[Prognostic impact of loss of sex chromosomes in children with acute myeloid leukemia subtype M2].

OBJECTIVE: To study the relationship between loss of sex chromosomes and prognosis in children with acute myeloid leukemia (AML) M2 subtype. METHODS: According to cytogenetic characteristics, 106 children with AML were divided into three groups: patients with normal karyotype (Group A, n=26), patients with abnormal karyotype who had no loss of sex chromosomes (Group B, n=52), and patients with abnormal karyotype who had loss of sex chromosomes (Group C, n=28). Prognosis was compared between the three groups. RESULTS: The 5-year event-free survival (EFS) rates of Groups A, B, and C were (38.9±11.2)%, (59.3±7.3)%, and (66.5±10.5)%, respectively; the EFS of Group C was significantly higher than that of Group A (P=0.035). The 5-year overall survival (OS) rates of Groups A, B, and C were (54.3±13.5)%, (68.1±7.7)%, and (77.9±9.8)%, respectively (P>0.05). The 5-year EFS of 58 patients with t(8;21) was (63.3±7.3)%, significantly higher than that of patients with normal karyotype (P=0.015). All the 28 cases in Group C had t(8;21), and their 5-year EFS was not significantly different from that of patients with t(8;21) in Group B (P>0.05). CONCLUSIONS: Loss of sex chromosomes is a favorable karyotype in children with AML M2 subtype and the patients in this group mostly have t(8;21). Why loss of sex chromosomes indicates a favorable prognosis is probably because it is accompanied by t(8;21) in the patients.

[A comparison of minimal residual disease in children with acute lymphoblastic leukemia of different genetic abnormalities].

OBJECTIVE: To study the changes of minimal residual disease (MRD) in children with B cell acute lymphoblastic leukemia (B-ALL) of different genetic abnormalities. METHODS: Between February 2004 and April 2013, 271 newly diagnosed B-ALL pediatric patients who had finished the induction chemotherapy were enrolled in the study. The characteristics of changes in MRD in patients with different genetic abnormalities on the 15th day and at the end of the induction therapy were analyzed. RESULTS: On the 15th day of the induction chemotherapy, the MRD positive proportion in patients with hyperdiploid was higher on all the three cut-off levels of MRD≥0.1%, 1% and 10% compared to patients without hyperdiploid (P<0.05), but there was no significant difference in the MRD positive proportion on the three levels of MRD between the TEL-AML1-positive and TEL-AML1-negative groups (P>0.05). On the end of induction chemotherapy, there was no significant difference in the MRD positive proportion on the three levels of MRD between the patients with and without hyperdiploid (P>0.05), neither between the BCR-ABL-positive and negative groups. The MRD positive proportion in TEL-AML1-negative patients was significantly higher than in TEL-AML1-positive patients on all three levels of MRD (P<0.05). The MRD positive proportion on two levels of MRD≥0.01% and 0.1% in E2A-PBX1-negative patients was significantly higher than in E2A-PBX1-positive patients (P<0.05). CONCLUSIONS: Children with B-ALL of different genetic abnormalities have different MRD levels during, and at the end of, induction therapy. The prognostic significance of MRD may be related to the genetic abnormalities.

[Therapeutic effect of clofarabine in children with relapsed or refractory acute lymphoblastic leukemia].

OBJECTIVE: To explore the efficacy and adverse effects of clofarabine for relapsed/refractory acute lymphoblastic leukemia in children. METHODS: Twenty-six pediatric patients with relapsed/refractory acute lymphoblastic leukemia were treated with clofarabine. There were 22 males and 4 females, with a mean age of 9.5 years (ranging from 4 to 17 years). They received clofarabine 52 mg/m2 intravenously over 2 hours daily for 5 days. Thirteen patients received two cycles and one patient received three cycles. RESULTS: In the first cycle of clofarabine, complete remission was obtained in 11 children (42%) and partial remission was obtained in 7 children (27%). Eight children (31%) were considered unresponsive. In the second cycle, 11 (85%) of the 13 children obtained complete remission, 1 (8%) partial remission and 1 (8%) was unresponsive. One child received three cycles and obtained complete remission in each cycle. The common adverse events were myelosuppression, infection, liver dysfunction and gastrointestinal adverse reactions. There were no chemotherapy-related deaths. CONCLUSIONS: Clofarabine is effective in the treatment of children with relapsed/refractory acute lymphoblastic leukemia and its adverse effects can be tolerated. Clofarabine could be a promising new treatment for relapsed/refractory acute lymphoblastic leukemia.

