Purpose: To examine cancer genes undergoing epigenetic inactivation in a set of ETV6/RUNX1-positive acute lymphoblastic leukemias in order to define the CpG island methylator phenotype (CIMP) in the disease and evaluate its relationship with clinical features and outcome.

Experimental Design: Methylation-specific PCR was used to analyze the methylation status of 38 genes involved in cell immortalization and transformation in 54 ETV6/RUNX1-positive samples in comparison with 190 ETV6/RUNX1-negative samples.

Results:ETV6/RUNX1-positive samples had at least one gene methylated in 89% of the cases. According to the number of methylated genes observed in each individual sample, 20 patients (37%) were included in the CIMP− group (0-2 methylated genes) and 34 (67%) in the CIMP+ group (>2 methylated genes). Remission rate did not differ significantly among either group of patients. Estimated disease-free survival and overall survival at 9 years were 92% and 100% for the CIMP− group and 33% and 73% for the CIMP+ group (P = 0.002 and P = 0.04, respectively). Multivariate analysis showed that methylation profile was an independent prognostic factor in predicting disease-free survival (P = 0.01) and overall survival (P = 0.05). A group of four genes (DKK3, sFRP2, PTEN, and P73) showed specificity for ETV6/RUNX1-positive subset of samples.

Conclusion: Our results suggest that methylation profile may be a potential new biomarker of risk prediction in ETV6/RUNX1-positive acute lymphoblastic leukemias.

The t(12;21)(p13;q22) translocation, which involves the ETV6 gene (previously TEL) located on 12p13 and the RUNX1 gene (previously AML1) on 21q22, is found in 20% to 30% of children with B cell precursor acute lymphoblastic leukemia (BCP-ALL; ref. 1). In general, t(12;21)-positive BCP-ALL is associated with a favorable prognosis, although conflicting results have been described. Relapses have been reported in ∼20% of cases, generally after a long remission period, although the relapse incidence varies and may depend on the treatment protocol (2–5). In a series of patients treated with the Dana-Farber Cancer Institute Acute Lymphoblastic Leukemia Consortium protocol between 1980 and 1991, none of the 22 patients with the ETV6/RUNX1 rearrangement relapsed (2). In the Berlin-Frankfurt-Munster clinical trials, however, 20% to 24% of patients with BCP-ALL studied during relapse had the ETV6/RUNX1 rearrangement, a frequency similar to that observed at diagnosis (3–5). Equally, there are isolated reports of event-free survivals measured in months only, or while patients were still on treatment (6, 7).

Because the t(12;21)-positive BCP-ALL group seems to be a heterogeneous group including patients that can be cured with conventional therapy and patients with dismal prognosis that need more intensive regimens, it would be beneficial to identify high-risk ETV6/RUNX1-positive patients at diagnosis or soon thereafter in order to modify their initial therapy with the goal of preventing treatment failure. However, conventionally applied epidemiologic features fail to identify these patients. There are no significant differences in event-free survival for t(12;21)-positive children based on age, presenting leukocyte blood cell count, or sex (8). Moreover, although t(12;21) is mostly associated with karyotypes characterized by modal numbers ranging from 45 to 52, trisomy 21, 12p aberrations, nonspecific chromosome deletions, deletion of the normal ETV6 allele, duplication of the fusion gene, and RUNX1 extra signal, these abnormalities secondary to the ETV6/RUNX1 do not seem to influence patient outcome (9). Therefore, useful molecular markers for risk-specific adjustments in therapeutic intensity are necessary in this disease.

DNA methylation is an essential mechanism for the regulation of gene expression in mammalian cells (10). Methylation occurs at cytosine residues within CpG dinucleotides and many genes are enriched with these dinucleotides in their promoters. These regions are known as CpG islands and are generally nonmethylated, a condition that allows genes to be transcriptionally competent. Methylation of CpG islands within gene promoters leads to transcriptional silencing through recruitment of methyl-CpG binding protein and histone deacetylases (11, 12). Hence, identification of the methylation patterns of CpG islands in mammalian cells is important for understanding normal and pathologic gene expression. Several reports have shown that abnormal methylation of CpG islands may contribute significantly to the pathogenesis of human leukemias providing an alternative route to gene mutation of cancer-related genes. We have recently shown that the methylation of cytosine nucleotides in ALL cells can help to inactivate tumor-suppressive apoptotic or growth-arresting responses and has a prognostic effect in B and T cell ALL (13, 14). The presence in individual tumors of multiple genes simultaneously methylated (a condition termed CpG island methylator phenotype or CIMP+) is an independent factor of poor prognosis in both childhood and adult ALL in terms of disease-free survival (DFS) and overall survival (OS). Moreover, methylation status was able to redefine the prognosis of selected ALL groups with well-established prognostic features. Lack of CIMP (CIMP−) improved the general poor outcome of patients presenting Philadelphia chromosome, high WBC count at diagnosis, or T cell phenotype (13, 14).

In order to determine whether methylation profile is also of clinical relevance in ETV6/RUNX1-positive ALL, we have examined multiple key cancer genes undergoing epigenetic inactivation in a set of de novo t(12;21)-positive BCP-ALLs of childhood with the aim of obtaining a map of this alteration in the disease and its possible correlation with clinical features and outcome of the patients.

