Ruidong Zhang

Gene expression-based classification and regulatory networks of pediatric acute lymphoblastic leukemia

Pediatric acute lymphoblastic leukemia (ALL) contains cytogenetically distinct subtypes that respond differently to cytotoxic drugs. Subtype classification can be also achieved through gene expression profiling. However, how to apply such classifiers to a single patient and correctly diagnose the disease subtype in an independent patient group has not been addressed. Furthermore, the underlying regulatory mechanisms responsible for the subtype-specific gene expression patterns are still largely unknown. Here, by combining 3 published microarray datasets on 535 mostly white childrens samples and generating a new dataset on 100 Chinese childrens ALL samples, we were able to (1) identify a 62-gene classifier with 97.6% accuracy from the white childrens samples and validated it on the completely independent set of 100 Chinese samples, and (2) uncover potential regulatory networks of ALL subtypes. The classifier we identified was, thus far, the only one that could be applied directly to a single sample and that sustained validation in a large independent patient group. Our results also suggest that the etiology of ALL is largely the same among different ethnic groups, and that the transcription factor hubs in the predicted regulatory network might play important roles in regulating gene expression and development of ALL.

FPGS rs1544105 polymorphism is associated with treatment outcome in pediatric B-cell precursor acute lymphoblastic leukemia

BackgroundFolypolyglutamate synthase (FPGS) catalyzes the polyglutamation of folates and antifolates, such as methotrexate (MTX), to produce highly active metabolites. FPGS tag SNP rs1544105C > T is located in the gene promoter. The aim of the present study was to investigate the impact of rs1544105 polymorphism on the treatment outcome in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL).MethodsThis study enrolled 164 children with BCP-ALL. We genotyped the FPGS SNP rs1544105, and analyzed the associations between its genotypes and treatment outcome. We also examined FPGS mRNA levels by real-time PCR in 64 of the 164 children, and investigated the function of this polymorphism on gene expression.ResultsWe found significantly poor relapse-free survival (RFS) (p = 0.010) and poor event-free survival (EFS) (p = 0.046) in carriers of CC genotype. Multivariable Cox regression analyses adjusted for possible confounding variables showed that, relative to the CT + TT genotypes, the CC genotype was an independent prognostic factor for poor RFS (hazard ratio [HR], 4.992.; 95% CI, 1.550-16.078; p = 0.007). No association was found between any toxicity and rs1544105 polymorphism. Quantitative PCR results showed that individuals with the T allele had lower levels of FPGS transcripts.ConclusionsOur study indicates that FPGS rs1544105C > T polymorphism might influence FPGS expression and affect treatment outcome in BCP-ALL patients.

NOTCH1 mutations are associated with favourable long-term prognosis in paediatric T-cell acute lymphoblastic leukaemia: a retrospective study of patients treated on BCH-2003 and CCLG-2008 protocol in China

Activating mutations of NOTCH1 are a common occurrence in T‐cell acute lymphoblastic leukaemia (T‐ALL), but its impact on T‐ALL treatment is still controversial. In this study, the incidence, clinical features, and prognosis of 92 Chinese children with T‐ALL treated using the Beijing Childrens Hospital‐2003 and Chinese Childhood Leukaemia Group‐2008 protocols were analysed. NOTCH1 mutations were found in 42% of T‐ALL patients and were not associated with clinical features, prednisone response, and minimal residual disease (MRD) at day 33 and 78. However, proline, glutamate, serine, threonine (PEST)/transactivation domain (TAD) mutations were associated with younger age (15/16 mutant vs. 48/76 wild‐type, P = 0·018) and more central nervous system involvement (4/16 mutant vs. 3/76 wild‐type, P = 0·016); while heterodimerization domain (HD) mutations were associated with KMT2A‐MLLT1 (MLL‐ENL; 4/30 mutant vs. 1/62 wild‐type, P = 0·037). Furthermore, prognosis was better in patients with NOTCH1 mutations than in those with wild‐type NOTCH1 (5‐year event‐free survival [EFS] 92·0 ± 4·5% vs. 64·0 ± 7·1%; P = 0·003). Long‐term outcome was better in patients carrying HD mutations than in patients with wild‐type HD (5‐year EFS 89·7 ± 5·6% vs. 69·3 ± 6·2%; P = 0·034). NOTCH1 mutations and MRD at day 78 were independent prognostic factors. These findings indicate that NOTCH1 mutation predicts a favourable outcome in Chinese paediatric patients with T‐ALL on the BCH‐2003 and CCLG‐2008 protocols, and may be considered a prognostic stratification factor.