[Clinical significance of the Wilms' tumor 1 mRNA expression in childhood myelodysplastic syndrome].

OBJECTIVE: To investigate the expression of the Wilms' tumor 1 (WT1) mRNA in childhood myelodysplastic syndrome (MDS), and to evaluate WT1 as a tool to differentiate MDS from aplastic anemia(AA). METHODS: The quantitative expression of WT1 transcript by using real-time quantitative polymerase chain reaction (RQ-PCR) was performed in the bone marrow samples of 36 childhood MDS and 49 childhood AA, the samples were collected from September 2008 to December 2011. RESULTS: (1) The positive rate of WT1 in severe AA (SAA) was 0, 14.3% in chronic AA (CAA), 58.6% in refractory cytopenia (RC), 100% in refractory anemia with excessive blast (RAEB) and 97.5% in acute myeloid leukemia (AML). The mean level of WT1 in SAA, CAA, RC, RAEB and AML was 0.041%, 0.357%, 7.037%, 12.680% and 24.210%, respectively. The positive rate of WT1 in RC patients was higher than that of SAA (P = 0.000) and CAA (P = 0.001). (2) The positive rate of WT1 in patients with hypoplastic MDS was 66.7% and was higher than that of SAA (P = 0.000) and CAA (P = 0.001). The mean level of WT1 in patients with hypoplastic MDS was (3.022 ± 5.040)% and higher than that of SAA \[(0.041 ± 0.047)%, P = 0.000\] and CAA\[(0.351 ± 0.479)%, P = 0.002\]. CONCLUSIONS: The level of WT1 in childhood MDS was higher than that of childhood AA. The degree of WT1 expression in MDS increased during disease progression. WT1 is a useful tool for differentiating the childhood hypoplastic MDS from AA.

[Clinical features and prognosis of t (8; 21)/AML1-ETO-positive childhood acute myeloid leukemia].

UNLABELLED: OBJECTIVE To study the clinical and biological characteristics and prognosis of t(8;21)/AML1-ETO-positive childhood acute myeloid leukemia (AML). METHODS: The clinical data of 55 children who were diagnosed as t (8; 21)/AML1-ETO-positive AML were retrospectively studied. Event-free survival (EFS), disease-free survival (DFS), and overall survival (OS) rates were estimated by the Kaplan-Meier method. Prognostic factors were evaluated by COX regression analysis software. RESULTS: Of the 55 patients, 4 patients gave up treatment after the diagnosis was confirmed and 4 patients were lost to follow-up after the first chemotherapy course. The remaining 47 patients received a double-induction therapy. The total complete remission (CR) rate was 71% and 94% after the first and second chemotherapy course, respectively. The disease was relapsed in 10 patients (21%). The 5-year EFS, DFS and OS rates were (56.1 ± 7.9)%, (59.8 ± 8.1)%, and (72.0 ± 8.1)%, respectively. Multivariate analysis showed that age was an independent risk factor for the long-term prognosis. The older children had a greater risk of experiencing an accident or death (P<0.05). The 5-year OS rate in 27 patients with regular consolidation chemotherapy was significantly higher than 13 patients with irregular chemotherapy after CRz [(47.5 ± 17.1)% vs (38.9 ± 17.3)%; P<0.01]. CONCLUSIONS: Childhood t(8;21)/AML1-ETO-positive AML is a highly heterogeneous disease, with a high CR rate and a good long-term prognosis. Age is one of the important factors affecting the long-term therapeutic effect. Regular consolidation chemotherapy applied after CR usually is helpful.

[Clinical features and etiological spectrum in children with pancytopenia].

