Abstract

In childhood acute lymphoblastic leukemia (ALL), early response to treatment is a powerful prognostic indicator. To identify genes associated with this response, we analyzed gene expression of diagnostic lymphoblasts from 189 children with ALL and compared the findings with minimal residual disease (MRD) levels on days 19 and 46 of remission induction treatment. After excluding genes associated with genetic subgroups, we identified 17 genes that were significantly associated with MRD. The caspase 8–associated protein 2 (CASP8AP2) gene was studied further because of its reported role in apoptosis and glucocorticoid signaling. In a separate cohort of 99 patients not included in the comparison of gene expression profiles and MRD, low levels of CASP8AP2 expression predicted a lower event-free survival (P = .02) and a higher rate of leukemia relapse (P = .01) and were an independent predictor of outcome. High levels of CASP8AP2 expression were associated with a greater propensity of leukemic lymphoblasts to undergo apoptosis. We conclude that measurement of CASP8AP2 expression at diagnosis offers a means to identify patients whose leukemic cells are highly susceptible to chemotherapy. Therefore, this gene is a strong candidate for inclusion in gene expression arrays specifically designed for leukemia diagnosis.

Introduction

Response to therapy in childhood acute lymphoblastic leukemia (ALL) is ultimately linked to the expression of genes that control cellular drug sensitivity and propensity to apoptosis. The discovery of such genes is important because it could provide a means to enhance classification systems based on relapse hazard and to identify signaling pathways that could be productively targeted with novel therapies. Genome-wide expression profiling technology promises to significantly facilitate these discoveries, as shown by studies determining gene expression changes in response to methotrexate and/or mercaptopurine,1,2  and by correlative studies based on drug sensitivity findings in vitro.3-5 

Minimal residual disease (MRD) assays provide a direct measure of treatment response in vivo that is likely to depend not only on the resistance of leukemic cells to individual drugs, but on other factors as well, including drug interactions and pharmacokinetic/pharmacogenetic variables.6-8  Such assays have revealed considerable heterogeneity in the response of childhood ALL patients to remission induction therapy, which was not appreciated from conventional microscopic analyses.9-14  While some patients can show profound reductions in their leukemia cell counts (to less than one leukemic cell among 10 000 normal bone marrow cells) after only 2 weeks of remission induction chemotherapy,13,15  others require additional remission induction chemotherapy to achieve a similar level of leukemia cytoreduction, or retain detectable MRD beyond the completion of remission induction and consolidation treatment.9-13  Thus, treatment response measured by MRD assays has consistently been the most powerful prognostic indicator in childhood ALL.7,16 

In this study, we sought to identify genes whose expression is closely associated with the in vivo response to multiagent chemotherapy. We therefore compared the gene expression profiles of ALL cells obtained at diagnosis from 189 children with MRD findings on days 19 and 46 of remission induction chemotherapy. Seventeen genes whose expression was specifically associated with MRD at both time points were identified, including CASP8AP2 (caspase 8–associated protein 2), which encodes a key mediator of apoptosis and glucocorticoid signaling,17-20  and whose expression in this study was inversely related to persistent MRD during remission induction therapy. In a separate cohort of 99 children with ALL, CASP8AP2 expression was a strong and independent predictor of treatment outcome.

Materials and methods

Patients and treatment

Bone marrow samples were collected at diagnosis from 288 children with ALL enrolled in St Jude Total Therapy Studies XIII, XIV, or XV. Samples were also collected during remission induction chemotherapy from 189 patients: MRD was studied on day 19 in 187 and on day 46 in 188 of these patients. At diagnosis, the immunophenotypic and karyotypic features of the leukemic cells were determined according to standard techniques.21,22  The presence of BCR-ABL, E2A-PBX1, and TEL-AML1 fusions or MLL gene rearrangements was detected by reverse transcriptase–polymerase chain reaction (RT-PCR).23  Among the 288 ALL cases studied (189 to identify genes associated with MRD and 99 to test the clinical significance of the identified genes), 47 were classified as T-lineage ALL and 241 as B-lineage ALL. The latter included 16 cases with BCR-ABL, 22 with E2A-PBX1, 18 with MLL rearrangements, 57 with TEL-AML1, 52 with hyperdiploidy (> 50 chromosomes), and 76 with other features.

Initial treatment consisted of methotrexate alone followed 4 days later by 6 weeks of remission induction therapy with prednisone, vincristine, daunorubicin, asparaginase, and etoposide plus cytarabine.24-26  Once they attained a complete clinical remission, all patients received 2 weeks of consolidation therapy with high-dose methotrexate and mercaptopurine, followed by risk-directed continuation therapy. The studies were approved by the St Jude institutional review board, with informed consent obtained from the parents or guardians of each child.

Gene expression profiling

Gene expression profiling studies were performed as previously described.27,28  Briefly, bone marrow mononuclear cells obtained at diagnosis were enriched with a density gradient, washed twice, and cryopreserved. We isolated total RNA from bone marrow mononuclear cells using the Trizol reagent (Invitrogen, Carlsbad, CA). After generating cDNA, we prepared biotin-labeled cRNA hybridization solutions according to the protocols of Affymetrix (Santa Clara, CA). The solutions were hybridized to HG-U133A oligonucleotide microarrays (Affymetrix). After staining with phycoerythrin-conjugated streptavidin, the arrays were read with a laser confocal scanner (Agilent, Palo Alto, CA). Signal values were computed from the image files using Affymetrix GeneChip Operating Software. Signal intensities were normalized to a standard target value of 500. Detection calls (present, marginal, or absent) were determined by default parameters. Intensity values for a total of 22 283 probe sets on the U133A microarray were obtained.

Minimal residual disease studies

Studies of MRD were performed by flow cytometry as previously described.9,12-14,29  Bone marrow mononuclear cells were labeled with various combinations of monoclonal antibodies conjugated to fluorescein isothiocyanate, phycoerythrin, peridinin chlorophyll protein, and allophycocyanin. For each case, optimal marker combinations specific for the leukemic clone were selected by labeling bone marrow mononuclear cells at diagnosis with antibody combinations previously shown to distinguish leukemic from normal cells; the combinations were then applied during clinical remission. Cell staining was analyzed using a dual laser FACSCalibur flow cytometer with Cell Quest software (Becton Dickinson, San Jose, CA). The flow cytometry protocol used for MRD detection has been described in detail elsewhere.12,13,30  In all samples, the data represent all mononuclear cells in each test tube (> 1 × 105). These detection methods allow the identification of one leukemic cell among 10 000 or more normal bone marrow cells,30  produce results that are highly concordant with those obtained by PCR analysis of antigen receptor genes,31  and are currently applicable to more than 95% of patients.