Patients. We studied 54 consecutive children (32 male and 22 female) with de novo ETV6/RUNX1-positive BCP-ALL who were enrolled in a multicenter study of the PETHEMA Spanish Study Group. All these patients were referred to the Reina Sofia Hospital of Cordoba, Spain, from January 1993 to December 2004. The median age at diagnosis was 5 years (range, 2-14 years). The study was approved by the Investigational Review Boards in accordance with the policies of the Department of Health and Human Services. Informed consent was obtained from the patient's guardians. Diagnosis was established according to standard morphologic, cytochemical, and immunophenotypic criteria. After diagnosis, patients were entered in ALL protocols for standard risk patients of the PETHEMA Spanish study group. All these patients were included in the specific PETHEMA ALL-93 treatment protocol. The design and results of this protocol have been previously reported (15, 16). Fifteen patients relapsed, whereas six patients received stem cell transplantation (two autologous and four allogeneic) in the second complete remission (CR). There are 47 patients currently alive. The clinical characteristics of the patients are listed in Table 1. Preliminary methylation data from 14 of these patients have been previously reported (13).

Table 1.

Clinical characteristics and outcome of 54 t(12;21)-positive BCP-ALL children according to gene methylation status

FeatureCIMP− (n = 20)CIMP+ (n = 34)P
Median age, range (y) 5 (2-14) 4.5 (2-14) NS 
Sex, M/F 11/9 20/14 NS 
WBC   NS 
    <50 × 109/L 18 30  
    >50 × 109/L 2 4  
Immunophenotype   NS 
    Common 18 31  
    Pre-B 2 3  
Bone marrow transplantation 1 5 NS 
Best response NS   
    CR 20 34  
Modal no.   NS 
    45-46 10 17  
    47-51 4 6  
    >51 0 0  
    Unknown 6 9  
Relapse 1 14 0.003 
Death 0 7 0.04 
FeatureCIMP− (n = 20)CIMP+ (n = 34)P
Median age, range (y) 5 (2-14) 4.5 (2-14) NS 
Sex, M/F 11/9 20/14 NS 
WBC   NS 
    <50 × 109/L 18 30  
    >50 × 109/L 2 4  
Immunophenotype   NS 
    Common 18 31  
    Pre-B 2 3  
Bone marrow transplantation 1 5 NS 
Best response NS   
    CR 20 34  
Modal no.   NS 
    45-46 10 17  
    47-51 4 6  
    >51 0 0  
    Unknown 6 9  
Relapse 1 14 0.003 
Death 0 7 0.04 

NOTE: CIMP−, patients with 0-2 methylated genes; CIMP+, patients with >2 methylated genes.

Abbreviation: NS, not significant.

View Large

In addition, we also studied 190 consecutive ETV6/RUNX1-negative BCP-ALL children diagnosed during the same period of time in order to compare the methylation profiles of both ETV6/RUNX1-positive and -negative BCP-ALL.

Cytogenetic investigations. Chromosome banding analyses of bone marrow samples were done using standard methods. All patients in the study cohort were analyzed with fluorescence in situ hybridization or reverse-transcriptase PCR or both, using standard methods for the presence of the cryptic translocation ETV6/RUNX1.

Gene selection. Bone marrow specimens were obtained from all the patients at the moment of diagnosis. High-molecular weight DNA was prepared from mononuclear diagnostic marrow cells using conventional methods, frozen at −80°C, and retrospectively analyzed to assess the role of methylation profile. In all the cases, the diagnostic bone marrow sample contained blast cells in the ratio of at least 70%. We studied 38 genes belonging to all of the molecular pathways involved in cell immortalization and transformation: cell cycle (FHIT, LATS2, p15, p16, p57, REPRIMO, and RIZ), cell adherence and metastasis process (ADAMTS1, ADAMTS5, CDH1, and CDH13), p53 network (ASPP1, p14, and p73), apoptosis (APAF1, ARTS, DAPK, DBC1, DIABLO, and TMS1), inhibitors of the oncogenic WNT signaling pathway (DKK3, HDPR1, sFRP1, sFRP2, sFRP4, sFRP5, and WIF1), differentiation regulation (NES1), folate carrier (hRFC), hormone receptor superfamily (PGR), ubiquitination (PACRG and PARK2), DNA repair (SMC1L1 and SMC1L2), tyrosine kinase with an essential role in signal transduction (SYK), negative regulator of the Jak/STAT signaling pathway (SHP1), and main tumor-suppressor genes (LATS1 and PTEN; Table 2). Different criteria were used for gene selection. ADAMTS1, ADAMTS5, APAF1, ASPP1, CDH1, CDH13, DAPK, DIABLO, DKK3, HDPR1, LATS1, LATS2, NES1, PACRG, PARK2, PTEN, p14, p16, p15, p57, p73, sFRP1, sFRP2, sFRP4, sFRP5, SHP1, SYK, TMS1, and WIF1 were selected because of their frequent methylation in ALL (13, 14). The other genes were studied because they have been found to be methylated in other malignancies including leukemic cell lines, and their abnormal expression could have potentially important roles in ALL (17–24). The regions in which these genes reside are not prone to mutations, deletions, or rearrangement in the majority of human leukemias; however, microsatellite markers from these regions have shown that most of them are common sites for loss of heterozygosity in ALL (25). Each of these genes possesses a CpG island in the 5′ region, which is normally unmethylated in corresponding normal tissues as expected for a typical CpG island. We and others have shown, in previous studies for such genes in individual tumor types, that when these CpG islands are hypermethylated in cancer cells, the expression of the corresponding gene is silenced and the silencing can be partially relieved by demethylation of the promoter region (13, 14, 17–24). For all these genes, we have analyzed, at least, 10 normal marrow and peripheral blood specimens, none of which showed methylation.

