bcr-abl translocation, poor response to prednisone, high bone marrow blasts at day 19 or after induction therapy, and high minimal residual disease (MRD) after induction therapy increased risk for recurrence. Multivariate analysis indicated that high MRD after induction therapy was associated with recurrence (RR = 2.20, 95%CI 1.26 - 3.84, P < 0.01). CONCLUSION: Survival has improved for children and adolescents with ALL by ALL-2005 protocol. Analysis of serum ferritin and bcr-abl translocation at diagnosis, early responses to treatment and MRD detection during therapy are powerful prognostic indicators.

[Antitumor effect of CpG oligonucleotides on human neuroblastoma xenografts in nude mice].

BACKGROUND AND OBJECTIVE: Synthetic CpG oligodeoxynucleotides(CpG ODN) containing unmethylated CpG dinucleotide motifs, which mimic the effects of bacterial DNA, can stimulate the host's immune defense to reject cancer cells as "non-self" signal. This study was to evaluate the antitumor effect of CpG ODN against human neuroblastoma xenografts in nude mice depending on the innate immunity. METHODS: The cytotoxicity of CpG ODN on neuroblastoma SK-N-MC cells was detected by WST-1 assay in vitro. Neuroblastoma xenografts were built subcutaneously in nude mice. When palpable tumor developed, the mice were randomized into normal saline (NS), non-CpG ODN, and CpG ODN groups (each group contained six mice), and were administered every other day for two weeks. When tumor grew to 5 cm3, the mice were killed to observe tumor morphology by histology and histochemistry. RESULTS: CpG ODN had no cytotoxicity on SK-N-MC cells in vitro. Tumor volume was significantly smaller in CpG ODN group than in NS and non-CpG ODN groups at the end of the observation [(0.14+/-0.03) cm3 vs. (2.97+/-0.40) cm3 and (3.80+/-1.12) cm3, P<0.01]. On HE-stained sections, the tumor tissues of CpG ODN group showed intratumoral infiltration of inflammatory cells and large areas of necrosis, whereas those of controls showed less infiltration and no necrosis. By immunohistochemistry, the tumor tissues of CpG ODN group showed more natural killer (NK) cells and macrophages as compared with those of control groups. CONCLUSION: CpG ODN may have therapeutic effect on neuroblastoma in nude mice via mediating the activity of NK cells and macrophages.

[Clinical characteristics and treatment outcome of 36 cases with non-Hodgkin's lymphoma arising from mediastinum in children].

OBJECTIVE: Non-Hodgkin's lymphoma (NHL) presenting as mediastinal mass is usually progressive and may cause severe respiratory distress and death. This study aimed to summarize the clinical features and prognosis of NHL arising from mediastinum. METHODS: Totally 36 patients with NHL arising from mediastinum reported herein were diagnosed between 1999 and 2007. Their clinical characteristics, pathologic classification, diagnosis, outcome of different treatment protocol were retrospectively analyzed. Of these 36 patients, 25 were male, 11 were female (2.2:1). The mean age was 7.9 (range 1 - 12) years. Diagnosis was established on pathology that was achieved by mediastinal mass or peripheral lymph nodes biopsy, while some were diagnosed based on bone marrow or pleural effusion cytology study and immunophenotyping. For staging, the St. Jude system was applied. Patients received T-NHL-CCCG97, T-NHL-2002 or B-NHL-2001 protocol according to morphology and immunophenotyping. Patients who experienced superior vena cava syndrome (SVCS) and/or superior mediastinum syndrome (SMS) received induction chemotherapy with cyclophosphamide (C), vincristine (O) and prednisone (P) for one week. RESULTS: Twenty-seven cases experienced mediastinal mass or peripheral lympho nodes biopsy and were diagnosed by histopathology and immunohistochemistry. Of them, 24 were lymphoblastic lymphoma and 3 were anaplastic large cell lymphoma. Nine patients were diagnosed by cytological study of bone marrow aspiration or pleural fluid. All the 36 cases were T-cell type. Twenty-four cases were in stage III, 12 in stage IV. Twenty-four patients had urgent situation of SVCS and airway obstruction, 22 patients reached good response after emergency management including COP induction chemotherapy and pleural effusion suction. Twenty-nine cases achieved complete remission (CR) while in 6 patients the disease relapsed. Thirteen patients died from disease progression, relapse or severe infection during chemotherapy. The Kaplan-Meier estimate of 5-year progression-free survival (PFS) was 61% +/- 8% (median follow up 35 months) for these 36 patients. CONCLUSION: Establishment of a diagnosis as soon as possible was important to reduce the mortality and improve long term survival of patients. Induction chemotherapy for emergency situation was efficacious. The regimen of T-NHL-CCCG97, T-NHL-2002, and B-NHL-2001 for NHL arising from mediastinum based on pathological classification is feasible.

Prognostic influence of minimal residual disease detected by flow cytometry and peripheral blood stem cell transplantation by CD34+ selection in childhood advanced neuroblastoma.

OBJECTIVE: To determine whether neuroblastoma (NB) minimal residual disease (MRD) in bone marrow (BM) detected by flow cytometry could predict prognosis and whether tumor cell purging by CD34(+) cell selection prior to transplantation will impact on disease-free survival. METHODS: NB MRD in BM was evaluated by flow cytometry with CD45-FITC-/CD81-PE+/CD56-PECy5+ monoclonal antibodies cocktail. Peripheral blood stem cell (PBSC) was enriched via positive CD34(+) cell selection by magnetic-activated cell separation system (MACS). RESULTS: Eleven of 31 patients with CD45(-)/CD81+/CD56+ cells by flow cytometry at diagnosis became negative after an average of four courses of chemotherapy. All 11 patients remained alive without evidence of disease. Thirteen of the 20 patients with positive MRD relapsed and 1 patient died from disease (mean 25.8 months). There was a significant difference between these two groups. MRD in BM was tested before PBSC transplantation (PBSCT) for 19 NB patients. Fourteen was negative, 4 of them relapsed and 10 patients remained alive without evidence of disease. Another 5 patients with positive MRD, all of them relapsed (mean 17 months after PBSCT) with a significant difference between these two groups. Fourteen of 19 PBSC were purged with CD34(+) selection procedure. Six of 14 relapsed (mean 18.43 months after PBSCT). Five patients did not purge for CD34(+) selection, and 3 of them relapsed with no significant difference between these two groups. CONCLUSIONS: Positive MRD in BM after an average of four courses of chemotherapy and before PBSCT is an unfavorable factor for stage IV NB. CD34(+) selection purging for PBSCT may not improve the prognosis for children with neuroblastoma in advanced stage.