CASP8AP2 expression studies

For detection of CASP8AP2 expression by RT-PCR, we obtained cDNA by reverse transcription of RNA of established ALL cell lines and primary ALL samples with random hexamers and amplified it using the primers 5′-GAAGGTAATCATCCTGCATT-3′ (sense) and 5′-GAGCTTCATTAGCTGCTGGA-3′ (antisense). PCR amplification was performed for 30 cycles (95°C for 45 seconds, 56°C for 45 seconds, and 72°C for 60 seconds). The PCR products were separated on a 2% agarose gel, and the DNA was visualized by ethidium bromide staining.

For flow cytometry, we used a specific rabbit polyclonal antibody anti-CASP8AP2 from ProSCI (Poway, CA). Cells were permeabilized with 8E, a reagent developed in our laboratory, and incubated with the antibody or with nonreactive rabbit immunoglobulin (as a control). After 2 washes in phosphate-buffered saline containing 0.2% serum albumin and 0.2% sodium azide, cells were incubated with a goat anti–rabbit Ig antibody conjugated to phycoerythrin (Jackson Laboratories, West Grove, PA). Tests in which the anti-CASP8AP2 antibody was preincubated with the immunizing peptide (ProSCI) were performed to ensure specificity of staining. Cells were analyzed with a FACSCalibur flow cytometer.

In vitro culture of leukemic lymphoblasts

In vitro cultures of leukemic lymphoblasts on bone marrow mesenchymal cells were performed as previously described.32  Briefly, the leukemic cells were resuspended in AIM-V medium (Gibco, Grand Island, NY) at a final concentration of 1.5 × 106/mL. Of the suspension, 200 μL was then placed in a 96-well tissue culture plate or seeded onto confluent bone marrow mesenchymal cell layers. In all samples, cell viability exceeded 80% by trypan-blue dye exclusion. All cell cultures were performed at 37°C under 5% CO2.

At the termination of cultures, cells were harvested by vigorous pipetting. B-lineage ALL samples were incubated with CD19 monoclonal antibody conjugated to FITC; T-ALL samples were incubated with FITC-conjugated CD7. All antibodies were from Becton Dickinson. Samples were analyzed with a FACScan flow cytometer with Cell Quest software, as previously described.32,33  After 7 days of culture, the percentage of cell recovery was calculated as follows: (no. of CD19+ or CD7+ lymphoblasts after 7 days of culture) × 100/(no. of CD19+ or CD7+ lymphoblasts after 1 hour of culture). Results are reported as the means of at least duplicate experiments. Leukemic cells were counted without knowledge of the patient's level of CASP8AP2 expression.

Statistical analysis

Individual genes associated with MRD adjusted for lineage and genetic subtypes were identified with an analysis-of-variance (ANOVA) model; t test analysis was used to identify individual genes associated with MRD without adjustment for other factors. Statistical significance and false discovery rate (FDR) estimates in this part of the analysis were determined using the profile information criterion and the FDR estimator, as described.34 

Correlations between gene expression, clinicobiologic features of ALL, and MRD status were performed with the Kruskal-Wallis test for multiple samples or Wilcoxon–Mann-Whitney tests for 2 samples. Event-free survival and cumulative incidence of relapse (where death in remission and second malignancy were treated as competing risks) were analyzed by a proportional hazard regression model, as well as by log rank and Gray test, respectively. Cumulative incidence of relapse in relation to competing known prognostic factors of childhood was analyzed with a Fine and Gray model.35  All analyses were performed with the R (The R Project, http://www.r-project.org/), SAS (SAS Institute, Cary, NC), and S-plus (Insightful, Seattle, WA) programs.

Results

Identification of individual genes associated with MRD during remission induction therapy

We compared gene expression data for the diagnostic bone marrow samples of 189 patients with results of MRD measurements obtained in 187 of these patients on day 19, and in 188 on day 46 of remission induction therapy. MRD positivity was defined as 0.01% or more cells expressing the leukemia-associated immunophenotype identified at diagnosis among bone marrow mononuclear cells. By this criterion, 109 (58.3%) of 187 patients were MRD positive on day 19, and 43 (22.9%) of 188 on day 46, in agreement with our previous findings in different patient cohorts.12,13  After eliminating the possible confounding influence of genetic subtypes known to be associated with treatment response (BCR-ABL, MLL gene rearrangements, TEL-AML1, and hyperdiploidy > 50 chromosomes), and applying a P value threshold of .001 by t test (estimated FDR, 17.1%),34  we identified 105 probe sets whose expression was associated with MRD on day 46. The probe sets corresponded to 85 named genes; 53 were overexpressed in diagnostic samples from patients with MRD on day 46, and 32 were underexpressed (Table 1). Expression of 17 of the 85 genes was also significantly (P < .02) related to the presence or absence of MRD at day 19: 10 such genes were overexpressed in diagnostic samples of patients who had MRD at days 19 and 46, whereas 7 were underexpressed (Table 1).

Table 1.

