[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.

[Evaluation on protocol SCMS-ALL-2005 for childhood B lineage acute lymphoblastic leukemia].

OBJECTIVE: To reduce the risk of therapy related complication during the treatment and keeps the long term event free survival, and to evaluate the results and risk factors of SCMC-lymphoblastic leukemia (ALL)-2005 protocol. METHODS: Designed the new protocol SCMC-ALL-2005 based on the previous protocol XH-99 for ALL. Divided the patients into low, median and high risk groups depends on risk factors including day 33 and 55 minimal residual disease (MRD) level. The higher risk group, the more intensive therapy was given. All the cases were registed on pediatric oncology network database (POND). All the abandonment patients were counted as event. From May 1(st) 2005 to April 30(th) 2009, 351 children who were newly diagnosed as B lineage ALL were enrolled in this study. The prognoses relating to risk grouping, age, mutation gene and MRD level were analyzed. RESULTS: Up to June 30, 2011, 273 patients were followed up with median time 49 months (range 26 to 74 months). Three hundred and forty-five patients (98.29%) achieved complete remission on day 35 induction. 12 cases were younger than 1 year old (3.42%), 285 cases between 1 and 9 years old (81.20%), 54 cases 10 to 18 years old (15.38%). Five year event-free survival (EFS) was 34%, 72% and 63%, respectively. One hundred and fifty-six cases belonged to lowered risk (44.44%), 177 to middle risk (50.43%) and 18 to higher risk (5.13%). Five year EFS was 78%, 64% and 30%, respectively. In this study, 18 patients were detected positive for BCR/ABL, 3 for MLL/AF4, 16 for PBX/E2A, and 36 for TEL/AML. The 5 year EFS were 11%, 66%, 75% and 74%, respectively. A total of 300 cases were tested for MRD levels on day 35. Of them, 241 cases were with MRD ≤ 0.01% (negative), and 59 cases > 0.01% (positive). The 5 year relapse free survival (RFS) was 79% and 58%, respectively. Total 6 patients died of complication (1.71%). 18 patients were abundant treatment with no disease progress. 70 patients relapsed (19.94%), including 52 bone marrow, 8 central nerve system (CNS), 1 both in bone marrow and CNS, 1 second cancer (M(4)) and 8 testis. Five year overall survival (OS) and EFS are 84% and 69%. CONCLUSIONS: The risk of therapy related death is low with the protocol SCMC-ALL-2005. MRD affects the prognosis. The long term prognosis is poor for high risk group, with BCR/ABL and positive MRD.

[Correlation between blood T-cell receptor rearrangement excision circles levels and severe infection in children with acute lymphoblastic leukemia].

OBJECTIVE: This study quantitatively examined signal joint T-cell receptor rearrangement excision circles (sjTRECs) levels in peripheral blood of children with acute lymphoblastic leukemia (ALL) at different stages in order to evaluate the role of sjTRECs in predicting severe infection postchemotherapy. METHODS: sjTRECs levels in peripheral blood were measured by fluorescent quantitation-polymerase chain reaction in 30 children with newly diagnosed ALL, 36 children with ALL who accepted chemotherapy but were not infected, 30 children with ALL who had severe infection after chemotherapy, and 50 normal children. RESULTS: Blood sjTRECs levels in the normal group (394 ± 270 copies/103 MNC) were significantly higher than those in the other three groups (P<0.05). Blood sjTRECs levels in the chemotherapy group without infection (96 ± 78 copies/103 MNC) were significantly lower than those in the newly diagnosed ALL group (210 ± 219 copies/103 MNC) (P<0.05). The chemotherapy group with severe infection showed the lowest blood sjTRECs levels (48 ± 40 copies/103 MNC) in the four groups. CONCLUSIONS: The measurement of blood sjTRECs levels might be helpful for predicting the occurrence of severe infection postchemotherapy in children with ALL.

Outcome of Chinese children with unrelated donor hematopoietic stem cell transplantation.

