ALBERT

Description: DAC is a potent hypomethylating agent with clinical activity in patients with myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML). VPA is a histone deacetylase inhibitor used as an antiepileptic agent. In vitro, the combination of DAC with VPA results in synergistic antileukemia activity at doses of VPA above 1mM. Based on this data, we have developed a phase I/II study of this combination for pts with leukemia. The phase I of the study followed a classic 3+3 design. The dose of DAC was fixed: 15 mg/m2 iv daily for 10 days. This was based on a previous phase I study (Blood2004;103:1635) that indicated that this schedule had an optimal toxicity-response profile in this population. Three dose levels of VPA were selected: 20, 35 and 50 mg/kg. VPA was given orally for 10 days concomitantly with DAC. 22 pts have completed the phase I portion of the study (median age 56 years, range 4–78, 20 pts AML, 2 MDS). At dose level 1 (20 mg/kg of VPA) no grade III-IV toxicity was observed. At dose level 2 (VPA 35 mg/kg), 2 out 6 pts developed grade III neurotoxicity. Both pts were receiving high doses of other neurotropic agents. After IRB approval, 3 mores pts were treated at this dose level with no significant toxicity. Subsequently, 3 pts were treated at the highest planned dose level (50 mg/kg) with no toxicity observed. This cohort was then expanded to a total of 10 pts. One pt developed grade III neurotoxicity. No other severe drug-related toxicities were observed, but 5 patients at all dose levels developed grade II sedation/somnolence. Pancytopenia was induced in all pts. At dose level 1, one pt with refractory AML achieved complete remission (CR) after the second course of therapy. This is now maintained for 5 courses. At dose level 2, a patient with HIV disease and relapsed AML achieved CR after the third course of therapy, and 2 pts with relapsed AML achieved complete marrow responses (marrow blasts less then 5%, no recovery of peripheral counts). Of 3 pts evaluable for response at dose level 3, 1 pt with MDS has achieved CR after 1 course, and 1 with relapsed AML a complete marrow response. Median free VPA levels pretreatment were 0, and 25 mg/L on both days 5 and 10 and returned to 0 prior to next course. Histone acetylation measured by Western blot was observed in 3 pts (25%), all at doses above 20 mg/kg of VPA. Reactivation of p21 expression was induced in 4 out 11 pts analyzed. Global hypomethylation measured using a bisulfite PCR LINE assay was induced in 1 out 3 pts so far studied. Based on the toxicity observed, the phase II portion of the study was initiated. This is restricted to pts with AML/MDS. Seven pts have been accrued to this phase, and 8 out the 10 pts at dose level 3 of the phase I are also evaluable. The response data of this pts will be updated at the meeting. In summary, the combination of low dose DAC and VPA up to doses of 50 mg/kg can be safely administered to pts with leukemia although it may be complicated by neurotoxicity. Clinical and biological activity was observed at all dose levels.

Description: The nucleosome is the basic structure of chromatin. Changes in the biochemical composition of nucleosome-associated histone tails are associated with specific gene activation states, and are the target of several antineoplastic agents such as histone deacetylase inhibitors (HDI). Nucleosomes are constrained into loops that are flanked by domains known as matrix-attached regions (MARs). MARs contain DNA topoisomerase II (Topo II) consensus sequences. Topo II is responsible for regulating and maintaining DNA topology and is the target of several antineoplastic agents such as the anthracycline IDA, an effect mediated by the induction of double strand DNA breaks (DSB). We hypothesized that the combination of a Topo II inhibitor and a HDI will have synergistic antileukemia activity. VPA and SAHA are two HDIs currently studied in several clinical trials with known antileukemia activity and tolerable toxicity. To test our hypothesis and to develop future clinical studies, we have analyzed the effect of the combination of IDA, a potent Topo II inhibitor, with VPA or SAHA. We treated the leukemic cells lines MOLT4 and HL60 with increasing doses of IDA (0.5-20nM), SAHA (0.3-3μM) or VPA (0.25-3mM) daily for 3 days. First, using trypan blue viability assays, we identified the IC10 of IDA to be 0.5nM for MOLT4 and 1.5nM for HL60. Doses in excess of 2μM of SAHA or 3mM of VPA resulted in more than 90% decrease in cell viability in both cell lines. Subsequently, SAHA at doses of 0.075-1μM and VPA at 1-3mM were used for the combination experiments with IDA at its specific cell line IC10. At low doses of SAHA (0.075-0.45 μM) and VPA (0.25-1 mM) the combination was shown to have synergistic antileukemia activity by the Fractional Product Method of Webb. These results were confirmed using Annexin V assays. Of importance, growth inhibition was independent of the sequence used. To analyze the effects of this combination on DSB generation, we analyzed using immonocytochemistry and western blot, the induction of γH2AX, a histone variant that has been identified as an early event after the DSBs. SAHA alone induced a modest increase in γH2AX compared to baseline, whereas IDA alone had a significant effect that was not potentiated by the addition of SAHA. Histone H3 and H4 acetylation increased in a dose-dependent manner (2.4–15 fold) with both SAHA and VPA, starting at 0.3μM of SAHA and 0.25mM of VPA. The addition of IDA had no significant effect on histone acetylation. Because of previous data indicating that HDIs may down-regulate the expression of Topo II-alpha, the target of IDA, we have studied using real-time PCR its levels prior and during exposure to the different combinations. SAHA or VPA had no effect on Topo II-alpha mRNA levels whereas IDA induced 2.0–3.5 fold its expression in a dose-independent manner, an effect no altered by the addition of SAHA or VPA. Expression of p21CIP1, that is silenced in both cell lines, was restored by single agent VPA, SAHA or IDA. The combination of these drugs resulted in an additive effect in terms of p21CIP1 induction. Despite this phenomenon, no changes in cell cycle status were observed in these cells. In summary, the combination of IDA and SAHA or VPA has potent in vitro antileukemia effect, and should be studied in clinical trials.