OBJECTIVE: To study the clinical features and etiological spectrum of pancytopenia in children. METHODS: The clinical data of 174 children with pancytopenia between September 2003 and January 2010 were retrospectively reviewed. RESULTS: Pale face was the most common clinical manifestation (147 cases, 84.5%), followed by bleeding (87 cases, 50.0%) and fever (41 cases, 23.6%). Mild to moderate anemia, severe thrombocytopenia and mild leucopenia were common in complete blood count. Of the 174 children, pancytopenia was attributed to hematopoietic system diseases in 155 cases (89.1%) and non-hematopoietic system diseases (virus infections, systemic lupus erythematosus, hypersplenism and neuroblastoma) in 6 cases (3.4%). Aplastic anemia (91 cases, 52.3%) was the most common cause of pancytopenia, followed by myelodysplastic syndrome (37 cases, 21.3%), acute leukemia and other hematological tumours (11 cases, 6.3%) and hemophagocytic syndrome (6 cases, 3.4%). The cause of pancytopenia was not identified in 13 cases (7.5%). CONCLUSIONS: Anemia, bleeding and fever are the main clinical manifestations of pancytopenia in children. Pancytopenia is mostly caused by aplastic anemia in children. Myelodysplastic syndrome, hematological tumours and hemophagocytic syndrome are also the common causes.

[Clinical characteristics of children with B cell type acute lymphoblastic leukemia carrying different fusion gene].

OBJECTIVE: To investigate whether there were differences in the clinical characteristics, cytogenetic characteristics, immunophenotype and prognosis in children with B cell type acute lymphoblastic leukemia (B-ALL) carrying different fusion genes. METHODS: The research included 80 children with B-ALL from Peking University People's Hospital between March 2006 and December 2008. Eighteen children were positive for TEL/AML1, 14 for E2A/PBX1, 11 for BCR/ABL,and 2 cases for MLL/AF4, and 35 cases were negative for all of the 4 fusion genes. Data including clinical characteristics, morphology, immunophenotype and cytogenetic characteristics were collected, and the disease-free survival (DFS) was evaluated. The children were followed up until April 2009. RESULTS: In the 18 children with TEL/AML1+B-ALL, 66.7% were younger than 5 years old. They had low tumor load. FAB-L2 morphology was commonly observed, but t(12;21) was often absence in these children. Up to now,17 children who survived were disease-free. In the 14 children with E2A/PBX1+B-ALL, the majority were female. Thirteen children showed FAB-L1 morphology. Twelve children showed pre-B-ALL immunophenotype. The EFS was close to 80%. In the 11 children with BCR/ABL+B-ALL, 10 children showed common B type immunophenotype. FAB-L1 and FAB-L2 morphology was found in 4 children respectively. The DFS was less than 20%. Two children with MLL/AF4 positive B-ALL had high tumor load. Their morphologic diagnosis was FAB-L1. Both showed the Pro-B-ALL immunophenotype. One child discontinued treatment at the early stage of chemotherapy, and the other child survived disease-free until now. CONCLUSIONS: The B-ALL children with different fusion genes have different clinical characteristics, immunophenotypes and prognosis.

[Identification of gene rearrangements in childhood leukemia by using a multiplex polymerase chain reaction-microarray approach].

The aim of study was to investigate the application of a novel microarray approach for the eight most common leukemia translocations in children for routine molecular diagnostic hematopathology practice. Bone marrow samples from 84 children with leukemia were analyzed by multiplex nested RT-PCR combined with oligonucleotide microarray. The results showed that out of 84 leukemic samples, 31 (36. 90%) carried 8 types of fusion genes including tel/aml1, e2a/pbx1, bcr/ablp190, bcr/ablp210, mll/af4, aml1/eto, pml/raralpha, cbfbeta/myh11. The sensitivity and specificity of the assay is comparable with the RT-PCR technique. In conclusion, this multiplex nested RT-PCR could quickly screen 8 types of chromosome structural aberrations at the same time. It can provide reliable and helpful information for risk stratification, therapy evaluation and prognosis prediction in childhood leukemia. There are both advantages and disadvantages in applying this new method. The microarray-based assay will be an effective and reliable tool in the clinical screening of leukemia patients for the presence of specific gene rearrangements with important diagnostic and prognostic implications.