Genes significantly associated with MRD on day 46


Probe set

Representative public ID

Gene title
Genes underexpressed in MRD+   
    206502_s_at  NM_002196  Insulinoma-associated 1  
    217281_x_at   AJ239383   IgM rheumatoid factor RF-TT9, variable heavy chain  
    217098_s_at   Z98745   Zinc finger protein 96  
    218736_s_at  NM_017734  Palmdelphin  
    207894_s_at  NM_020552  T-cell leukemia/lymphoma 6  
    220657_at  NM_018143  Kelch-like 11 (Drosophila)  
    206142_at  NM_003436  Zinc finger protein 135 (clone pHZ-17)  
    203702_s_at   AL043927   Tubulin tyrosine ligase-like family, member 4* 
    207940_x_at  NM_001840  Cannabinoid receptor 1 (brain)  
    207979_s_at  NM_004931  CD8 beta polypeptide 1* 
    218986_s_at  NM_017631  Hypothetical protein FLJ20035 
    215717_s_at   X62009   Fibrillin 2 (congenital contractural arachnodactyly)  
    212419_at   AA131324   Chromosome 10 open reading frame 56  
    219364_at  NM_024119  Likely ortholog of mouse D11Igp2  
    209760_at   AL136932   KIAA0922 protein* 
    218033_s_at  NM_003498  Stannin  
    216444_at   AK024138   SMAD-specific E3 ubiquitin protein ligase 2  
    203276_at  NM_005573  Lamin B1* 
    209502_s_at   BC002495   BAI1-associated protein 2  
    205888_s_at   AI962693   Jak and microtubule interacting protein 2  
    203422_at  NM_002691  Polymerase (DNA directed), delta 1, catalytic subunit 125 kDa  
    211717_at   BC005853   Ankyrin repeat domain 40  
    203963_at  NM_001218  Carbonic anhydrase XII  
    218115_at  NM_018154  ASF1 antisilencing function 1 homolog B (S cerevisiae)* 
    219165_at  NM_021630  PDZ and LIM domain 2 (mystique)  
    209499_x_at   BF448647   Tumor necrosis factor (ligand) superfamily, member 13  
    202326_at  NM_006709  Euchromatic histone-lysine N-methyltransferase 2* 
    222201_s_at   AB037736   CASP8-associated protein 2* 
    38158_at   D79987   Extra spindle poles-like 1 (S cerevisiae) 
    218586_at  NM_018270  Chromosome 20 open reading frame 20  
    204804_at  NM_003141  Tripartite motif-containing 21  
    204599_s_at  NM_006428  Mitochondrial ribosomal protein L28  
Genes overexpressed in MRD+   
    201429_s_at  NM_000998  Ribosomal protein L37a  
    200025_s_at  NM_000988  Ribosomal protein L27  
    200034_s_at  NM_000970  Ribosomal protein L6  
    200038_s_at  NM_000985  Ribosomal protein L17  
    200949_x_at  NM_001023  Ribosomal protein S20  
    212042_x_at   BG389744   Ribosomal protein L7  
    200716_x_at  NM_012423  Ribosomal protein L13a  
    208904_s_at   BC000354   Ribosomal protein S28  
    208856_x_at   BC003655   Ribosomal protein, large, P0  
    216520_s_at   AF072098   Tumor protein, translationally controlled 1  
    200019_s_at  NM_001997  Ribosomal protein S30  
    200927_s_at   AA919115   RAB14, member RAS oncogene family  
    202649_x_at  NM_001022  Ribosomal protein S19  
    200937_s_at  NM_000969  Ribosomal protein L5  
    211927_x_at   BE963164   Eukaryotic translation elongation factor 1 gamma  
    212773_s_at   BG165094   Translocase of outer mitochondrial membrane 20 homolog (yeast)  
    213890_x_at   AI200589   Ribosomal protein S16  
    208724_s_at   BC000905   RAB1A, member RAS oncogene family  
    200081_s_at   BE741754   Ribosomal protein S6  
    200005_at  NM_003753  Eukaryotic translation initiation factor 3, subunit 7 zeta, 66/67 kDa  
    217915_s_at  NM_016304  Chromosome 15 open reading frame 15  
    218268_at  NM_022771  TBC1 domain family, member 15  
    217747_s_at  NM_001013  Ribosomal protein S9  
    201259_s_at   AI768845   Synaptophysin-like 1  
    217719_at  NM_016091  Eukaryotic translation initiation factor 3, subunit 6 interacting protein  
    211937_at  NM_001417  Eukaryotic translation initiation factor 4B  
    218041_x_at  NM_018573  Solute carrier family 38, member 2  
    214351_x_at   AA789278   Ribosomal protein L13  
    221726_at   BE250348   Ribosomal protein L22  
    204102_s_at  NM_001961  Eukaryotic translation elongation factor 2  
    209510_at   AF064801   Ring finger protein 139  
    200024_at  NM_001009  Ribosomal protein S5  
    201337_s_at  NM_004781  Vesicle-associated membrane protein 3 (cellubrevin)* 
    200632_s_at  NM_006096  N-myc downstream regulated gene 1  
    218084_x_at  NM_014164  FXYD domain containing ion transport regulator 5* 
    212202_s_at   BG493972   DKFZP564G2022 protein  
    221646_s_at   AF267859   Zinc finger, DHHC-type containing 11  
    203544_s_at  NM_003473  Signal transducing adaptor molecule 1* 
    218562_s_at  NM_018202  Transmembrane protein 57  
    206890_at  NM_005535  Interleukin 12 receptor, beta 1  
    204426_at  NM_006815  Coated vesicle membrane protein  
    208330_at  NM_021926  Aristaless-like homeobox 4  
    221718_s_at   M90360   A kinase (PRKA) anchor protein 13  
    209288_s_at   AL136842   CDC42 effector protein (Rho GTPase binding) 3* 
    202393_s_at  NM_005655  Kruppel-like factor 10  
    209732_at   BC005254   C-type lectin domain family 2, member B* 
    209795_at   L07555   CD69* 
    212509_s_at   BF968134   Matrix-remodeling associated 7* 
    209543_s_at   M81104   CD34  
    213075_at   AL050002   Olfactomedin-like 2A* 
    201904_s_at   BF031714   CTD small phosphatase-like  
    215177_s_at   AV733308   Integrin, alpha 6* 
    201325_s_at
 