Table 2.

Genes studied for methylation in t(12;21)-positive BCP-ALL

GeneLocationFunctionReference for MSP primers
ADAMTS1 21q21.2 Metalloprotease Roman-Gomez et al. (14) 
ADAMTS5 21q21.3 Metalloprotease Roman-Gomez et al. (14) 
APAF1 12q23 Apoptosis regulation Roman-Gomez et al. (13) 
ASPP1 14q32-33 P53 costimulator, apoptosis regulation Roman-Gomez et al. (14) 
CDH1 16q22 Cell-cell adhesion Roman-Gomez et al. (13) 
CDH13 16q24 Cell-cell adhesion Roman-Gomez et al. (13) 
DAPK 9q34 Apoptosis regulation Roman-Gomez et al. (13) 
DBC1 9q32-33 Apoptosis regulation Izumi et al. (18) 
DIABLO 12q24.31 Apoptosis regulation Roman-Gomez et al. (14) 
DKK3 11p15 Wnt signaling pathway antagonist Roman-Gomez et al. (14) 
FHIT 3p14.2 TSG, purine metabolism Iwai et al. (19) 
HDPR1 14q23.1 Wnt signaling pathway antagonist Roman-Gomez et al. (14) 
hRFC 21q22.3 Folate carrier Rothem et al. (20) 
LATS1 6q23-25 TSG, G2-M cell cycle control Roman-Gomez et al. (13) 
LATS2 13q11-12 G1-S cell cycle control Roman-Gomez et al. (14) 
NES1 19q13 Growth and differentiation control Roman-Gomez et al. (13) 
P14 9p21 Cell cycle control, apoptosis regulation Roman-Gomez et al. (13) 
P15 9p21 G1-S cell cycle control Roman-Gomez et al. (13) 
P16 9p21 TSG, G1-S cell cycle control Roman-Gomez et al. (13) 
P57 11p15 G1-S cell cycle control Roman-Gomez et al. (13) 
P73 1p36 G1-S cell cycle control Roman-Gomez et al. (13) 
PACRG 6q26 Ubiquitination Roman-Gomez et al. (13) 
PARK2 6q25-27 Ubiquitination Roman-Gomez et al. (13) 
PTEN 10q23 TSG, cell adhesion/motility, apoptosis, angiogenesis, G1 cell cycle regulation, signal transduction Roman-Gomez et al. (13) 
PGR 11q22-23 Progesterone receptor Liu et al. (21) 
REPRIMO 2q23.3 G2-M cell cycle control Takahashi et al. (22) 
RIZ 1p36 TSG, retinoblastoma pathway Matsushita et al. (23) 
sFRP1 8p12-11.1 Wnt signaling pathway antagonist Roman-Gomez et al. (14) 
sFRP2 4q31.3 Wnt signaling pathway antagonist Roman-Gomez et al. (14) 
sFRP4 7p14.1 Wnt signaling pathway antagonist Roman-Gomez et al. (14) 
sFRP5 10q24.1 Wnt signaling pathway antagonist Roman-Gomez et al. (14) 
SHP1 12p13 Jak/STAT signaling pathway inhibitor Roman-Gomez et al. (14) 
SMC1L1 Xp11.22 DNA repair Shar et al. (24) 
SMC1L2 22q13.31 DNA repair Shar et al. (24) 
SYK 9q22 Signal transduction Roman-Gomez et al. (14) 
TMS1 16p11-12 Apoptosis regulation Roman-Gomez et al. (13) 
WIF1 12q14.3 Wnt signaling pathway antagonist Roman-Gomez et al. (14) 
GeneLocationFunctionReference for MSP primers
ADAMTS1 21q21.2 Metalloprotease Roman-Gomez et al. (14) 
ADAMTS5 21q21.3 Metalloprotease Roman-Gomez et al. (14) 
APAF1 12q23 Apoptosis regulation Roman-Gomez et al. (13) 
ASPP1 14q32-33 P53 costimulator, apoptosis regulation Roman-Gomez et al. (14) 
CDH1 16q22 Cell-cell adhesion Roman-Gomez et al. (13) 
CDH13 16q24 Cell-cell adhesion Roman-Gomez et al. (13) 
DAPK 9q34 Apoptosis regulation Roman-Gomez et al. (13) 
DBC1 9q32-33 Apoptosis regulation Izumi et al. (18) 
DIABLO 12q24.31 Apoptosis regulation Roman-Gomez et al. (14) 
DKK3 11p15 Wnt signaling pathway antagonist Roman-Gomez et al. (14) 
FHIT 3p14.2 TSG, purine metabolism Iwai et al. (19) 
HDPR1 14q23.1 Wnt signaling pathway antagonist Roman-Gomez et al. (14) 
hRFC 21q22.3 Folate carrier Rothem et al. (20) 
LATS1 6q23-25 TSG, G2-M cell cycle control Roman-Gomez et al. (13) 
LATS2 13q11-12 G1-S cell cycle control Roman-Gomez et al. (14) 
NES1 19q13 Growth and differentiation control Roman-Gomez et al. (13) 
P14 9p21 Cell cycle control, apoptosis regulation Roman-Gomez et al. (13) 
P15 9p21 G1-S cell cycle control Roman-Gomez et al. (13) 
P16 9p21 TSG, G1-S cell cycle control Roman-Gomez et al. (13) 
P57 11p15 G1-S cell cycle control Roman-Gomez et al. (13) 
P73 1p36 G1-S cell cycle control Roman-Gomez et al. (13) 
PACRG 6q26 Ubiquitination Roman-Gomez et al. (13) 
PARK2 6q25-27 Ubiquitination Roman-Gomez et al. (13) 
PTEN 10q23 TSG, cell adhesion/motility, apoptosis, angiogenesis, G1 cell cycle regulation, signal transduction Roman-Gomez et al. (13) 
PGR 11q22-23 Progesterone receptor Liu et al. (21) 
REPRIMO 2q23.3 G2-M cell cycle control Takahashi et al. (22) 
RIZ 1p36 TSG, retinoblastoma pathway Matsushita et al. (23) 
sFRP1 8p12-11.1 Wnt signaling pathway antagonist Roman-Gomez et al. (14) 
sFRP2 4q31.3 Wnt signaling pathway antagonist Roman-Gomez et al. (14) 
sFRP4 7p14.1 Wnt signaling pathway antagonist Roman-Gomez et al. (14) 
sFRP5 10q24.1 Wnt signaling pathway antagonist Roman-Gomez et al. (14) 
SHP1 12p13 Jak/STAT signaling pathway inhibitor Roman-Gomez et al. (14) 
SMC1L1 Xp11.22 DNA repair Shar et al. (24) 
SMC1L2 22q13.31 DNA repair Shar et al. (24) 
SYK 9q22 Signal transduction Roman-Gomez et al. (14) 
TMS1 16p11-12 Apoptosis regulation Roman-Gomez et al. (13) 
WIF1 12q14.3 Wnt signaling pathway antagonist Roman-Gomez et al. (14) 