[Can As2O3 improve the prognosis of childhood acute promyelocytic leukemia?--A single center experience].

OBJECTIVE: To retrospectively analyze the treatment outcomes and side effects of childhood acute promyelocytic leukemia (APL) treated with all-trans retinoic acid (ATRA) or ATRA + arsenic trioxide (As2O3). METHODS: From 1992 to 2006, 45 patients with newly diagnosed APL were enrolled. All of them were PML-RAR alpha positive. 24 patients were induced with ATRA (group A) and 21 with ATRA + As2O3 (group B). The remission rate and side effects were observed. RESULTS: 1) 19 (79.2%) patients in group A achieved CR, while 21(100%) patients in group B achieved CR. The CR rate in group A was lower than that in group B (P=0.027). 2) The recovery time of coagulation parameters and PLT count in group B was shorter than that in group A. 3) The overall survival (OS) and event-free survival(EFS) in group A were 77.8% and 66.9% at 41 months of follow-up, and in group B were 100% and 100% respectively at 34 months of followup. Group A had a significant lower EFS (P=0.0357)than group B. 4) The time of PML-RAR alpha fusion gene converting to negative in group A was longer (P=0.026) than that in group B. CONCLUSIONS: ATRA + As2O3 for patients with newly diagnosed childhood APL is a feasible treatment with higher CR rate, less side effects and longer long-term survival.

Integrated treatment of traditional Chinese medicine and western medicine for early- and intermediate-stage diabetic nephropathy / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the therapeutic effect of traditional Chinese herbal medicinal preparation Tangshenqing (TSQ) combined with alprostadil in the treatment of early- and intermediate-stage diabetic nephropathy (DN).</p><p><b>METHODS</b>One hundred and twenty DN patients were randomized into 3 groups for different treatment protocols. The patients in the control group were given the basic treatment (low-protein diabetic diet and rigorous control of blood glucose, blood pressure, and blood fatty acid), and those in treatment group A received TSQ (containing Astragalus membranaceus, Panax notoginseng, Epimedium brevicornum, etc) in addition to the basic treatment, and those in treatment group B were treated with alprostadil injections (for 14 consecutive days) in addition to the treatment given in group A. Therapeutic effect evaluation was carried out after a 30-day treatment in all the patients.</p><p><b>RESULTS</b>The overall efficaey rate of the treatment was 78.37% in the control group, 88.57% in the treatment group A, and 94.44% in treatment group B, suggesting better therapeutic effect in the latter two groups than in the control group (P<0.05). Patients in all the 3 groups exhibited symptomatic improvement of various degrees, but the treatment group B had the best results. After the treatments, the patients' blood glucose and fatty acids were lowered, without obvious difference between the 3 groups. Compared with the control group, the patients in the two treatment groups showed significant reduction in fibrinogen, 24-h urine microprotein and urine protein after the treatment (P<0.01 or 0.05).</p><p><b>CONCLUSION</b>Combined use of traditional Chinese herbal medicine TSQ and alprostadil injections produces definite therapeutic effect on early- to intermediate-stage DN.</p>

[Cloning and expression of the genes encoding glycerol dehydratase reactivase and identification of its biological activity].

The gdrA, gdrB gene coding glycerol dehydratase reactivase factor were amplified by using the genomic DNA of Klebsiella pneumoniae as the template. The gdrA and gdrB were inserted in pMD-18T to yield the recombinant cloning vector pMD-gdrAB. After the DNA sequence was determined, the gdrAB gene was subcloned into expression vector pET-28a(+) to yield the recombinant expression vector pET-28gdrAB. Under screening pressure by ampicillin and kanamycin simultaneously, the activity of glycerol dehydratase reactivase was characterized by coexpression of pET-32gldABC, which carry the gldABC gene encoding glycerol dehydratase, and pET-28gdrAB in E. coli BL21(DE3).

[Leukemia-associated immunophenotypes in 415 childhood and adult patients with B lineage acute lymphoblastic leukemia by multiparametric flow cytometry analysis].