BACKGROUND: To evaluate the prognostic factors and outcomes in Chinese children undergoing unrelated donor hematopoietic stem cell transplantation (UDT). METHODS: Retrospective analysis of clinical data from 53 consecutive children who received UDT from November 2002 to December 2007 in our center. RESULTS: The median recipient age was 8.4 years (range 1.5-21). With a median follow-up of 36 months (range 18-80), the probability of 3-year overall survival (OS) was 71.5%. Treatment-related mortality (TRM) was 19.0%, and 9.5% died after post-transplant leukemia relapse. Incidence of grades I-II, III-IV acute and chronic graft versus host disease (GVHD) was 63%, 29%, and 46%. There was significant difference in OS between patients older or younger than 10 years (50.0% vs. 84.8%, P = 0.003), patients with different underlying diseases (ALL, AML, CML, and non-malignant disease: 36.4%, 80.0%, 61.5%, and 100%, P = 0.001) and patients receiving either HLA 0-1 versus 2-3 loci high-resolution mismatched UDT (83.3% vs. 53.3%, P = 0.034). The OS was not affected by the stem cell source (peripheral stem cell 70.3%, bone marrow 87.5% vs. cord blood 62.5%, P = 0.542) or the severity of acute GVHD (grade 0-II 77.8% vs. grade III-IV 60.0%, P = 0.140). CONCLUSIONS: The important prognostic factors for OS after UDT were the degree of HLA match, the age of patient and the type of underlying disease. Patients < 10-year with non-malignant disease receiving 0-1 locus high-resolution mismatched UDT had the most favorable outcomes.

[Relationship between the antileukemic activity of L-asparaginase and Asn level around leukemic cells].

OBJECTIVE: To study the antileukemic activity of L-asparaginase through determining the changes of 4 kinds of amino acids (Asn, Aspa, Glu and Gln) in cell culture medium. METHODS: Following L-Asp treatment with designed concentrations and duration, the IC50 (inhibitory concentration 50%) of 8 kinds of common leukemia cell lines (U937, HL-60, Jurkat, NB4, THP-1, Namalwa, Karpass299, K562) were determined by CCK-8 assay. The changes of the 4 kinds of amino acids mentioned above were detected by high performance liquid chromatography (HPLC). RESULTS: The asparagines in cell culture medium were rapidly exhausted when treated with 0.01 U/mL L-Asp for 4 hrs or 1 U/mL L-Asp for 5 minutes. There were significant differences in the sensitivities to L-Asp of different leukemia cell lines. The sensitivities to L-Asp of various cell lines were dose-dependent. Low concentration of L-Asp resulted in a low IC50 and the IC50 increased following the L-Asp concentration increased. CONCLUSIONS: Different leukemia cell lines have different sensitivities to L-Asp, suggesting that exhaustion of asparagines around leukemia cells could not reflect the treatment efficacy of L-Asp. L-Asp antileukemic activity is dose-dependent, which suggests the importance of high-dose L-Asp on childhood acute lymphoblastic leukemia.

[Effect of protocol RS-99 for childhood rhabdomyosarcoma].

OBJECTIVE: To develop a reasonable protocol for childhood rhabdomyosarcoma (RS) to improve the prognosis of this disease. METHODS: The protocol RS-99, developed on the base of pathological examination, clinical staging and grouping was used for 30 RS patients, 15 male and 15 female, aged 53 months (15-180 months). For the low-risk patients the regimen VCP and IeV were used alternately for 6 treatment courses and then local radiotherapy was given, for the median and high risk patients, regimen AVCP and IEV were used alternately for 6 courses, local radiotherapy and selective operation were performed, and then DEV and IeV were used alternately for 6 courses, and for the high-risk patients DEV and IeV were used alternately for 18 courses in total and then hematopoietic stem cell transplantation was conducted. RESULTS: Twenty-three of the 30 patients reached complete response (CR) 17 of which remained in CR for 37 (32-92) months, and 7 of the 30 patients achieved partial response (PR) but their condition worsened later. Relapse was seen in 6 patients 15 (7-38) months later. The 5 year event-free survival (EFS) rate was 47% and the overall survival (OS) rate was 52%. All the 10 stage I and II patients, 5 of the 8 stage III patients, and only 2 of the 12 stage IV patients still remained in CR. CONCLUSION: The protocol RS-99 is effective on the RS at stages I, II, and III, however, is ineffective on the disease at stage IV.

[Relationship between immunological characteristics and prognosis in children with acute myeloid leukemia].