Description: Pretreatment aberrant DNA methylation patterns are stable at time of relapse in acute lymphocytic leukemia (ALL). We hypothesized that the detection of residual methylation alterations at the time of morphologic remission may predict for worse prognosis. We developed a real-time bisulfite polymerase chain reaction assay and analyzed the methylation levels of p73, p15, and p57KIP2 at the time of initial remission in 199 patients with Philadelphia chromosome-negative and MLL− ALL. Residual p73 methylation was detected in 18 (9.5%) patients, p15 in 33 (17.4%), and p57KIP2 in 7 (3.7%); 140 (74%) patients had methylation of 0 genes and 48 (25%) of more than or equal to 1 gene. In 123 (65%) patients, matched pretreatment samples were also studied and compared with remission ones: in 82 of those with initial aberrant methylation of at least one gene, 59 (72%) had no detectable methylation at remission and 23 (28%) had detectable residual methylation. By multivariate analysis, the presence of residual p73 methylation was associated with a significant shorter duration of first complete remission (hazard ratio = 2.68, P = .003) and overall survival (hazard ratio = 2.69, P = .002). In conclusion, detection of epigenetic alterations allows the identification of patients with ALL with standard risk but with poor prognosis.

Description: Aberrant DNA methylation of multiple promoter CpG islands is a common feature of ALL. Specific DNA methylation patterns involving a triad of hierarchical clustered cell cycle controlling genes including p73, p15 and p57KIP2 predict for poor prognosis in Ph negative ALL (Garcia-Manero, Blood, 2003). Of importance, this aberrant DNA methylation patterns are stable at the time of relapse in a majority of patients. This indicates that aberrant DNA methylation is a fundamental molecular feature of the ALL blast cell (Garcia-Manero, Clinical Cancer Research, 2002). Based on this, we hypothesized that the detection of residual DNA methylation in patients with standard risk (Ph negative) ALL could predict for shorter disease free survival, and worse overall survival (OS) in this patient population. To test this hypothesis, we have analyzed the frequency of p73, p15 and p57KIP2 DNA methylation at the time of initial documentation of complete remission (CR) (days 14 to 21) in 199 patients in CR with Ph negative ALL. All patients had been treated with hyperCVAD-based chemotherapy at our institution. To perform this analysis, we have developed a sensitive and specific novel real-time methylation specific PCR assay to detect DNA methylation. This method requires bisulfite DNA conversion. It uses the interferon-gamma gene as a DNA loading control and can be performed in less than 24 hours. The characteristics of the 199 patients evaluated are: median age 38 years (range 15–83), cytogenetics: 32% diploid, 5% hyper or hypodiploid, 20% insufficient metaphases and 3% MLL+, the rest others. 16% were of T cell phenotype. Using this assay, p73 methylation was detected in 18 (10%) patients, p15 in 33 (16%) and p57KIP2 in 7 (3.5%). Methylation of at least 1 gene of this triad was observed in 48 (24%) patients. The median OS of the whole group was 209 weeks (95% CI 158–514) and the disease free survival (DFS) 146 weeks (95% CI 110–335). These were representative of the overall experience with hyperCVAD at MDACC. By multivariate analysis, the presence of residual p73 methylation on days 14 to 21 in patients with Ph negative ALL was associated with a shorter DFS (hazard ratio 2.68, p=0.003) and a worse OS (hazard ratio 2.69, p=0.002). This was independent of any other patient characteristic. Other characteristic included in these models included age and platelet count for OS, and age, b2-microglobulin, LDH and platelets for DFS. The presence of p15, p57KIP2 methylation or the combination of multiple genes was not associated with shorter OS or DFS. In summary, the detection of residual p73 methylation allows the identification of a subset of patients with ALL in remission and worse prognosis. This data may allow the development of specific therapeutic interventions, such as the use of hypomethylating agents, or early allogeneic stem cell transplantation for this at-risk patient population, and suggest that inactivation of p73 is a key event in ALL.