Abbreviation: TSG, tumor suppressor gene.

View Large

Methylation-specific PCR. Aberrant promoter methylation of these genes was determined by a methylation-specific PCR (MSP) method after bisulfite treatment of DNA as reported by Herman et al. (26). The primer sequences of each gene for the unmethylated and methylated reactions have been reported elsewhere (13, 14, 17–24). “Hot start” PCR was done for 30 cycles consisting of denaturation at 95°C for 1 minute, annealing at 60°C for 1 minute, and extension at 72°C for 1 minute, followed by a final 7-minute extension for all primer sets. The products were separated by electrophoresis on 2% agarose gel. Bone marrow DNA from healthy donors was used as a negative control for methylation-specific assays. Human male genomic DNA universally methylated for all genes (Intergen Company, Purchase, NY) was used as a positive control for methylated alleles. Water blanks were included with each assay. The presence of a clearly visible band in the MSP using primers for the methylated alleles was considered as a positive result for methylation. This result was always confirmed by repeat MSP assays after an independently done bisulfite treatment. In the sporadic cases in which only faint bands were observed in both analyses, methylation results were validated by Southern blot and/or sequencing and/or association with lack of expression assessed by reverse-transcriptase PCR as appropriate.

Statistical analysis. Although there is, as yet, no consensus definition for CIMP+, patients with ETV6/RUNX1-positive BCP-ALL were arbitrarily classified into two different methylation groups: cases showing methylation at three or more loci were defined as CIMP+, whereas those in which methylation was low occurring at two or fewer loci were defined as CIMP−. P values for comparisons of continuous variables between groups of patients were two-tailed and based on the Wilcoxon rank sum test. P values for dichotomous variables were based on the Fisher exact test. The remaining P values were based on the Pearson χ2 test. OS was measured from the day of diagnosis until death from any cause and was censored only for patients known to be alive at last contact. DFS was measured from the day that CR was established until either relapse or death without relapse, and it was censored only for patients who were alive without evidence of relapse at the last follow-up. The distributions of OS and DFS curves were estimated by the method of Kaplan and Meier, with 95% confidence intervals calculated by means of Greenwood's formula. Comparisons of OS or DFS between groups were based on the log-rank test. Comparisons adjusted for significant prognostic factors were based on Cox regression models and hazard regression models. All relapse and survival data were updated on March 2005, and all follow-up data were censored at that point.