Low expressions of ARS2 and CASP8AP2 predict relapse and poor prognosis in pediatric acute lymphoblastic leukemia patients treated on China CCLG-ALL 2008 protocol

ARS2 protein is important to early development and cell proliferation, in which ARS2-CASP8AP2 interaction is implicated. However, the predictive significance of ARS2 in childhood acute lymphoblastic leukemia (ALL) is unknown. Here we evaluate the predictive values of ARS2 expression and combined ARS2 and CASP8AP2 expression in relapse. We showed that ARS2 expression in ALL bone marrow samples at initial diagnosis was markedly lower than that in complete remission (CR). Likewise, the levels of ARS2 expression in the patients suffering from relapse were significantly lower than that of patients in continuous CR. Furthermore, low expression of ARS2 was closely correlated to poor treatment response including poor prednisone response and high minimal residual disease (MRD), and the patients with high MRD (≥10(-4)) and low ARS2 were more subject to relapse. The multivariate analyses for relapse free survival and event free survival revealed that ARS2 expression remained an independent prognostic factor after adjusting other risk factors. In addition, combined assessment of ARS2 and CASP8AP2 expression was more accurate to predict relapse, based on which an algorithm composed of ARS2 and CASP8AP2 expression, prednisone response and MRD (day 78) was proposed. Together, ARS2 and CASP8AP2 expressions can precisely predict high-risk of relapse and ALL prognosis.

Infections during induction therapy of protocol CCLG-2008 in childhood acute lymphoblastic leukemia: a single-center experience with 256 cases in China.

Background:Infections remain a major cause of therapy-associated morbidity and mortality in children with acute lymphoblastic leukemia (ALL). Methods:We retrospectively analyzed the medical charts of 256 children treated for ALL under the CCLG-2008 protocol in Beijing Childrens Hospital. Results:There were 65 infectious complications in 50 patients during vincristine, daunorubicin, L-asparaginase and dexamethasone induction therapy, including microbiologically documented infections (n = 12; 18.5%), clinically documented infections (n = 23; 35.3%) and fever of unknown origin (n = 30; 46.2%). Neutropenia was present in 83.1% of the infectious episodes. In all, most infections occurred around the 15th day of induction treatment (n = 28), and no patients died of infection-associated complications. Conclusions:The infections in this study was independent of treatment response, minimal residual diseases at the end of induction therapy, gender, immunophenotype, infection at first visit, risk stratification at diagnosis, unfavorable karyotypes at diagnosis and morphologic type. The infection rate of CCLG-2008 induction therapy is low, and the outcome of patients is favorable.

DNMT3A mutations and prognostic significance in childhood acute lymphoblastic leukemia

Abstract Little is known about DNMT3A mutations in childhood acute lymphoblastic leukemia (ALL). We screened for DNMT3A mutations in exon 23 and its adjacent intron regions in diagnostic samples of 201 children with ALL. The cDNA samples from 82 patients were also sequenced to identify other mutations in the entire coding region. DNMT3A mutations were detected in exon 23 and its adjacent intron regions only in five patients (2.5%). There was only one mutation in exon 23 in two patients, respectively. In the other three patients, five intronic mutations were found. None of the mutations was found in the five corresponding complete remission samples. DNMT3A mutations were correlated with higher minimal residual disease at the end of remission induction (p = 0.078). Treatment outcome was obviously worse in patients with DNMT3A mutations than in other patients (p < 0.05). Thus, DNMT3A mutations can be found in a few children with ALL, and may have an adverse impact on prognosis.