OBJECTIVE: The prognostic significance of immunophenotyping in acute myeloid leukemia (AML) has been controversial. This study investigated the relationship of immunophenotypes with French-American-British (FAB) subtypes and chromosomal abnormalities and assessed the prognostic value of immunophenotyping in children with AML. METHODS: From January 1998 to May 2003, 75 children with newly diagnosed AML were enrolled on protocol AML-XH-99. Immunophenotypes were measured with the flow cytometry. According to the McAbs used, the patients were classified into five groups: panmyeloid antigens (CD13, CD33, and MPO), myeloid-lineage associated antigens (CD14, CD15), lineage-specific antigens (CD41, GlyA), progenitor-associated antigens (CD34, HLA-DR) and lymphoid-associated antigens (CD19, CD7). The probability of event-free survival (EFS) was estimated by Kaplan-Meier analysis. The distributions of EFS were compared using the log-rank test. Chi-square analysis or Fisher exact test was used to compare the differences in the distribution of biologic presenting features. A Cox proportional hazards model was used to identify independent prognostic factors. RESULTS: At least one of panmyeloid antigens CD13, CD33 and MPO was expressed in 72 patents (97.3%). Two or more panmyeloid antigens were expressed in 45 patients (60.8%). The proportion of children with AML expressing one or more of the lymphoid-associated antigens was 24.3%. Lymphoid-associated antigen CD19 was expressed by blast cells in most of FAB M2 patients. The patients with acute promyelocytic leukemia were characterized by the absence of HLA-DR and lymphoid-associated antigens CD19 and CD7. Monovariate analysis showed immunophenotypes were not related to the complete remission rate after the first induction course and the 5-year-EFS. Multivariate analysis suggested immunophenotyping had no independent prognostic value in AML. CONCLUSIONS: Immunophenotyping can not be used independently in the evaluation of risk classification in children with AML. However, it is useful in the reorganization of special types of AML.

[Tandem application of flow cytometry and polymerase chain reaction for choice targets of minimal residual disease in childhood acute lymphoblastic leukemia].

OBJECTIVE: Minimal residual disease (MRD) is one of the most important prognostic factors in childhood acute lymphoblastic leukemia (ALL). Flow cytometry and PCR are two common techniques for examining MRD in ALL. This study aimed to identify MRD targets by tandem application of both techniques in children with ALL. METHODS: From September 2001 to October 2003, 126 children with newly diagnosed ALL were enrolled on the treatment protocol ALL-XH-99. Tandem application of flow cytometry and PCR was performed to identify MRD targets in these patients. RESULTS: 1. Using sets of combined antibodies, immunophenotypic expression of leukemia cells was observed in 95 of 106 B-lineage ALL cases (89.6%). Only one aberrant immunophenotype was observed in 11 cases (11.6%) and most patients with B-lineage ALL (88.4%) expressed at least two suitable targets. 2. Using PCR technique, T-cell receptor (TCR) or immunoglobulin gene rearrangements were identified in 26 of 27 patients (96.3%). Two or more monoclonal/ bi-allelic gene rearrangements were identified in 17 cases (65.4%). The majority (70%) of T-lineage ALL cases contained TCRVgammaI-Jgamma1.3/2.3. Cross-lineage TCR rearrangements were found in 57.1% of cases with B-lineage ALL. 3. Suitable MRD targets of immunophenotypic abnormalities or antigen receptor gene rearrangements were detected in 121 patients (96.0%). CONCLUSIONS: MRD targets were identified using tandem application of flow cytometry and PCR in almost of children with ALL. Cross-lineage TCR rearrangements and bi-allelic gene rearrangements were observed in many patients.

[Clinical observation on the treatment outcome of 14 children with hepatoblastoma in a single medical center].

OBJECTIVE: To evaluate the efficacy and safety of the ICE regimen (iphosphamide + carboplatin + etoposide) used in treating children with hepatoblastoma in the Shanghai Children's Medical Center. METHODS: From June 2000 to June 2008, 14 children with newly diagnosed hepatoblastoma (7 males and 7 females) were enrolled. Their median age on diagnosis was 1.33 years (range: 0.25-8.25 years). Six patients had stage I disease, 1 had stage II, 5 had stage III, and 2 had stage IV diseases. Thirteen children had markedly increased serum AFP level, and 1 had normal serum AFP level. Multidisciplinary co-operation treatment was performed. Eight patients had primary surgery while 3 patients had pre-operation chemotherapy before surgery. ICE chemotherapy regimen was used. Totally, 73 courses of chemotherapy were administered for the 14 children and 25 out of the 73 courses were performed before operation. RESULTS: Twelve patients responded to the treatment (85.7%) and 2 failed. Ten patients (71.4%) achieved complete remission after treatment, and two had partial remission. By July 31st, 2008, 9 patients survived without any event, with a median follow-up duration of 35 months (range: 16-96 months). The 5-year overall survival rate was 70.71+/-12.37%, and the 5-year event-free survival rate was 64.29+/-12.81%. One patient had disease relapse and two patients were lost to follow-up after they achieved partial remission. CONCLUSIONS: The ICE regimen combined with operation is effective and safe in treating children with hepatoblastoma.