To evaluate the significance of FCM in minimal residual disease (MRD) detection, the immunophenotyping and leukemia-associated immunophenotypes (LAIP) of leukemia cells from 273 adult and 142 childhood patients with B lineage acute lymphoblastic leukemia (B-ALL) were detected by four to six antibody combinations of 4-color CD45/SSC gating multiparametric flow cytometry (FCM). The results showed that the B-ALL patients could be classified into 4 subtypes based on different expression CD34 and CD10: subtype I (CD34(+)/CD10(-)), subtype II (CD34(+)/CD10(+)), subtype III (CD34(-)/CD10(+)), subtype IV (CD34(-)/CD10(-)). The LAIP was observed in 100% and 92% patients of subtype I and subtype II, respectively, whereas only 79.2% in subtype III. The incidence of LAIP in total B-ALL cases was 90% by using the antibodies detected in this investigation. There was no significantce different for incidence of LAIP between adult and pediatric patients. LAIP was observed in 77.6% of patients by labeling only CD34/CD10/CD19/CD45 4-color antibody combination. It is concluded that in 90% of childhood and adult B-ALL patients LAIP can be found, which suits MRD detection by multiparameter flow cytometry.

[Clinical significance for minimal residual disease detection by 4 color flow cytometry in adult and childhood B lineage acute lymphoblastic leukemia].

OBJECTIVE: To evaluate the clinical significance for minimal residual disease (MRD) detection by 4 color flow cytometry in B lineage acute lymphoblastic leukemia (B-ALL). METHODS: MRD was analyzed and followed up by using two panels of 4 color antibodies, mainly CD34/CD10/CD45/CD19, in 671 consecutive bone marrow specimens and 1 cerebrospinal fluid from 98 B-ALL patients. In 26 cases of them the immunophenotyping informations at diagnosis were not available. RESULTS: Of 671 bone marrow samples, 579 were MRD negative with leukemic cells below 0.0001 and 93 were MRD positive with leukemic cells over 0.0001. Of 93 MRD positive samples, leukemic cells below 0.05 were found in 64 bone marrow samples, meanwhile in the other 29 samples leukemic cells were over 0.05. Twenty patients relapsed, 19 were bone marrow relapse and one center nerves system. Fifteen of them were found MRD positive 7 - 17 weeks before relapse including 6 patients having no immunophenotyping data at diagnosis. The percentages of leukemia cells in these 15 patients were all over 0.0001. Two relapsed patients were MRD negative in 3 and 9 months before relapse, respectively. Two relapsed after MRD monitoring stopped. If MRD level was > 0.0001 at the end of induction chemotherapy and 12 weeks of treatment, the rate of relapse was 50% (6/12), while, it was 7.5% (3/40) in MRD negative patients (P = 0.000). CONCLUSION: Relapses can be predicted by MRD monitoring, if MRD was positive in the early phase of treatment, the risk of relapse was higher. Based on the characteristics of B cells ontogeny, MRD detection can be done independently of immunophenotypic information at diagnosis.

[Comparison of the immunophenotype of patients with B lineage acute lymphoblastic leukemia at diagnosis and relapse].

OBJECTIVE: To compare the leukemia-associated immunophenotypes (LAIP) in patients with B lineage acute lymphoblastic leukemia (B-ALL) at diagnosis and relapse, and investigate its implications for minimal residual disease (MRD) detection. METHODS: The immunophenotype of leukemia cells from 410 newly diagnosed and 6 relapsed patients with B-ALL were detected by four to six antibody combination, mainly CD34/CD10/CD45/CD19 of 4-color CD45/SSC gating flow cytometry (FCM). RESULTS: The proportion of CD45 under-expressed or negative in relapsed patients was much higher than that in newly diagnosed patients, being 69.2% and 37.8% respectively. Immunophenotypic changes occurred in 9 relapsed patients (including 8 hematological relapse and 1 central nerves system relapse) when analyzed by paired samples analysis at diagnosis vs at relapse: 4 cases showed CD45 down-modulation and 2 up-modulation; 4 CD34 down-modulation and 2 CD10 up-modulation, while the expression of CD19 remained no change. MRD was observed in all 7 cases of hematological relapse 2 - 4 months before relapse, and the immunophenotype of MRD cells was the same as that in relapse. CONCLUSION: A high frequency of immunophenotypic changes occurred at relapse and even in MRD before relapse, however the accuracy of MRD monitoring seemed not affected by the FCM strategy used in this investigation.