Frequency of methylation in ETV6/RUNX1-positive BCP-ALL. Gene methylation frequencies varied from 2% to 58%. Twenty-three genes showed a relatively high frequency of aberrant methylation: NES1 (58%), ADAMTS1 (47%), PGR (45%), DKK3 (42%), sFRP1 (36%), CDH1 (36%), SMC1L2 (36%), ADAMTS5 (34%), CDH13 (33%), REPRIMO (33%), RIZ (31%), PARK2 (31%), PACRG (31%), P73 (30%), PTEN (29%), HDPR1 (28%), WIF1 (27%), LATS2 (27%), FHIT (21%), hRFC (21%), DIABLO (21%), sFRP2 (20%), and sFRP5 (20%). The other 15 genes studied showed a low frequency (2-18%) of methylation (Table 3). No methylated genes were found in 6 of 54 patients (11%), whereas most t(12;21)-positive BCP-ALLs (48 of 54, 89%) had methylation of at least one gene, ranging from 1 to 20 methylated genes. No case was found to have methylation of >20 genes. According to the number of methylated genes observed in each individual sample, 20 patients (37%) were included in the CIMP− group (0-2 methylated genes) and 34 (63%) in the CIMP+ group (>2 methylated genes).

Table 3.

Methylation profile in BCP-ALL

FeatureETV6/RUNX1-positive (%)ETV6/RUNX1-negative (%)P
Methylated genes    
    ADAMTS1 47 53 NS 
    ADAMTS5 34 28 NS 
    APAF1 16 12 NS 
    ARTS 14 36 0.05 
    ASPP1 9 15 NS 
    CDH1 36 40 NS 
    CDH13 33 40 NS 
    DAPK 11 12 NS 
    DBC1 13 13 NS 
    DIABLO 21 24 NS 
    DKK3 42 27 0.05 
    FHIT 21 20 NS 
    HDPR1 28 30 NS 
    hRFC 21 40 0.05 
    LATS1 13 33 0.05 
    LATS2 27 28 NS 
    NES1 58 52 NS 
    P14 3 5 NS 
    P15 18 20 NS 
    P16 15 15 NS 
    P57 2 2 NS 
    P73 30 14 0.05 
    PACRG 31 32 NS 
    PARK2 31 32 NS 
    PTEN 29 13 0.05 
    PGR 45 52 NS 
    REPRIMO 33 30 NS 
    RIZ 31 35 NS 
    sFRP1 31 36 NS 
    sFRP2 20 7 0.05 
    sFRP4 8 30 0.04 
    sFRP5 20 21 NS 
    SHP1 3 12 0.05 
    SMC1L1 5 0 NS 
    SMC1L2 36 53 0.01 
    SYK 9 7 NS 
    TMS1 4 7 NS 
    WIF1 27 31 NS 
Methylation profile    
    CIMP+ patients 63 65 NS 
No. of methylated genes    
    Mean methylated genes 6.8 4.8 0.008 
FeatureETV6/RUNX1-positive (%)ETV6/RUNX1-negative (%)P
Methylated genes    
    ADAMTS1 47 53 NS 
    ADAMTS5 34 28 NS 
    APAF1 16 12 NS 
    ARTS 14 36 0.05 
    ASPP1 9 15 NS 
    CDH1 36 40 NS 
    CDH13 33 40 NS 
    DAPK 11 12 NS 
    DBC1 13 13 NS 
    DIABLO 21 24 NS 
    DKK3 42 27 0.05 
    FHIT 21 20 NS 
    HDPR1 28 30 NS 
    hRFC 21 40 0.05 
    LATS1 13 33 0.05 
    LATS2 27 28 NS 
    NES1 58 52 NS 
    P14 3 5 NS 
    P15 18 20 NS 
    P16 15 15 NS 
    P57 2 2 NS 
    P73 30 14 0.05 
    PACRG 31 32 NS 
    PARK2 31 32 NS 
    PTEN 29 13 0.05 
    PGR 45 52 NS 
    REPRIMO 33 30 NS 
    RIZ 31 35 NS 
    sFRP1 31 36 NS 
    sFRP2 20 7 0.05 
    sFRP4 8 30 0.04 
    sFRP5 20 21 NS 
    SHP1 3 12 0.05 
    SMC1L1 5 0 NS 
    SMC1L2 36 53 0.01 
    SYK 9 7 NS 
    TMS1 4 7 NS 
    WIF1 27 31 NS 
Methylation profile    
    CIMP+ patients 63 65 NS 
No. of methylated genes    
    Mean methylated genes 6.8 4.8 0.008 

NOTE: CIMP+, patients with >2 methylated genes.

View Large

Comparative analysis of the methylation frequencies between children with ETV6/RUNX1-positive and ETV6/RUNX1-negative BCP-ALL classifies the 38 genes examined into three groups. The first group is that of genes showing significantly higher frequencies of methylation in ETV6/RUNX1-positive compared with ETV6/RUNX1-negative BCP-ALL (DKK3, sFRP2, PTEN, and P73; Table 3). The second group was that of genes showing significantly higher frequencies of methylation in ETV6/RUNX1-negative compared with ETV6/RUNX1-positive BCP-ALL (ARTS, hRFC, SMC1L2, sFRP4, SHP1, and LATS1), and the third group was that of genes demonstrating a similar frequency of methylation in both subtypes of BPC-ALL (the remaining 28 genes). Furthermore, although CIMP+ was equally distributed among ETV6/RUNX1-positive BCP-ALL (63%) and ETV6/RUNX1-negative BCP-ALL (65%), the density of gene methylation in ETV6/RUNX1-positive BCP-ALL (mean of methylated genes, 6.8 ± 0.8) was significantly higher compared with ETV6/RUNX1-negative BCP-ALL (mean of methylated genes, 4.8 ± 0.5; P = 0.008).