NM_001423
 
Epithelial membrane protein 1*
 

Probe set

Representative public ID

Gene title
Genes underexpressed in MRD+   
    206502_s_at  NM_002196  Insulinoma-associated 1  
    217281_x_at   AJ239383   IgM rheumatoid factor RF-TT9, variable heavy chain  
    217098_s_at   Z98745   Zinc finger protein 96  
    218736_s_at  NM_017734  Palmdelphin  
    207894_s_at  NM_020552  T-cell leukemia/lymphoma 6  
    220657_at  NM_018143  Kelch-like 11 (Drosophila)  
    206142_at  NM_003436  Zinc finger protein 135 (clone pHZ-17)  
    203702_s_at   AL043927   Tubulin tyrosine ligase-like family, member 4* 
    207940_x_at  NM_001840  Cannabinoid receptor 1 (brain)  
    207979_s_at  NM_004931  CD8 beta polypeptide 1* 
    218986_s_at  NM_017631  Hypothetical protein FLJ20035 
    215717_s_at   X62009   Fibrillin 2 (congenital contractural arachnodactyly)  
    212419_at   AA131324   Chromosome 10 open reading frame 56  
    219364_at  NM_024119  Likely ortholog of mouse D11Igp2  
    209760_at   AL136932   KIAA0922 protein* 
    218033_s_at  NM_003498  Stannin  
    216444_at   AK024138   SMAD-specific E3 ubiquitin protein ligase 2  
    203276_at  NM_005573  Lamin B1* 
    209502_s_at   BC002495   BAI1-associated protein 2  
    205888_s_at   AI962693   Jak and microtubule interacting protein 2  
    203422_at  NM_002691  Polymerase (DNA directed), delta 1, catalytic subunit 125 kDa  
    211717_at   BC005853   Ankyrin repeat domain 40  
    203963_at  NM_001218  Carbonic anhydrase XII  
    218115_at  NM_018154  ASF1 antisilencing function 1 homolog B (S cerevisiae)* 
    219165_at  NM_021630  PDZ and LIM domain 2 (mystique)  
    209499_x_at   BF448647   Tumor necrosis factor (ligand) superfamily, member 13  
    202326_at  NM_006709  Euchromatic histone-lysine N-methyltransferase 2* 
    222201_s_at   AB037736   CASP8-associated protein 2* 
    38158_at   D79987   Extra spindle poles-like 1 (S cerevisiae) 
    218586_at  NM_018270  Chromosome 20 open reading frame 20  
    204804_at  NM_003141  Tripartite motif-containing 21  
    204599_s_at  NM_006428  Mitochondrial ribosomal protein L28  
Genes overexpressed in MRD+   
    201429_s_at  NM_000998  Ribosomal protein L37a  
    200025_s_at  NM_000988  Ribosomal protein L27  
    200034_s_at  NM_000970  Ribosomal protein L6  
    200038_s_at  NM_000985  Ribosomal protein L17  
    200949_x_at  NM_001023  Ribosomal protein S20  
    212042_x_at   BG389744   Ribosomal protein L7  
    200716_x_at  NM_012423  Ribosomal protein L13a  
    208904_s_at   BC000354   Ribosomal protein S28  
    208856_x_at   BC003655   Ribosomal protein, large, P0  
    216520_s_at   AF072098   Tumor protein, translationally controlled 1  
    200019_s_at  NM_001997  Ribosomal protein S30  
    200927_s_at   AA919115   RAB14, member RAS oncogene family  
    202649_x_at  NM_001022  Ribosomal protein S19  
    200937_s_at  NM_000969  Ribosomal protein L5  
    211927_x_at   BE963164   Eukaryotic translation elongation factor 1 gamma  
    212773_s_at   BG165094   Translocase of outer mitochondrial membrane 20 homolog (yeast)  
    213890_x_at   AI200589   Ribosomal protein S16  
    208724_s_at   BC000905   RAB1A, member RAS oncogene family  
    200081_s_at   BE741754   Ribosomal protein S6  
    200005_at  NM_003753  Eukaryotic translation initiation factor 3, subunit 7 zeta, 66/67 kDa  
    217915_s_at  NM_016304  Chromosome 15 open reading frame 15  
    218268_at  NM_022771  TBC1 domain family, member 15  
    217747_s_at  NM_001013  Ribosomal protein S9  
    201259_s_at   AI768845   Synaptophysin-like 1  
    217719_at  NM_016091  Eukaryotic translation initiation factor 3, subunit 6 interacting protein  
    211937_at  NM_001417  Eukaryotic translation initiation factor 4B  
    218041_x_at  NM_018573  Solute carrier family 38, member 2  
    214351_x_at   AA789278   Ribosomal protein L13  
    221726_at   BE250348   Ribosomal protein L22  
    204102_s_at  NM_001961  Eukaryotic translation elongation factor 2  
    209510_at   AF064801   Ring finger protein 139  
    200024_at  NM_001009  Ribosomal protein S5  
    201337_s_at  NM_004781  Vesicle-associated membrane protein 3 (cellubrevin)* 
    200632_s_at  NM_006096  N-myc downstream regulated gene 1  
    218084_x_at  NM_014164  FXYD domain containing ion transport regulator 5* 
    212202_s_at   BG493972   DKFZP564G2022 protein  
    221646_s_at   AF267859   Zinc finger, DHHC-type containing 11  
    203544_s_at  NM_003473  Signal transducing adaptor molecule 1* 
    218562_s_at  NM_018202  Transmembrane protein 57  
    206890_at  NM_005535  Interleukin 12 receptor, beta 1  
    204426_at  NM_006815  Coated vesicle membrane protein  
    208330_at  NM_021926  Aristaless-like homeobox 4  
    221718_s_at   M90360   A kinase (PRKA) anchor protein 13  
    209288_s_at   AL136842   CDC42 effector protein (Rho GTPase binding) 3* 
    202393_s_at  NM_005655  Kruppel-like factor 10  
    209732_at   BC005254   C-type lectin domain family 2, member B* 
    209795_at   L07555   CD69* 
    212509_s_at   BF968134   Matrix-remodeling associated 7* 
    209543_s_at   M81104   CD34  
    213075_at   AL050002   Olfactomedin-like 2A* 
    201904_s_at   BF031714   CTD small phosphatase-like  
    215177_s_at   AV733308   Integrin, alpha 6* 
    201325_s_at
 
NM_001423
 
Epithelial membrane protein 1*
 
*

Genes also associated with MRD on day 19.

Figure 1.

Expression ofCASP8AP2in leukemic lymphoblasts. (A) Expression of CASP8AP2 transcripts by RT-PCR in ALL cell lines and 10 primary ALL samples. (B) Expression of CASP8AP2 protein by flow cytometry in 2 cases of ALL. Overlay histograms indicate staining with anti-CASP8AP2 antibody and with nonreactive rabbit immunoglobulin (Control Ig) in each sample. The corresponding CASP8AP2 signal by gene array in each sample is shown. (C) Relation between CASP8AP2 transcript expression by gene array and protein expression by flow cytometry. Spearman correlation coefficient is shown.

Figure 1.

Expression ofCASP8AP2in leukemic lymphoblasts. (A) Expression of CASP8AP2 transcripts by RT-PCR in ALL cell lines and 10 primary ALL samples. (B) Expression of CASP8AP2 protein by flow cytometry in 2 cases of ALL. Overlay histograms indicate staining with anti-CASP8AP2 antibody and with nonreactive rabbit immunoglobulin (Control Ig) in each sample. The corresponding CASP8AP2 signal by gene array in each sample is shown. (C) Relation between CASP8AP2 transcript expression by gene array and protein expression by flow cytometry. Spearman correlation coefficient is shown.

A review of the reported functions of the 17 genes associated with MRD at both time points led us to select CASP8AP2 (caspase 8–associated protein 2), also known as FLASH (FLICE-associated huge protein), for further study. This gene encodes a protein that interacts with caspase 8, a key mediator of apoptosis,17,18  and has been shown to be a determinant of glucocorticoid signaling as well.19,20  The reported function of CASP8AP2 together with its underexpression in leukemic cells from patients with a poor initial early response to remission induction therapy (as demonstrated by the presence of MRD on days 19 and 46) provided a compelling rationale for assessing its clinical significance in childhood ALL.