[Prognostic value of early treatment response in children with acute lymphoblastic leukemia: a single institution experience in Shanghai, China].

OBJECTIVE: Early response to therapy is one of the most important prognostic factors in childhood acute lymphoblastic leukemia (ALL). This study aimed to assess the prognostic value of morphological assessment of bone marrow blasts during remission induction and determination of minimal residual disease (MRD) after remission induction. METHODS: From January 1998 to May 2003, 193 children with newly diagnosed ALL were enrolled on the ALL-XH-99 protocol. Blast cell count in the bone marrow was examined on day 19 of remission induction and by the completion of remission induction. MRD was measured with the flow cytometry. Event-free survival (EFS) was estimated by Kaplan-Meier analysis and the distributions of EFS were compared using the log-rank test. A Cox proportional hazards model was used to identify independent prognostic factors. RESULTS: The 4-year EFS was significantly worse in patients with > or = 5% lymphoblasts in the bone marrow on day 19 as compared to those with <5% lymphoblasts on that date (42.59%+/- 14.28% vs 74.24%+/- 6.67%; p< 0.01). The 4-year EFS was significantly worse in patients with any amount of lymphoblasts in the bone marrow on the remission date as compared to that of other patients with no morphologically identifiable blasts (63.47%+/-9.23% vs 76.41%+/- 6.09%; p<0.05). The patients with MRD <0.01 had significantly better outcome than those with a level > or = 0.01% (15-month EFS:94.44%+/-5.40% vs 23.81%+/- 20.26%; p<0.01). CONCLUSIONS: Early treatment response as assessed by morphological examination or minimal residual leukemia determination by flow cytometry has important prognostic significance, and can be performed in a resource-poor patient population.

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.

Comparative proteomic analysis of differentially expressed proteins between K562 and K562/ADM cells.

BACKGROUND: Multidrug resistance to chemotherapeutic agents is an important clinical problem during the treatment of leukemia. The resistance process is multifactorial. To realize the total factors involved in multidrug resistance, we analyzed the differentially expressed proteins of K562 and K562/ADM cells and we investigated one of the up-regulated proteins (CRKL) using siRNA to determine its role in K562/ADM cells. METHODS: Altered protein expressions between K562/S (K562 ADM-sensitive cell line) and K562/ADM (K562 multidrug resistant cell line induced by adriamycin) were identified by 2D-DIGE coupled with mass spectrometry. Meanwhile, we confirmed the differential expression of CRKL and Stathmin in both K562 and K562/ADM cells by Western blot analysis. Furthermore, we used RNA interference to silence the CRKL gene expression. RESULTS: Among the 9 differentially expressed proteins, 3 were up-regulated in K562/ADM cells, while 6 were down-regulated in the K562/ADM cells compared with its parent cell line. The expression of CRKL was up-regulated significantly in K562/ADM cells, and it can be decreased by recombinant lentivirus. Moreover, the multidrug resistance of K562/ADM cells was efficiently reversed by silence of CRKL gene expression. CONCLUSIONS: The data provided the differentially expressed proteins in K562 and its resistant cell line and highlights the power of 2D-DIGE for the discovery of resistance markers in cancer. We found CRKL may be a new protein involved in the multidrug resistance of leukaemia cells.

[Prognostic factors for childhood acute non-mature B-lymphoblastic leukemia].