Clinical outcome of ETV6/RUNX1-positive BCP-ALL and methylation profile. As shown in Table 1, clinical and laboratory characteristics did not differ significantly between methylation groups. Table 1 also details the relapse history, CR rates and mortality for patients included in the different methylation groups. CR rates of patients in both CIMP− and CIMP+ groups were 100%. However, patients in the CIMP− group had a lower relapse rate than patients in the CIMP+ group (5% versus 44%, P = 0.003). Mortality rate was also lower for the CIMP− group compared with the CIMP+ group (0% versus 19%, P = 0.04).

We analyzed the DFS among patients who achieved CR according to the methylation profile. Estimated DFS rates at 9 years were 92% and 33% for CIMP− and CIMP+ groups, respectively (P = 0.002; Fig. 1A). The actuarial OS at 9 years calculated for all leukemic patients was 100% for CIMP− patients and 73% for CIMP+ patients (P = 0.04; Fig. 1B).

Fig. 1.
Fig. 1. Kaplan-Meier survivor function for ETV6/RUNX1-positive BCP-ALL patients. DFS (A) and OS (B) curves for all the patients enrolled in this study according to the methylation profile. CIMP− (patients with 0-2 methylated genes, n = 20); CIMP+ (patients with >2 methylated genes, n = 34). Log-rank test was significant for both DFS (P = 0.002) and OS (P = 0.04).
View largeDownload slide

Kaplan-Meier survivor function for ETV6/RUNX1-positive BCP-ALL patients. DFS (A) and OS (B) curves for all the patients enrolled in this study according to the methylation profile. CIMP− (patients with 0-2 methylated genes, n = 20); CIMP+ (patients with >2 methylated genes, n = 34). Log-rank test was significant for both DFS (P = 0.002) and OS (P = 0.04).

Fig. 1.
Fig. 1. Kaplan-Meier survivor function for ETV6/RUNX1-positive BCP-ALL patients. DFS (A) and OS (B) curves for all the patients enrolled in this study according to the methylation profile. CIMP− (patients with 0-2 methylated genes, n = 20); CIMP+ (patients with >2 methylated genes, n = 34). Log-rank test was significant for both DFS (P = 0.002) and OS (P = 0.04).
View largeDownload slide

Kaplan-Meier survivor function for ETV6/RUNX1-positive BCP-ALL patients. DFS (A) and OS (B) curves for all the patients enrolled in this study according to the methylation profile. CIMP− (patients with 0-2 methylated genes, n = 20); CIMP+ (patients with >2 methylated genes, n = 34). Log-rank test was significant for both DFS (P = 0.002) and OS (P = 0.04).

Close modal

A multivariate analysis of potential prognostic factors showed that methylation profile was the only independent prognostic factor in predicting DFS (P = 0.01) and OS (P = 0.05; Table 4).

Table 4.

Multivariate Cox model for DFS and OS in t(9;22)-ALL

FeatureUnivariate analysis (P)Multivariate analysis (P)
DFS   
    Methylation profile 0.002 0.01 
    WBC >50,000 mm3 0.20 — 
    Age >6 y 0.46 — 
Immunologic subtype 0.52 — 
Modal no. (>46) 0.23 — 
OS   
    Methylation profile 0.04 0.05 
    WBC >50,000 mm3 0.11 — 
    Age >6 y 0.64 — 
    Immunologic subtype 0.87 — 
    Modal no. (>46) 0.65 — 
FeatureUnivariate analysis (P)Multivariate analysis (P)
DFS   
    Methylation profile 0.002 0.01 
    WBC >50,000 mm3 0.20 — 
    Age >6 y 0.46 — 
Immunologic subtype 0.52 — 
Modal no. (>46) 0.23 — 
OS   
    Methylation profile 0.04 0.05 
    WBC >50,000 mm3 0.11 — 
    Age >6 y 0.64 — 
    Immunologic subtype 0.87 — 
    Modal no. (>46) 0.65 — 
View Large

Epigenetic gene silencing is increasingly being recognized as a common way in which cancer cells inactivate cancer-related genes (11, 12). In addition to its pathogenic implications, promoter hypermethylation and transcriptional repression of functionally important cancer-related genes may also influence tumor behavior, affecting clinical outcomes. The epigenetic silencing of genes that determine tumor invasiveness, growth patterns, and apoptosis, in particular, may dictate tumor recurrence after treatment and affect OS. Because each tumor may harbor multiple genes susceptible to promoter hypermethylation, individual tumors would exhibit different frequencies of methylation profile potentially predictive of a patient's clinical outcome (13, 14). This methylation profile could be of relevance in those tumors in which additional clinical and biological features of prognostic importance are not easily available, as occurs in ETV6/RUNX1-positive BCP-ALL.