CASP8AP2 transcripts were detectable in ALL cells by RT-PCR. In all 9 ALL cell lines (B-lineage: 380, OP-1, NALM6, RS4;11, REH, and 697; T-lineage: CEM-C1, CEM-C7, and Jurkat) and in 10 primary ALL samples (not included in the gene expression array study), a transcript of approximately 700 kb corresponding to CASP8AP2 was clearly detectable (Figure 1A). To demonstrate that the CASP8AP2 transcript was translated into the encoded protein in ALL cells, we used a rabbit polyclonal antibody anti-CASP8AP2. Labeling of the ALL cell line REH with this antibody after cell membrane permeabilization stained virtually all cells; staining was prevented by preincubating the antibody with the immunizing peptide corresponding to amino acids 1966 to 1981 of the human CASP8AP2 protein (not shown). In general, levels of protein expression correlated with those of the CASP8AP2 transcripts, as shown by staining of 12 primary ALL samples selected among those studied by microarray (r = 0.839, P < .001 by Spearman correlation test; Figure 1B-C). We noted, however, that differences in levels of transcript expression were generally higher than those measured by flow cytometry. It is unclear whether this was due to the low affinity of the polyclonal antibody used or to posttranslational regulatory mechanisms. Such a discrepancy has been reported for other molecules expressed in ALL cells.36 

Relation of CASP8AP2 expression to selected clinicobiologic features of ALL and treatment outcome

To assess the relationship of CASP8AP2 expression at diagnosis to the clinical and biologic features of ALL in 288 children with ALL, we divided the patients into 3 groups of 96 patients each according to level of CASP8AP2 expression measured by gene array. Among clinical features, low levels of expression were significantly more prevalent among patients younger than 1 year of age, a known adverse prognostic feature (P = .016),6  whereas patients with hyperdiploidy (> 50 chromosomes), a feature associated with favorable outcome,6  generally had higher levels of CASP8AP2 expression (P < .001) (Table 2). Using CASP8AP2 expression as a continuous variable, we found that low levels of expression were again significantly associated with age younger than 1 year (P = .012) and less favorable genetic subtypes (P < .001). No significant associations between CASP8AP2 and other presenting clinicobiologic features were apparent (Table 2). Levels of CASP8AP2 were not different among patients enrolled in the 3 sequential treatment protocols (Total XIII, XIV, and XV; P = .16 by Kruskal-Wallis test).

Table 2.

Correlation between CASP8AP2 gene expression and presenting clinicobiologic features




CASP8AP2 expression, no.

Presenting feature
Patients studied, no.
Lower
Middle
Higher
P*
Age      
    Younger than 1 y   9   7   1   1   .016  
    1 to 9 y   203   58   71   74   
    10 y or older   76   31   24   21   
Race      
    White   214   74   67   73   .401  
    Black   55   19   21   15   
    Other   19   3   8   8   
Sex      
    Male   169   58   56   55   .905  
    Female   119   38   40   41   
WBC, × 109/L      
    Less than 10   90   27   30   33   .328  
    10 to 50   85   27   26   32   
    50 to 100   52   16   17   19   
    More than 100   61   26   23   12   
BCR-ABL      
    Present   16   6   5   5   .936  
    Absent   272   90   91   91   
TEL-AML1      
    Present   57   15   19   23   .352  
    Absent   231   81   77   73   
MLL-AF4      
    Present   18   10   5   3   .099  
    Absent   270   86   91   93   
Ploidy      
    Hyperdiploid  52   8   15   29   < .001  
    Others   236   88   81   67   
DNA index      
    1.16 or higher   47   8   11   28   < .001  
    Less than 1.16
 
241
 
88
 
85
 
68
 

 



CASP8AP2 expression, no.

Presenting feature
Patients studied, no.
Lower
Middle
Higher
P*
Age      
    Younger than 1 y   9   7   1   1   .016  
    1 to 9 y   203   58   71   74   
    10 y or older   76   31   24   21   
Race      
    White   214   74   67   73   .401  
    Black   55   19   21   15   
    Other   19   3   8   8   
Sex      
    Male   169   58   56   55   .905  
    Female   119   38   40   41   
WBC, × 109/L      
    Less than 10   90   27   30   33   .328  
    10 to 50   85   27   26   32   
    50 to 100   52   16   17   19   
    More than 100   61   26   23   12   
BCR-ABL      
    Present   16   6   5   5   .936  
    Absent   272   90   91   91   
TEL-AML1      
    Present   57   15   19   23   .352  
    Absent   231   81   77   73   
MLL-AF4      
    Present   18   10   5   3   .099  
    Absent   270   86   91   93   
Ploidy      
    Hyperdiploid  52   8   15   29   < .001  
    Others   236   88   81   67   
DNA index      
    1.16 or higher   47   8   11   28   < .001  
    Less than 1.16
 
241
 
88
 
85
 
68
 

 

WBC indicates white blood cell count.

*

Calculated by general association test.

More than 50 chromosomes.

As expected, low CASP8AP2 expression was significantly associated with slow early treatment response as defined by the presence of MRD at day 19 (P = .006) and at day 46 (P < .001; Wilcoxon–Mann-Whitney test; data not shown). Figure 2 illustrates the prevalence of MRD on days 19 and 46 in 2 groups of patients with the lowest and highest levels of CASP8AP2 expression, respectively. On day 19, MRD was detected in 36 of the 50 patients with low expression of this gene versus 20 of the 50 with high expression; on day 46, these prevalence rates were 22 of 50 versus 5 of 50, respectively. Among the positive samples, higher levels of MRD were found primarily in cases with low CASP8AP2 expression. For example, 7 patients with low CASP8AP2 expression, compared with 1 with high expression, had an MRD level higher than 1% at day 46, a feature typically associated with a dismal outcome and an indication for hematopoietic stem cell transplantation.12  Indeed, all but 1 of the 7 patients with this finding and low CASP8AP2 expression have relapsed or shown persistent MRD after day 46 of remission induction therapy.

Figure 2.

Prevalence of MRD (by level) on days 19 and 46 in 2 groups of patients with the lowest and highest expression ofCASP8AP2among 189 patients studied. Solid circles denote persistent MRD and/or hematologic relapse after day 46, while open circles denote continuous complete remission without MRD

Figure 2.

Prevalence of MRD (by level) on days 19 and 46 in 2 groups of patients with the lowest and highest expression ofCASP8AP2among 189 patients studied. Solid circles denote persistent MRD and/or hematologic relapse after day 46, while open circles denote continuous complete remission without MRD

To test the suggestive relationship of CASP8AP2 level with clinical outcome in Figure 2, we focused our analysis on a separate group of 99 patients enrolled in St Jude Total Therapy Study XIII.24  As a continuous variable, CASP8AP2 expression measured by gene array at diagnosis was significantly associated with event-free survival (P = .023) and with cumulative incidence of ALL relapse (P = .013) in a proportional hazard regression model. When the patients were divided into 3 groups of 33 each according to level of CASP8AP2 expression, those with high levels of expression had a significantly better event-free survival rate than those with intermediate or low levels (P = .011 by log rank test; Figure 3A), and a lower cumulative incidence of relapse (P = .043 by Gray test; Figure 3B). In a cumulative incidence regression model including all major presenting features associated with prognosis in childhood ALL, expression of CASP8AP2 remained a significant predictor of outcome (Table 3). Note that age older than 10 years was the only factor that increased the relapse hazard more than low CASP8AP2 expression did in this analysis. The results indicate that CASP8AP2 levels measured in diagnostic samples of leukemic blasts are a powerful predictor of treatment response in childhood ALL.

Table 3.