[Detection of minimal residual disease in B lineage acute lymphoblastic leukemia by 4-color flow cytometry].

OBJECTIVE: To evaluate the method and value of detecting bone marrow minimal residual disease (MRD) in B lineage acute lymphoblastic leukemia (B-ALL) by multiparameter flow cytometry (FCM). METHODS: The FCM immunophenotyping of B-lymphocyte precursors in regenerating stage and MRD was analyzed by using two sets of 4-color antibody panels, including mainly CD34, CD10, CD45, CD19, in 26 regenerating bone marrow samples after chemotherapy and 297 consecutive bone marrow specimens from 50 patients with B-ALL, respectively. The immunophenotype of leukemia cells of B-ALL was also detected by four to six antibodies combination of 4-color CD45/SSC gating FCM. The CD19, CD10, CD34 fluorescence intensity of B-cell precursors in normal and leukemic bone marrow was compared by relatively quantitative method. RESULTS: Twelve patients were MRD positive (MRD(+)) among 50 patients during MRD monitoring, the percentages of residual leukemia cells were 0.06% to 7.73%. Eleven cases displayed CD45, CD34, CD19, CD10 antigens aberrant expression. Five patients were bone marrow relapsed and 4 of them were MRD (+) 7-17 weeks before relapse. The percentages of leukemia cells in all the 4 patients were over 0.1%. Only one patient relapsed with MRD negative. The regenerated B precursors could be divided into 3 stages according to the expression of CD45, CD34, CD19, CD10, CD22, and CD20 antigens. Abnormal expression of CD45, CD34, CD19, and CD10 were detected in 71.8% of 213 patients with B-ALL, the percentage of only aberrant expression of CD38 and myeloid antigen was 8.1%. CONCLUSION: Detection of MRD by multiparameter flow cytometry is a rapid, quantitative and sensitive approach, and has a higher predictability for relapse.

[The clinical significance of detecting minimal residual disease in acute childhood B-cell lymphoblastic leukemia with flow cytometry].

OBJECTIVE: Flow cytometry may be used to detect minimal residual disease (MRD) in acute lymphoblastic leukemia because leukemic cells often display aberrant phenotypes when compared to normal cells. The present study was designed to establish a flow cytometric method for detecting MRD in children with B-ALL and evaluate its clinical prognostic value. The investigators also aimed to study the value of the detection of MRD by flow cytometry in childhood B-ALL without effective antibody combinations. METHODS: Thirty-six cases of childhood B-ALL with effective antibody combinations were performed MRD analysis after induction therapy. The authors detected MRD in 6 cases without effective antibody combinations by the four-color antibody combinations consisting of CD(45)/CD(19)/CD(10)/CD(34) and CD(45)/CD(19)/CD(20)/CD(22) and detected the aberrance of the minor subsets of CD(19)(+) cells. RESULTS: (1) Forty-two cases of childhood B-ALL were screened for antibody combinations of interest and were identified in 86% (36/42) of the cases. The sensitivity of this method was 0.01%. (2) Patients with MRD levels > or = 0.01% at 9 and 12 months of therapy had significantly low disease-free survival compared with patients with MRD levels < 0.01%. (3) Six out of seven patients with recurrence in the BM had MRD levels > or = 0.1% prior to recurrence. Patients with MRD levels > or = 0.1% during chemotherapy had significantly low disease-free survival as compared with patients with MRD values < 0.1%. (4) Two out of seven patients with recurrence had positive results of the qualitative PCR prior to recurrence. (5) Five patients with recurrence had no shift of antigen expression at relapse except that a patient missed CD(13). (6) Detectable MRD was not found in six patients without effective antibody combinations. CONCLUSION: (1) Flow cytometry is a sensitive and specific method for detecting MRD of childhood ALL, and could predict the coming relapse. (2) Patients with MRD levels > 10(-3) had poor prognosis. (3) The levels of MRD at month 9 and 12 had prognostic value. (4) The value of antibody combinations consisting of CD(45)/CD(19)/CD(10)/CD(34) and CD(45)/CD(19)/CD(20)/CD(22) should be further investigated in patients without effective antibody combinations.