Polymorphisms in methotrexate transporters and their relationship to plasma methotrexate levels, toxicity of high-dose methotrexate, and outcome of pediatric acute lymphoblastic leukemia

High-dose methotrexate (HDMTX) plays an important role in the treatment of acute lymphoblastic leukemia (ALL) although there is great inter-patient variability in the efficacy and toxicity of MTX. The relationship between polymorphisms in genes encoding MTX transporters and MTX response is controversial. In the present study, 322 Chinese children with standard- and intermediate-risk ALL were genotyped for 12 polymorphisms. SLCO1B1 rs10841753 showed a significant association with plasma MTX levels at 48 h (P = 0.017). Patients who had the ABCB1 rs1128503 C allele had longer duration of hospitalization than did those with the TT genotype (P = 0.006). No association was found between oral mucositis and any polymorphism. Long-term outcome was worse in patients with the SLCO1B1 rs4149056 CC genotype than in patients with TT or TC (5-year event-free survival [EFS] 33.3 ± 19.2% vs. 90.5 ± 1.7%, P < 0.001), and was worse in patients with the SCL19A1 rs2838958 AA genotype than in patients with AG or GG (5-year EFS 78.5 ± 4.6% vs. 92.2 ± 1.8%, P = 0.008). Multiple Cox regression analyses revealed associations of minimal residual disease (MRD) at day 33 (hazard ratio 3.458; P = 0.002), MRD at day 78 (hazard ratio 6.330; P = 0.001), SLCO1B1 rs4149056 (hazard ratio 12.242; P < 0.001), and SCL19A1 rs2838958 (hazard ratio 2.324; P = 0.019) with EFS. Our findings show that polymorphisms in genes encoding MTX transporters substantially influence the kinetics and response to HDMTX therapy in childhood ALL.

MRD analysis and treatment outcome in three children with SET-NUP214-positive hematological malignancies