Cumulative risk of ALL relapse according to CASP8AP2 expression in relation to that of other selected clinical and biologic variables




95% Cl of hazard ratio

Variable
Hazard ratio
Lower
Upper
P
CASP8AP2 expression     
    Low vs high*  7.98‡   1.45   43.83   .017  
    Log-transformed signal  6.32‡   1.83   21.79   .004  
Age, y     
    Younger than 1 vs 1 to 10   5.63   0.71   44.61   .100  
    Older than 10 vs 1 to 10   10.33‡   3.00   35.61   <.001  
WBC, × 109/L     
    More than 10 to 49 vs 10 or less   0.86   0.22   3.38   .830  
    50 to 99 vs 10 or less   2.72   0.50   14.72   .240  
    100 or more vs 10 or less   4.15   0.95   18.22   .059  
Genotype (B-lineage only)     
    BCR-ABL vs other   6.45‡   1.69   24.65   .001  
    E2A-PBX vs other   0.96   0.26   3.47   .940  
    MLL-AF4 vs other   1.70   0.54   5.38   .370  
    TEL-AML1 vs other   5.18   0.96   27.92   .056  
    Hyperdiploidy vs other   0.37   0.03   4.62   .440  
Lineage     
    T vs B
 
6.12‡
 
1.58
 
23.78
 
.009
 



95% Cl of hazard ratio

Variable
Hazard ratio
Lower
Upper
P
CASP8AP2 expression     
    Low vs high*  7.98‡   1.45   43.83   .017  
    Log-transformed signal  6.32‡   1.83   21.79   .004  
Age, y     
    Younger than 1 vs 1 to 10   5.63   0.71   44.61   .100  
    Older than 10 vs 1 to 10   10.33‡   3.00   35.61   <.001  
WBC, × 109/L     
    More than 10 to 49 vs 10 or less   0.86   0.22   3.38   .830  
    50 to 99 vs 10 or less   2.72   0.50   14.72   .240  
    100 or more vs 10 or less   4.15   0.95   18.22   .059  
Genotype (B-lineage only)     
    BCR-ABL vs other   6.45‡   1.69   24.65   .001  
    E2A-PBX vs other   0.96   0.26   3.47   .940  
    MLL-AF4 vs other   1.70   0.54   5.38   .370  
    TEL-AML1 vs other   5.18   0.96   27.92   .056  
    Hyperdiploidy vs other   0.37   0.03   4.62   .440  
Lineage     
    T vs B
 
6.12‡
 
1.58
 
23.78
 
.009
 

Analyzed using Fine and Gray's estimator.35 

*

Comparisons of relapse hazards for 33 patients with the lowest levels of CASP8AP2 expression versus the 33 patients with the highest levels.

Increase of risk per unit decrease in log CASP8AP2 expression.

We performed similar analyses with 2 other genes included in the group of 17 genes associated with MRD: integrin α6 and tissue matrix remodeling-like gene (MXRA7; Table 1). Both genes were overexpressed in patients with a positive MRD assay at days 19 and 46. However, we found that expression of neither gene was significantly associated with ALL relapse in the independent cohort of 99 patients (not shown).

CASP8AP2 expression is associated with the capacity of ALL cells to grow in vitro

To begin to define the role of CASP8AP2 in ALL cell biology, we determined the association between CASP8AP2 expression and the capacity of leukemic cells to survive and grow in vitro. For this purpose, we used a 7-day culture assay in which the survival of leukemic cells is supported by bone marrow mesenchymal cells.37  This assay is well suited to test the growth potential of leukemic cells, a feature that correlates with treatment outcome.38  We previously found that cells from approximately 50% of ALL cases expand in vitro when grown on mesenchymal cell layers, whereas in the remaining cases, the leukemic cells undergo apoptosis.32 

After dividing 24 cases of ALL studied by expression array into 2 equal groups based on the expression of CASP8AP2, we compared recovery of leukemic cells after 7 days of culture. As shown in Figure 4, the recovery of lymphoblasts was significantly lower in cases with higher CASP8AP2 expression (P = .018), in agreement with morphologic and flow cytometric evidence of apoptosis (not shown). This result together with the reported function of CASP8AP2 suggests that the lower prevalence of MRD and better outcome in patients with high CASP8AP2 expression could be related to a higher propensity of the cells to undergo apoptosis and a lower capacity for expansion.

Discussion

The wealth of information generated by microarray studies provides unprecedented opportunities for identifying molecules that influence the propensity of leukemic cells to undergo apoptosis and hence their susceptibility to multiagent chemotherapy in vivo. We postulated that genes whose expression is associated with the presence of MRD during remission induction should have significant prognostic impact. After adjusting for associations with known ALL subtypes and using P values below .001 as a cutoff, we found 85 genes whose expression level was associated with MRD on day 46. Comparison of the reported functions of these genes led us to select CASP8AP2 (FLASH), a member of the apoptosis signaling complex that activates caspase 8 and facilitates Fas-induced apoptosis,17  as a candidate for further study. CASP8AP2 participates in apoptosis,17,18  and its enforced expression in brain cells increases glucocorticoid receptor–mediated transactivation.20  The proapoptotic function of CASP8AP2, together with the down-regulation of CASP8AP2 in the leukemic lymphoblasts of patients with persistent MRD during remission induction therapy, strongly suggested that this gene may be an important prognostic factor in ALL.

Figure 3.

Prognostic impact ofCASP8AP2expression in a group of 99 patients enrolled in St Jude Total Therapy Study XIII. Patients were divided into 3 equal groups according to level of CASP8AP2 expression measured by gene array at diagnosis. Event-free survival and cumulative incidence of relapse are shown.

Figure 3.

Prognostic impact ofCASP8AP2expression in a group of 99 patients enrolled in St Jude Total Therapy Study XIII. Patients were divided into 3 equal groups according to level of CASP8AP2 expression measured by gene array at diagnosis. Event-free survival and cumulative incidence of relapse are shown.

In an independent cohort of children with ALL, we found a striking association between low levels of CASP8AP2 expression and a high rate of leukemia relapse. In this cohort, low expression of CASP8AP2 was a very strong predictor of ALL relapse, second only to age older than 10 years. Low CASP8AP2 expression was more prevalent among patients younger than 1 year of age, a subgroup with generally poor response to therapy,39  whereas high CASP8AP2 expression was more prevalent among patients with hyperdiploidy, a favorable genetic abnormality.40-42  However, the relation between CASP8AP2 expression with age and chromosome number was not absolute, and, in a multivariate analysis, it remained a significant predictor of outcome.