Our results indicate that the methylation of multiple genes is a common phenomenon in ETV6/RUNX1-positive BCP-ALL and may be the most important way to inactivate cancer-related genes in this disease; 89% of cases had at least one gene methylated, whereas 63% of cases had three or more genes methylated. Moreover, ETV6/RUNX1-positive patients showed a higher degree of genes simultaneously methylated than ETV6/RUNX1-negative patients, suggesting that methylation plays a more important role in t(12;21) leukemogenesis than in other forms of BCP-ALLs. This issue is interesting in view of recent backtracking studies in twins and triplets, as well as studies of newborn Guthrie blood spots, indicate that the ETV6/RUNX1 fusion occurs in utero and it is necessary but insufficient for leukemogenesis (27). Subsequent unidentified molecular events in early childhood seem necessary for the clinical development of BCP-ALL. Our data suggest that an epigenetic mechanism could be involved in this process.

Our data also show that the methylation in human ETV6/RUNX1-positive cells can participate in the loss of regulation of four key cellular pathways: (a) growth-deregulating events comprising those that target the principal late-G1 cell cycle checkpoint (LATS2, NES1, PTEN, and p73 inactivation); (b) the apoptotic program through inactivation of DIABLO, PTEN, and REPRIMO; (c) the cell-cell adhesion by the inactivation of some members of the cadherin (CDH13 and CDH1) and metalloprotease (ADAMTS1 and ADAMTS5) families; and (d) deregulation of the WNT signaling pathway by inactivation of its antagonists, DKK3, sFRP1, sFRP2, sFRP5, HDPR1, and WIF1. Because the frequencies of methylation of the majority of these genes were similar in ETV6/RUNX1-positive and -negative BCP-ALL, one could speculate that the disruption of these oncogenic pathways is a common phenomenon in all types of childhood lymphoid leukemogenesis. However, a group of four genes (DKK3, sFRP2, PTEN, and P73) showed specificity for ETV6/RUNX1-positive BCP-ALL, suggesting that they play an important role in t(12;21) leukemogenesis.

Some studies have indicated that patients with t(12;21) have an excellent prognosis (2). However, in patients treated with the Berlin-Frankfurt-Munster group protocols, ETV6/RUNX1-positive patients displayed better outcome in short-term follow-up, but seemed to have more late relapses (3–5). Although one possible explanation for these diverging results is that the prognosis effect of this translocation is dependent on therapy, they also indicate that the biological nature of leukemias carrying the ETV6/RUNX1 fusion gene is heterogeneous. However, a global view of gene expression in ETV6/RUNX1-positive BCP-ALL using microarrays was associated with a characteristic and very homogeneous gene expression signature (28). Therefore, it is very difficult to obtain data of prognostic significance using this procedure. In contrast to this, our results show that ETV6/RUNX1-positive BCP-ALL is very heterogeneous from an epigenetic point of view. Aberrant methylation of CpG islands is quantitatively different in individual tumors within the same tumor type, and this patient-specific methylation profile provides important prognostic information in ETV6/RUNX1-positive BCP-ALL patients treated with the same therapeutic protocol and with a long follow-up. The presence in individual tumors of multiple epigenetic events that affect each of the pathways discussed above is a factor of poor prognosis in this disease. Patients with methylation of three or more genes had a poorer DFS and OS than patients with two or less methylated genes. Multivariate analysis confirmed that methylation profile was associated with a shorter DFS and OS. Therefore, methylation profiling in ETV6/RUNX1-positive BCP-ALL could have important clinical information for guiding the selection of therapy and also providing a basis for developing novel therapies, such as demethylation treatment. Because the number of samples analyzed is relatively small, our results should be independently confirmed in a larger series.

In summary, our results indicate that simultaneously aberrant methylation affecting key molecular pathways is a common phenomenon in ETV6/RUNX1-positive BCP-ALL. The methylation profile seems to be an important factor in predicting the clinical outcome of these patients.

Grant support: Fondo de Investigacion Sanitaria (Spain) 03/0141, 01/0013-01, 01/F018, 02/1299; Navarra Goverment (31/2002); RETIC C03/10, Junta de Andalucia 03/143; 03/144 and funds from IMABIS (Malaga, Spain), “UTE project CIMA,” Fundación de Investigación Médica Mutua Madrileña Automovilista, and Asociacion Medicina e Investigacion.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1
Loh ML, Rubnitz JE. TEL/AML1-positive pediatric leukemia: prognostic significance and therapeutic approaches.
Curr Opin Hematol
 
2002
;
9
:
345
–52.
2
McLean TW, Ringold S, Neuberg D, et al. TEL/AML1 dimerizes and is associated with a favorable outcome in childhood acute lymphoblastic leukemia.
Blood
 
1996
;
88
:
4252
–8.
3
Borkhardt A, Cazzaniga G, Viehmann S, et al. Associazione Italiana Ematologia Oncologia Pediatrica and the Berlin-Frankfurt-Munster Study Group. Incidence and clinical relevance of TEL/AML1 fusion genes in children with acute lymphoblastic leukemia enrolled in the German and Italian multicenter therapy trials.
Blood
 
1997
;
90
:
571
–7.
4
Harbott J, Viehmann S, Borkhardt A, Henze G, Lampert F. Incidence of TEL/AML1 fusion gene analyzed consecutively in children with acute lymphoblastic leukemia in relapse.
Blood
 
1997
;
90
:
4933
–7.
5
Seeger K, Adams HP, Buchwald D, et al. TEL-AML1 fusion transcript in relapsed childhood acute lymphoblastic leukemia. The Berlin-Frankfurt-Munster Study Group.
Blood
 