OBJECTIVE: To study the prognostic factors for events-free survival (EFS) in children with acute non-mature B-lymphoblastic leukemia. METHODS: One hundred and sixty-one children with newly diagnosed acute non-mature B-lymphoblastic leukemia received the ALL-XH-99 protocol treatment. Their medical data, including clinical, biological and molecule features, early responses to treatment (bone marrow evaluation on the 19th day of induction therapy), minimal residual disease (MRD) in bone marrow after remission induction therapy, the risk grade of disease before the beginning of chemotherapy and the outcome, were retrospectively studied. RESULTS: Univariable analysis indicated that the gender and P170 levels before therapy had no effect on the outcome. Age, initial white blood cell count (WBC), prednisone response, early response to treatment, fusion genes (BCR/ABL or MLL/AF4) and MRD level were significantly related to the EFS (P<0.01). Immunophenotype, myeloid-associated antigen and the risk grade of disease were also related to the EFS (P<0.05). Multivariable analysis showed that WBC >or=50 x 10(9)/L, Cmu positive, BCR/ABL or MLL/AF4 positive and MRD positive (>or=0.01%) were risk factors for the poor prognosis (P<0.01). The early response to treatment was important to modify the therapy protocol. CONCLUSIONS: WBC >or=50 x 10(9)/L, Cmu positive, BCR/ABL or MLL/AF4 positive and MRD positive have important prognostic values in childhood acute non-mature B-lymphoblastic leukemia. Early response to treatment is an important index for modifying the chemotherapy protocol.

[Clinical importance of minimal residual disease testing in the therapy of childhood B-cell acute lymphoblastic leukemia].

OBJECTIVE: To study the role of minimal residual disease (MRD) in the evaluation of therapeutic effectiveness of childhood B-cell acute lymphoblastic leukemia (ALL). METHODS: MRD testing was performed in 124 children with B-cell ALL, who were newly diagnosed and enrolled in the ALL-XH-99 treatment protocol from September 2001 to April 2005MRD was determined by 4-color flow cytometry in the different time points during the treatment period. RESULTS: After induction therapy, 103, 13 and 8 patients showed MRD <0.01%, 0.01%-0.1% and >0.1%, respectively. The 5-year relapse-free survival (RFS) in the patients with MRD <0.01%, 0.01%-0.1% and >0.1% was 88.9+/-3.9%, 70.0+/-14.5% and 0%, respectively and the 5-year event-free survival (EFS) was 82.4+/-4.4%, 21.2+/-18.0% and 0%, respectively. There were significant differences in the RFS and EFS among the patients with different MRD levels (P<0.01). Within half a year after induction remission, the 5-year RFS in patients with MRD negative (<0.01%) and positive was 87.7+/-4.1% and 58.3+/-14.2%, respectively (P<0.01) and the 5-year RFS was 80.7+/-4.6% and 25.6+/-13.8%, respectively (P<0.01). After half a year with induction remission, the patients with MRD negative and positive also showed statistical differences in the 5-year RFS (92.0+/-3.6% vs 48.5+/-15.5%; P<0.01) and EFS (85.6+/-4.5% vs 21.4+/-11.0%; P<0.01). Multivariate analysis confirmed that the MRD level after induction chemotherapy together with the reaction to prednisone, the bone marrow features on the 19th day of induction, and the fusion gene with BCR-ABL or MLL-AF4 had prognostic significance in childhood B-cell ALL. CONCLUSIONS: The MRD level in the whole course of therapy is an important outcome indicator in childhood B cell ALL.

[Effectiveness of the AML-XH-99-M3 protocol for treatment of acute promyelocytic leukemia in children].

OBJECTIVE: To evaluate the effectiveness of AML-XH-99-M3 protocol for treatment of acute promyelocytic leukemia (APL) in children. METHODS: Thirty-three children with APL received AML-XH-99-M3 protocol treatment. The event-free survival (EFS), disease-free survival (DFS), and overall survival (OS) were evaluated by the Kaplan-Meier medthod with SPSS13.0 software. RESULTS: Thirty patients (90.9%) achieved a complete remission (CR) after one course of treatment. The total CR rate was 100%. Six patients (18.2%) relapsed in an average of 29.17 months (16-38 months). Two patients (6.1%) died. The 7-year EFS and DFS in the 30 patients were 73.4+/-9.4%. The overall survival rate was 91.2+/-6.0%. The difference of EFS was observed in patients receiving intermittent all-trans-retinoic acid (ATRA) administration or not in the maintenance therapy (88.9+/-10.5% vs 62.5+/-13.6%) (P<0.05). CONCLUSIONS: The AML-XH-99-M3 protocol for the treatment of APL produced a higher CR rate and higher EFS, DFS and OS rates in children. Intermittent administration of ATRA in the maintenance therapy can improve EFS rate.