Recently, Holleman et al4  reported correlations between expression of apoptosis-related genes and drug sensitivity in vitro in samples of childhood ALL cells. Their study focused on the prognostic significance of another apoptosis regulator, BCL2L13, but we noted that in their analysis CASP8AP2 expression was, on average, twice as high in prednisone-sensitive cases and was associated with higher sensitivity to asparaginase and daunorubicin. In our study, higher CASP8AP2 levels were related to a reduced capacity of leukemic lymphoblasts to grow in vitro, suggesting that this feature confers a general propensity to undergo apoptosis. CASP8AP2 was not detected in 2 other screenings of genes associated with early treatment response in ALL. The study by Chiaretti et al43  analyzed genes associated with early treatment response in 33 adult patients with T-ALL and was performed with the HG-U95A Affymetrix GeneChip, which does not include probes for CASP8AP2. The study of Cario et al44  analyzed gene expression in 51 patients classified as either “MRD standard risk” or “MRD high risk” according to the BFM2000 criteria but was performed on a different microarray platform.45  Both groups of investigators noted that low expression of TTK (a gene encoding a kinase involved in cell-cycle regulation)46  was associated with poorer treatment response. Remarkably, low expression of TTK was also associated with the presence of MRD on both days 19 and 46 in our series, although its association with MRD was weaker than that of CASP8AP2 and of the other genes listed in Table 1. Nevertheless, the consistent association of this gene with treatment response clearly merits further investigation.

Figure 4.

Cell recovery of primary ALL cells after culture on mesenchymal cell layers according toCASP8AP2expression. Primary ALL cells, selected from cases with known CASP8AP2 levels by GeneChip, were cultured on confluent mesenchymal cells for 7 days in serum-free medium. The number of viable ALL cells recovered at the end of the cultures was compared with the number of cells originally seeded. The dashed line indicates median cell recovery for the 24 cases. P value was calculated by Wilcoxon 2-sample test.

Figure 4.

Cell recovery of primary ALL cells after culture on mesenchymal cell layers according toCASP8AP2expression. Primary ALL cells, selected from cases with known CASP8AP2 levels by GeneChip, were cultured on confluent mesenchymal cells for 7 days in serum-free medium. The number of viable ALL cells recovered at the end of the cultures was compared with the number of cells originally seeded. The dashed line indicates median cell recovery for the 24 cases. P value was calculated by Wilcoxon 2-sample test.

Further progress in the treatment of childhood ALL will require optimization of risk assignment to avoid overtreatment and under-treatment of patients as well as the development of new antileukemic agents capable of overcoming drug resistance.6,47  We suggest that measurements of CASP8AP2 expression could help to identify patients whose leukemic cells are highly susceptible or highly resistant to chemotherapy. Indeed, this gene is a strong candidate for inclusion in gene arrays specifically designed for leukemia diagnosis. Since patients with detectable MRD during remission induction therapy appeared to have a much higher risk of relapse if their leukemic cells had low levels of CASP8AP2, measurements of this gene's levels could also be used to augment the informative power of MRD studies.

Prepublished online as Blood First Edition Paper, April 20, 2006; DOI 10.1182/blood-2006-01-0322.

Supported by grants CA60419 and CA21765 from the National Cancer Institute, and by the American Lebanese Syrian Associated Charities (ALSAC). C.-.H.P. is an American Cancer Society–F.M. Kirby Clinical Research Professor.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

We thank Chris Clark, Peixin Liu, and Mo Mehrpooya for technical assistance, and John Gilbert for critical review of the paper.