[Clinical significance of detection of AML1/ETO fusion transcripts in childhood AML using real-time quantitative reverse transcription polymerase chain reaction].

This study was aimed to investigate the clinical value of quantification of AML1/ETO fusion transcripts using real-time reverse transcription PCR. Fourteen AML1/ETO positive children out of 52 AML children were selected. A serial dilution of AML1/ETO plasmid was used as a template for the AML1/ETO real-time PCR. AML1/ETO was quantified according to the expression of the GAPDH housekeeping gene at new diagnosis and during/after chemotherapy and transplantation. SPSS statistics was used to analyze the data. The results showed that the ratio of AML1/ETO: GAPDH expression level at new diagnosis varied in the range 0.219-2.080 (median 0.648) among the patients, without relevance with percentage of blasts. The detection sensitivity was up to the dilution of 1:10(5). Six patients showed a slight decline of AML1/ETO (higher than 5 x 10(-2)) at 1 month, three of whom relapsed in the early stage and one later. Five patients had a higher level than 5 x 10(-3) at 3 months, three of whom relapsed. Four patients with always a higher level than 5 x 10(-3) all relapsed in early stage. After six months, four out of them with constant low-level expression (10(-4) - 10(-6)) were in continuous complete hematological remission (CCR). In another patient, a rapid rise of AML1/ETO transcripts could be detected at CR stage and he relapsed 5 months later. The AML1/ETO gene expression leveling off by 10(-5) - 10(-6) could be detected in 3 patients at their complete remission after 9 months. It is concluded that real-time RT-PCR is a suitable approach for quantifying AML1/ETO transcripts in monitoring of AML patients with t(8;21) during/after chemotherapy and provides data of diagnostic relevance.

[Real-time quantitative study of minimal residual disease in childhood B cell acute lymphoblastic leukemia].

OBJECTIVE: The study was aimed to investigate the feasibility and clinical significance of quantitative detection of minimal residual disease (MRD) in childhood acute lymphoblastic leukemia (ALL) by real-time quantitative polymerase chain reaction (RQ-PCR). METHODS: Clonal IgH gene rearrangements of samples at diagnosis were identified by standard PCR assay with consensus primers. Monoclonal IgH gene rearrangements were analyzed using DNAPLOT software. Upstream primers were designed with the Primer Express software and allele specific oligonucleotide developed complementary to the V-D or D-J junction. Samples at diagnosis were serially diluted to generate the patient specific standard curves. RQ-PCR method was used to quantify the MRD of the follow up samples collected at five time points during chemotherapy. To check the quantity and quality of DNA, the investigators used RQ-PCR analysis for the albumin gene. RESULTS: Totally 16 monoclonal IgH gene rearrangements were identified from 34 patients with B-ALL. The analysis of the 16 monoclonal rearrangements showed that the most frequently used V segment was from V3 family and J segment from J4 and J6. The RQ-PCR sensitivity of 10(-4) to 10(-5) was mostly reached. Non-specific amplification was seen in 6 patients. The number of inserted and deleted nucleotides did not appear to be related to the sensitivity (P > 0.05). The correlation coefficients of all 16 standard curves were excellent (> or = 0.99). The mean slope of the standard curves was -3.4 +/- 0.37 and the mean intercept was 24.3 +/- 2.95. MRD analysis of follow up samples from the 16 patients showed an association between high degree of MRD and relapse. There was no apparent relationship between MRD degree at the end of induction chemotherapy and other high risk factors of ALL (P > 0.05). CONCLUSION: The study showed that the above approach with RQ-PCR was applicable to clinical detection of MRD in childhood ALL. Quantitative and dynamic study of MRD was of prognostic importance.