NOTCH1 mutations in T-cell acute lymphoblastic leukemia: prognostic significance and implication in multifactorial leukemogenesis.

PURPOSE: NOTCH signaling pathway is essential in T-cell development and NOTCH1 mutations are frequently present in T-cell acute lymphoblastic leukemia (T-ALL). To gain insight into its clinical significance, NOTCH1 mutation was investigated in 77 patients with T-ALL. EXPERIMENTAL DESIGN: Detection of NOTCH1 mutation was done using reverse transcription-PCR amplification and direct sequencing, and thereby compared according to the clinical/biological data of the patients. RESULTS: Thirty-two mutations were identified in 29 patients (with dual mutations in 3 cases), involving not only the heterodimerization and proline/glutamic acid/serine/threonine domains as previously reported but also the transcription activation and ankyrin repeat domains revealed for the first time. These mutations were significantly associated with elevated WBC count at diagnosis and independently linked to short survival time. Interestingly, the statistically significant difference of survival according to NOTCH1 mutations was only observed in adult patients (>18 years) but not in pediatric patients (< or = 18 years), possibly due to the relatively good overall response of childhood T-ALL to the current chemotherapy. NOTCH1 mutations could coexist with HOX11, HOX11L2, or SIL-TAL1 expression. The negative effect of NOTCH1 mutation on prognosis was potentiated by HOX11L2 but was attenuated by HOX11. CONCLUSION: NOTCH1 mutation is an important prognostic marker in T-ALL and its predictive value could be even further increased if coevaluated with other T-cell-related regulatory genes. NOTCH pathway thus acts combinatorially with oncogenic transcriptional factors on T-ALL pathogenesis.

[Prognostic value of early treatment response in children with acute myeloid leukemia].

OBJECTIVE: To assess the prognostic value of early treatment response in children with acute myeloid leukemia (AML). METHODS: Sixty-one children with AML, 37 male and 24 female, aged 96 months (6 - 154 months), underwent treatment according to the protocol AML-XH-99 with a total treatment course of 15 months and were followed up for 12 months (1 - 74 months). Bone marrow smear was made 48 hours after the end of the first course of induction treatment. Then the children were divided into 2 groups according to the number of bone marrow blast cells: group with the number of blast cells > or = 0.15 and group with the number of blast cells < 0.15. Second bone marrow smear was made when complete remission was achieved after the end of the treatment course. Then the children were divided into 2 groups according to the number of bone marrow blast cells: group with the number of blast cells of 0.00 and group with the number of blast cells between 0.00 and 0.05. The probability of event-free survival (EFS) was estimated by Kaplan-Meier analysis. Log-rank test was used to compare the 5-year EFS (pEFS) of different groups. The differences in the biological features were compared by Chi-square analysis or Fisher exact test. RESULTS: The pEFS of the group with the number of blast cells > or = 0.15 was 18% +/- 15%, significantly shorter than that of the group with the number of blast cells < 0.15 (49% +/- 11%, P = 0.079). The 3 patients without morphologically identifiable blast all survived 5 years after complete remission had been achieved, and the pEFS of the 39 patients with the number of blast cells between 0.00 and 0.05 was 53% +/- 10%. The pEFS of the patients among which complete remission was achieved after the first course of treatment (n = 39) was 54% +/- 10%, significantly higher than that of the patients without complete remission after the first course of treatment (10% +/- 9%, P = 0.0002). Multiple factor analysis showed that achievement of complete remission after the first course of treatment and existence of central nervous system leukemia were both independent prognostic factors with the hazard ratios of 4.007 and 7.050 respectively and the 95% confidential intervals of 1.019 to 6.163 and 0.018 to 0.547 respectively (P = 0.045 and P = 0.008). The number of blasts 48 hours after the end of the first course of induction treatment was highly correlated with the rate of complete remission after the first treatment course (P = 0.000 028 8). CONCLUSION: With important prognostic significance, early treatment response, such as the number of blasts 48 hours after the end of the first course of induction treatment can predict whether complete remission can be achieved.

[Asparagine synthetase activity in pediatric acute lymphoblastic leukemia].