1
Cheok MH, Yang W, Pui CH, et al. Treatment-specific changes in gene expression discriminate in vivo drug response in human leukemia cells.
Nat Genet.
2003
;
34
:
85
-90.
2
Zaza G, Cheok M, Yang W, et al. Gene expression and thioguanine nucleotide disposition in acute lymphoblastic leukemia after in vivo mercaptopurine treatment.
Blood.
2005
;
106
:
1778
-1785.
3
Holleman A, Cheok MH, den Boer ML, et al. Gene-expression patterns in drug-resistant acute lymphoblastic leukemia cells and response to treatment.
N Engl J Med.
2004
;
351
:
533
-542.
4
Holleman A, den Boer ML, Menezes RX, et al. The expression of 70 apoptosis genes in relation to lineage, genetic subtype, cellular drug resistance, and outcome in childhood acute lymphoblastic leukemia.
Blood.
2006
;
107
:
769
-776.
5
Lugthart S, Cheok MH, den Boer ML, et al. Identification of genes associated with chemotherapy crossresistance and treatment response in childhood acute lymphoblastic leukemia.
Cancer Cell.
2005
;
7
:
375
-386.
6
Pui CH, Campana D, Evans WE. Childhood acute lymphoblastic leukemia: current status and future perspectives.
Lancet Oncol.
2001
;
2
:
597
-607.
7
Campana D. Determination of minimal residual disease in leukemia patients.
Br J Haematol.
2003
;
121
:
823
-838.
8
Pui CH, Relling MV, Downing JR. Acute lymphoblastic leukemia.
N Engl J Med.
2004
;
350
:
1535
-1548.
9
Coustan-Smith E, Behm FG, Sanchez J, et al. Immunological detection of minimal residual disease in children with acute lymphoblastic leukaemia.
Lancet.
1998
;
351
:
550
-554.
10
van Dongen JJ, Seriu T, Panzer-Grumayer ER, et al. Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in childhood.
Lancet.
1998
;
352
:
1731
-1738.
11
Cave H, van der Werff ten Bosch, Suciu S, et al. Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia: European Organization for Research and Treatment of Cancer: Childhood Leukemia Cooperative Group.
N Engl J Med.
1998
;
339
:
591
-598.
12
Coustan-Smith E, Sancho J, Hancock ML, et al. Clinical importance of minimal residual disease in childhood acute lymphoblastic leukemia.
Blood.
2000
;
96
:
2691
-2696.
13
Coustan-Smith E, Sancho J, Behm FG, et al. Prognostic importance of measuring early clearance of leukemic cells by flow cytometry in childhood acute lymphoblastic leukemia.
Blood.
2002
;
100
:
52
-58.
14
Coustan-Smith E, Gajjar A, Hijiha N, et al. Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia after first relapse.
Leukemia.
2004
;
18
:
499
-504.
15
Panzer-Grumayer ER, Schneider M, Panzer S, Fasching K, Gadner H. Rapid molecular response during early induction chemotherapy predicts a good outcome in childhood acute lymphoblastic leukemia.
Blood.
2000
;
95
:
790
-794.
16
Szczepanski T, Orfao A, van der Velden VH, San Miguel JF, van Dongen JJ. Minimal residual disease in leukaemia patients.
Lancet Oncol.
2001
;
2
:
409
-417.
17
Imai Y, Kimura T, Murakami A, et al. The CED-4-homologous protein FLASH is involved in Fas-mediated activation of caspase-8 during apoptosis.
Nature.
1999
;
398
:
777
-785.
18
Jun JI, Chung CW, Lee HJ, et al. Role of FLASH in caspase-8-mediated activation of NF-kappaB: dominant-negative function of FLASH mutant in NF-kappaB signaling pathway.
Oncogene.
2005
;
24
:
688
-696.
19
Kino T, Chrousos GP. Tumor necrosis factor alpha receptor- and Fas-associated FLASH inhibit transcriptional activity of the glucocorticoid receptor by binding to and interfering with its interaction with p160 type nuclear receptor coactivators.
J Biol Chem.
2003
;
278
:
3023
-3029.
20
Obradovic D, Tirard M, Nemethy Z, et al. DAXX, FLASH, and FAF-1 modulate mineralocorticoid and glucocorticoid receptor-mediated transcription in hippocampal cells: toward a basis for the opposite actions elicited by two nuclear receptors?
Mol Pharmacol.
2004
;
65
:
761
-769.
21
Campana D, Behm FG. Immunophenotyping of leukemia.
J Immunol Methods.
2000
;
243
:
59
-75.
22
Raimondi SC, Mathew S, Pui CH. Cytogenetics as a diagnostic aid for childhood hematological disorders: conventional cytogenetic techniques, fluorescence in situ hybridization, comparative genomic hybridization. In: Hanausek M, Walaszek Z, eds.
Methods in Molecular Biology.
Totowa, NJ: Humana Press;
1998
:
209
-227.
23
Scurto P, Hsu RM, Kane JR, et al. A multiplex RT-PCR assay for the detection of chimeric transcripts encoded by the risk-stratifying translocations of pediatric acute lymphoblastic leukemia.
Leukemia.
1998
;
12
:
1994
-2005.
24
Pui CH, Sandlund JT, Pei D, et al. Improved outcome for children with acute lymphoblastic leukemia: results of Total Therapy Study XIIIB at St Jude Children's Research Hospital.
Blood.
2004
;
104
:
2690
-2696.
25
Pui CH, Pei D, Sandlund JT, et al. Risk of adverse events after completion of therapy for childhood acute lymphoblastic leukemia.
J Clin Oncol.
2005
;
23
:
7936
-7941.
26
Pui CH, Relling MV, Sandlund JT, et al.. Total Therapy Study XV for newly diagnosed childhood acute lymphoblastic leukemia: study design and preliminary results.
Ann Hematol.
2006
;
85
(
suppl 13
):
88
-91.
27
Yeoh EJ, Ross ME, Shurtleff SA, et al. Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling.
Cancer Cell.
2002
;
1
:
133
-143.
28
Ross ME, Zhou X, Song G, et al. Classification of pediatric acute lymphoblastic leukemia by gene expression profiling.
Blood.
2003
;
102
:
2951
-2959.
29
Coustan-Smith E, Sancho J, Hancock ML, et al. Use of peripheral blood instead of bone marrow to monitor residual disease in children with acute lymphoblastic leukemia.
Blood.
2002
;
100
:
2399
-2402.
30
Campana D, Coustan-Smith E. Advances in the immunological monitoring of childhood acute lymphoblastic leukaemia.
Best Pract Res Clin Haematol.
2002
;
15
:
1
-19.
31
Neale GA, Coustan-Smith E, Stow P, et al. Comparative analysis of flow cytometry and polymerase chain reaction for the detection of minimal residual disease in childhood acute lymphoblastic leukemia.
Leukemia.
2004
;
18
:
934
-938.
32
Ito C, Kumagai M, Manabe A, et al. Hyperdiploid acute lymphoblastic leukemia with 51 to 65 chromosomes: a distinct biological entity with a marked propensity to undergo apoptosis.
Blood.
1999
;
93
:
315
-320.
33
Srivannaboon K, Shanafelt AB, Todisco E, et al. Interleukin-4 variant (BAY 36-1677) selectively induces apoptosis in acute lymphoblastic leukemia cells.
Blood.
2001
;
97
:
752
-758.
34
Cheng C, Pounds S, Boyett JM, et al. Statistical significance threshold criteria for analysis of microarray gene expression data.
Stat Appl Genet Mol Biol.
2004
;
3
:
Article 36
.
35
Fine JP, Gray J. A proportional hazards model for the subdistributionof a competing risk.
JASA.
1999
;
94
:
496
-509.
36
Kern W, Kohlmann A, Wuchter C, et al. Correlation of protein expression and gene expression in acute leukemia.
Cytometry B Clin Cytom.
2003
;
55
:
29
-36.
37
Manabe A, Coustan-Smith E, Behm FG, Raimondi SC, Campana D. Bone marrow-derived stromal cells prevent apoptotic cell death in B-lineage acute lymphoblastic leukemia.
Blood.
1992
;
79
:
2370
-2377.
38
Kumagai M, Manabe A, Pui CH, et al. Stroma-supported culture in childhood B-lineage acute lymphoblastic leukemia cells predicts treatment outcome.
J Clin Invest.
1996
;
97
:
755
-760.
39
Pui CH, Chessells JM, Camitta BA, et al. Clinical heterogeneity in childhood acute lymphoblastic leukemia with 11q23 rearrangements.
Leukemia.
2003
;
17
:
700
-706.
40
Williams DL, Tsiatis A, Brodeur GM, et al. Prognostic importance of chromosome number in 136 untreated children with acute lymphoblastic leukemia.
Blood.
1982
;
60
:
864
-871.
41
Trueworthy R, Shuster J, Look T, et al. Ploidy of lymphoblasts is the strongest predictor of treatment outcome in B-progenitor cell acute lymphoblastic leukemia of childhood: a Pediatric Oncology Group study.
J Clin Oncol.
1992
;
10
:
606
-613.
42
Secker-Walker LM, Prentice HG, Durrant J, et al. Cytogenetics adds independent prognostic information in adults with acute lymphoblastic leukaemia on MRC trial UKALL XA. MRC Adult Leukaemia Working Party.
Br J Haematol.
1997
;
96
:
601
-610.
43
Chiaretti S, Li X, Gentleman R, et al. Gene expression profile of adult T-cell acute lymphocytic leukemia identifies distinct subsets of patients with different response to therapy and survival.
Blood.
2004
;
103
:
2771
-2778.
44
Cario G, Stanulla M, Fine BM, et al. Distinct gene expression profiles determine molecular treatment response in childhood acute lymphoblastic leukemia.
Blood.
2005
;
105
:
821
-826.
45
Kuo WP, Jenssen TK, Butte AJ, Ohno-Machado L, Kohane IS. Analysis of matched mRNA measurements from two different microarray technologies.
Bioinformatics.
2002
;
18
:
405
-412.
46
Hogg D, Guidos C, Bailey D, et al. Cell cycle dependent regulation of the protein kinase TTK.
Oncogene.
1994
;
9
:
89
-96.
47
Silverman LB, Sallan SE. Newly diagnosed childhood acute lymphoblastic leukemia: update on prognostic factors and treatment.
Curr Opin Hematol.
2003
;
10
:
290
-296.