1998
;
91
:
1716
–22.
6
Ford AM, Fasching K, Panzer-Grumayer ER, Koenig M, Hass OA, Greaves MF. Origins of “late” relapse in childhood acute lymphoblastic leukemia with TEL-AML1 fusion gene.
Blood
 
2001
;
98
:
558
–64.
7
Weston VJ, McConville CM, Mann JR, et al. Molecular analysis of single colonies reveals a diverse origin of initial clonal proliferation in B-precursor acute lymphoblastic leukemia that can precede the t(12;21) translocation.
Cancer Res
 
2001
;
61
:
8547
–53.
8
Sawinska M, Ladon D. Mechanism, detection and clinical significance of the reciprocal translocation t(12;21)(p12;q22) in the children suffering from acute lymphoblastic leukaemia.
Leuk Res
 
2004
;
28
:
35
–42.
9
Alvarez Y, Coll MD, Ortega JJ, et al. Genetic abnormalities associated with the t(12;21) and their impact in the outcome of 56 patients with B-precursor acute lymphoblastic leukemia.
Cancer Genet Cytogenet
 
2005
;
162
:
21
–9.
10
Bird A. DNA methylation patterns and epigenetic memory.
Genes Dev
 
2002
;
16
:
6
–21.
11
Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation.
N Engl J Med
 
2003
;
349
:
2042
–54.
12
Esteller M. Relevance of DNA methylation in the management of cancer.
Lancet Oncol
 
2003
;
4
:
351
–8.
13
Roman-Gomez J, Jimenez-Velasco A, Castillejo JA, et al. Promoter hypermethylation of cancer-related genes: a strong independent prognostic factor in acute lymphoblastic leukemia.
Blood
 
2004
;
104
:
2492
–8.
14
Roman-Gomez J, Jimenez-Velasco A, Agirre X, Prosper F, Heiniger A, Torres A. Lack of CpG island methylator phenotype defines a clinical subtype of T-cell acute lymphoblastic leukemia associated with good prognosis.
J Clin Oncol
 
2005
;
23
:
7043
–9.
15
Ortega JJ. Spanish acute lymphoblastic leukemia trials.
Int J Pediatr Hematol Oncol
 
1998
;
5
:
163
–76.
16
Ribera JM, Ortega JJ, Oriol A, et al. Prognostic value of karyotypic analysis in children and adults with high-risk acute lymphoblastic leukemia included in the PETHEMA ALL-93 trial.
Haematologica
 
2002
;
87
:
154
–66.
17
Elhasid R, Sahar D, Merling A, et al. Mitochondrial pro-apoptotic ARTS protein is lost in the majority of acute lymphoblastic leukemia patients.
Oncogene
 
2004
;
23
:
5468
–75.
18
Izumi H, Inoue J, Yokoi S, et al. Frequent silencing of DBC1 is by genetic or epigenetic mechanisms in non-small cell lung cancers.
Hum Mol Genet
 
2005
;
14
:
997
–1007.
19
Iwai M, Kiyoi H, Ozeki K, et al. Expression and methylation status of the FHIT gene in acute myeloid leukemia and myelodisplastic syndrome.
Leukemia
 
2005
;
19
:
1367
–75.
20
Rothem L, Stark M, Kaufman Y, Mayo L, Assaraf YG. Reduced folate carrier gene silencing in multiple antifolate-resistant tumor cell lines is due to a simultaneous loss of function of multiple transcription factors but not promoter methylation.
J Biol Chem
 
2004
;
279
:
374
–84.
21
Liu ZJ, Zhang XB, Zhang Y, Yang X. Progesterone receptor gene inactivation and CpG island hypermethylation in human leukemia cancer cells.
FEBS Lett
 
2004
;
567
:
327
–32.
22
Takahashi T, Suzuki M, Shigematsu H, et al. Aberrant methylation of Reprimo in human malignancies.
Int J Cancer
 
2005
;
115
:
503
–10.
23
Matsushita C, Yang Y, Takeuchi S, et al. Aberrant methylation in promoter-associated CpG islands of multiple genes in relapsed childhood acute lymphoblastic leukemia.
Oncol Rep
 
2004
;
12
:
97
–9.
24
Schar P, Fasi M, Jessberger R. SMC1 coordinated DNA double-strand break repair pathways.
Nucleic Acids Res
 
2004
;
32
:
3921
–9.
25
Takeuchi S, Bartram CR, Wada M, et al. Allelotype analysis of childhood acute lymphoblastic leukemia.
Cancer Res
 
1995
;
55
:
5377
–82.
26
Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.
Proc Natl Acad Sci U S A
 
1996
;
93
:
9821
–6.
27
Morrow M, Horton S, Kioussis D, Brady HJ, Williams O. TEL-AML1 promotes development of specific hematopoietic lineages consistent with preleukemic activity.
Blood
 
2004
;
103
:
3890
–6.
28
Fine BM, Stanulla M, Schrappe M, et al. Gene expression patterns associated with recurrent chromosomal translocation in acute lymphoblastic leukemia.
Blood
 
2004
;
103
:
1043
–9.
American Association for Cancer Research
2006