OBJECTIVE: To study the cellular activity of asparagine synthetase in different types of childhood acute lymphoblastic leukemia (ALL). METHODS: The cellular activity of asparagine synthetase was detected by HPLC-FLD and Protein measurement in 28 ALL children (7 cases of T-ALL and 21 cases of B-lymphoid lineage ALL) before chemotherapy. RESULTS: The asparagines synthetase activity levels in T-ALL children were significantly higher than those of the B-lymphoid lineage ALL patients, with the median activity level of 9.3 nM Asn/mg protein/hr vs 5.2 nM Asn/mg protein/hr (P < 0.05). The distribution of the asparagine synthetase activity demonstrated a polymorphism in either T-ALL or B-lymphoid lineage ALL patients. CONCLUSIONS: The cellular activity of asparagines synthetase in ALL patients is presented with a polymorphism distribution. The asparagines synthetase activity levels in T-ALL are significantly higher than in B-lymphoid lineage ALL.

Genomic Profiling of Adult and Pediatric B-cell Acute Lymphoblastic Leukemia.

Genomic landscapes of 92 adult and 111 pediatric patients with B-cell acute lymphoblastic leukemia (B-ALL) were investigated using next-generation sequencing and copy number alteration analysis. Recurrent gene mutations and fusions were tested in an additional 87 adult and 93 pediatric patients. Among the 29 newly identified in-frame gene fusions, those involving MEF2D and ZNF384 were clinically relevant and were demonstrated to perturb B-cell differentiation, with EP300-ZNF384 inducing leukemia in mice. Eight gene expression subgroups associated with characteristic genetic abnormalities were identified, including leukemia with MEF2D and ZNF384 fusions in two distinct clusters. In subgroup G4 which was characterized by ERG deletion, DUX4-IGH fusion was detected in most cases. This comprehensive dataset allowed us to compare the features of molecular pathogenesis between adult and pediatric B-ALL and to identify signatures possibly related to the inferior outcome of adults to that of children. We found that, besides the known discrepancies in frequencies of prognostic markers, adult patients had more cooperative mutations and greater enrichment for alterations of epigenetic modifiers and genes linked to B-cell development, suggesting difference in the target cells of transformation between adult and pediatric patients and may explain in part the disparity in their responses to treatment.

[Analysis on GST-Pi genetic polymorphism in children with acute leukemia].

OBJECTIVES: To study the frequency distribution patterns of the genetic polymorphisms for glutathione S-transferase Pi (GST-Pi) in children with acute leukemia, and explore the possible relationship of GST-Pi gene mutation to the vulnerability of children with leukemia and the chemotherapeutic response. METHOD: Direct DNA sequencing was applied to detect the genotype polymorphism in 85 healthy children as the control group and 120 children with acute leukemia. The distribution difference of the genotypes between them was analyzed. RESULTS: Gene mutation rate of GST-Pi exon5 was 47.5% in children with acute leukemia, significantly higher than that of 31.8% in the control group (OR = 1.944, 95% CI 1.088-3.473), and this polymorphism distribution pattern was similar to that reported abroad. The mutation rate was much higher in ALL group than in others groups and was not significantly between the AML children and the control group. The overall mutation rate of B-lineage ALL (60.3%) was markedly higher than that of T-ALL (47.1%), but the homozygous mutation rate of the latter group (17.6%) was much higher than that of the former group (3.4%) (P < 0.05). The average survival time of the children with wild type exon5 was 24 months, longer than that of the mutation group (17.6 months), however with no statistical difference (P > 0.05). The genotype of exon5 had no effect on the survival time of AML children. No Ala(114)Val variant genotype of GST-Pi exon6 reported in literatures was found in this study, but two new mutant genotypes were discovered. A/G hybridity at 99 loci of exon6 was found in one healthy child and such hybrid genotype did not result in amino acid alteration. G-->T/G bases hybridity at 103 loci of exon6 occurred in two children with leukemia, leading to GAC of Asp (aspartic acid) replaced by TAC of Try (tyrosine) at 147 loci of the protein peptide chain, producing Asp(147) Try hybrid mutation with a genotypic frequency of 1.7%. CONCLUSION: The gene mutation of GST-Pi exon5 is one of the potential vulnerable factors in leukemogenesis of the Chinese children and the genetic polymorphism of exon6 in Chinese is greatly different from that in other races. The role of the newly discovered variant genotype Asp(147) Try in leukemogenesis remains to be further studied.