Sir, Chromosome aberrations that generate fusion genes are common in hematological malignancies. The SETNUP214 fusion gene resulting from balanced t(9;9) (q34;q34) or del(9)(q34.11q34.13) is rare in hematological malignancies. The SET-NUP214-positive cases were reported as patients with acute undifferentiated leukemia (AUL), T-cell acute lymphoblastic leukemia (T-ALL), or acute myelomonocytic leukemia (von Lindern et al., 1992; Van Vlierberghe et al., 2008) with estimated incidence of 2.9% in adult T-ALL (Gorello et al., 2008). However, there is no conclusive evidence in the aspect of minimal residual disease (MRD) and prognosis for the SETNUP214-positive pediatric hematological malignancies till now. Here, we reported the dynamics of MRD and outcomes of three children with SET-NUP214-positive leukemia/lymphoma. Of the 803 children with hematological malignancies (including 534 cases of B cell precursor ALL, 48 cases of T-ALL, 149 cases of acute myeloid leukemia, 27 cases of chronic myeloid leukemia, and 45 cases of lymphoma) enrolled in Beijing Children’s Hospital (BCH) from January 2007 to October 2010, we found three (0.4%) children with SET-NUP214-positive leukemia/lymphoma by nested multiplex PCR (Pallisgaard et al., 1998). Identical fusion transcripts with a fusion between exon 7 of SET and exon 18 of NUP214 were confirmed by sequence analysis in the three patients. Morphology analysis demonstrated lymphoid characteristics in all of the three patients. However, immunophenotyping showed some differences: CD34+/CD13+/ CD33+/CD117+/CD41+, HLA-DR+/CD33+/CD117+/CD2+, CD34+/CD10+ in the three patient respectively, in addition to CD5+/CD7+/cytoplasmic CD3+ in all of the three patients. Chromosomal karyotyping only succeeded in patient 1, which showed complex chromosomal abnormality: 49,XX,+6,+8,-17,+19,+21[3]/47,XX,+14[1]/47,XX, +20[1]/45,XX,-15[1]/46,XX[11]. Finally, these three patients were diagnosed as hybrid acute leukemia (HAL), T-ALL, and T lymphoblastic lymphoma, respectively, and treated in accordance with BCH-2003 protocol for ALL medium risk (MR) according to BFM risk criteria (Table 1). We quantified MRD at six time points (TP) (Figure 1) with two types of markers. Clonal Ig/TCR rearrangements at diagnosis were analyzed using BIOMED-2 standard multiplex PCR protocols (van Dongen et al., 2003). Allele-specific oligonucleotides were designed and used in combination with the germline TaqMan probes and reverse primers to quantify MRD according to European MRD laboratory guidelines (van der Velden et al., 2007; Liu et al., 2008; Cui et al., 2010). Quantitative range of 10 and sensitivity of 10 10 were achieved in all the RQ-PCR reactions (Figure 1a). We chose TRD/TRB, TRD, and TRB rearrangement as MRD targets for the three patients, respectively. To quantify MRD with SET-NUP214 fusion transcript, we had tried to design TaqMan probe and primers but failed to reach good sensitivity and reproducibility. At last, the SYBR GREEN I chemistry was chosen for the detection of the fusion transcript. A standard curve (Slope: )3.35, R:0.99 and Intercept: 36.77) was established using serial dilutions of recombinant plasmids containing SET-NUP214 fusion fragment. The quantitative range and sensitivity were 10 copies and one copy, respectively (Figure 1b). ABL gene was used as an internal control (Beillard et al., 2003). In all of the 15 follow-up bone marrow (BM) samples, there was a strong correlation between MRD detected by fusion transcript and Ig/TCR rearrangements (Spearman r = 0.9034, P < 0.0001). This indicated that the two methods were largely consistent for each patient. So, the SET-NUP214 fusion transcript could be used as an alternative MRD marker for this type of hematological malignancy. We noted tremendous differences in MRD and outcome among the three patients. Patient 1 with HAL reached complete remission (CR) at the 5th month and underwent allogeneic HSCT after 45 days; accordingly, MRD was always above 10. She finally suffered BM relapse at the 12th month from the beginning of chemotherapy. Patient 2 with T-ALL achieved clinical CR at LETTER TO THE EDITOR INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY

Low CREBBP expression is associated with adverse long-term outcomes in paediatric acute lymphoblastic leukaemia

CREBBP alterations are associated with many diseases including leukaemia. However, CREBBP expression and its clinical relevance in paediatric acute lymphoblastic leukaemia have not been elucidated.

An advanced fragment analysis-based individualized subtype classification of pediatric acute lymphoblastic leukemia

Pediatric acute lymphoblastic leukemia (ALL) is the most common neoplasm and one of the primary causes of death in children. Its treatment is highly dependent on the correct classification of subtype. Previously, we developed a microarray-based subtype classifier based on the relative expression levels of 62 marker genes, which can predict 7 different ALL subtypes with an accuracy as high as 97% in completely independent samples. Because the classifier is based on gene expression rank values rather than actual values, the classifier enables an individualized diagnosis, without the need to reference the background distribution of the marker genes in a large number of other samples, and also enables cross platform application. Here, we demonstrate that the classifier can be extended from a microarray-based technology to a multiplex qPCR-based technology using the same set of marker genes as the advanced fragment analysis (AFA). Compared to microarray assays, the new assay system makes the convenient, low cost and individualized subtype diagnosis of pediatric ALL a reality and is clinically applicable, particularly in developing countries.