Description: Aberrant DNA methylation of promoter-associated CpG islands is a frequent phenomenon in human leukemias, and in particular in adult ALL. Hck is a member of the Src family of tyrosine kinases, and functionally is located downstream of BCR-ABL signaling in chronic myelogenous leukemia (CML). Hck expression is limitedly to myeloid cells and B cell lymphocytes. Although some evidence indicates that Hck is required for malignant transformation and apoptosis, its role in leukemia is not fully understood. Here we analyze the role of aberrant DNA methylation of Hck in leukemia cell lines and patients. Using BLAT, we first identified the presence of a canonical CpG island in the near proximity of the transcription start site of HcK. To detect and measure DNA methylation, we designed a combined bisulfite restriction PCR assay. Using this assay, we found that Hck was methylated in 13 out of 23 hematopoietic and 8 out of 10 non-hematopoietic cell lines, but not in the bone marrow from 6 healthy individuals. We subsequently studied Hck expression by real-time PCR using GAPDH expression as an internal control. Hck expression was lower (dCT = −14.2± 3.6) in 7 Hck methylated cell lines than in 8 Hck unmethylated ones (dCT= −9.0± 3.5), p=0.017. All the cell lines studied were of myeloid or B cell origin. We then treated the Raji cell line with the hypomethylating agent 5-aza-2-deoxycytidine (DAC). DAC treatment resulted in partial hypomethylation of Hck and in an increment of Hck expression (dCT: −19.37 to −8.47). Subsequently, the effects of DAC treatment on Hck protein expression levels were analyzed using Western blot. These experiments showed a strong correlation between hypomethylation, gene re-expression and protein expression levels. These data therefore indicates that DNA methylation is an important aberrant regulator of Hck expression in leukemia cell lines. Based on the relevance of these findings, we then analyzed the frequency of Hck methylation in patients with leukemia. Using a cut-off of 10%, Hck was found to be methylated in 15 out of 44 (34%) patients with ALL, 9 out 23 pts (39%) with CML, and 3 out 10 pts (30%) with AML. Of importance, the density of Hck methylation was significantly higher in patients with ALL (mean 11.3%; range 0–76) compared to those with CML(5.2%; range 0–12) or AML ( 7.5%, range 0–14), p=0.02. Hck methylation was not associated with a B cell phenotype or the presence of the Philadelphia chromosome in patients with ALL. Nine ALL pts out of 15 with Hck methylation had died compared to 7 out 29 unmethylated (total ALL group n=34). Median survival had not been reached for the group of patients with no Hck methylation (n=29) compared to 116 weeks for those with Hck methylation (n=15) (p=0.08). All pts had been treated with hyperCVAD based chemotherapy. These data indicates that Hck methylation is a frequent phenomenon in human leukemia that maybe associated with a worse prognosis in ALL and suggests that Hck has a tumor suppressor like function in these disorders.

Description: We studied the cellular and molecular effects of the combination of an anthracycline with 2 different histone deacetylase inhibitors (HDACIs): vorinostat (suberoylanilide hydroxamic acid) and valproic acid (VPA). The 10% inhibitory concentration (IC10) of idarubicin was 0.5 nM in MOLT4 and 1.5 nM in HL60 cells. Concentrations above 0.675 μM of vorinostat resulted in at least 80% loss of cell viability in both cell lines. Concentrations of 1.5 to 3 mM of VPA induced 50% to 60% loss in viability in HL60 and 80% in MOLT4 cells. The combination of idarubicin with vorinostat at 0.075 μM or VPA at 0.25 mM resulted in at least an additive loss of cell viability in both lines. Vorinostat (0.35 μM) and VPA (0.25 mM) in combination with idarubicin (0.5 nM) resulted in a significant increase in apoptotic cells in MOLT4 cells. The combination resulted in an increase in histone H3 and H4 acetylation at 24 hours, phosphorylated H2AX, as well as in the induction of p21CIP1 mRNA. No effect on cell cycle transition was observed. Of importance, the cellular and molecular effects observed were independent of the sequence used. In summary, the combination of an anthracycline with an HDACI should have significant clinical activity in patients with leukemia.

Description: Most patients with acute promyelocytic leukemia (APL) exhibit a characteristic t(15;17) translocation that fuses the PML on 15q22 to the RARα on 17q12. In a small subset of APL, RARα fuses to several other genes including the PLZF on 11q23, NPM1 on 5q35, NUMA1 on 11q13, STAT5b on 17q11, and PRKAR1α on 17q23. The STAT5b-RARα chimeric protein was delocalized from the cytoplasm to the nucleus, where it displayed a microspeckled pattern, implicating that this APL might result from dysregulation of the JAK/STAT5 signal transduction pathway. However, only two patients with APL harboring STAT5b-RARα fusion gene have been reported, and clinical features and response to therapy as well as the pathogenesis in this subgroup remain to be determined. We examined 8 PML-RARα-negative APL patients for the presence of the above alternative fusion genes by RT-PCR. We here present the third patient with APL harboring a STAT5b-RARα fusion gene and a missense mutation G596V in the SH2 domain of the STAT5b. A 41-year old Japanese man admitted to our hospital because of petechiae, fever and leukocytosis (77.8 x 109/L). His promyelocytes were relatively mature with dense nuclei and abundant azurophilic granules, while faggot cells were observed. Promyelocytes were positive for CD13 and CD33, but negative for CD34, CD117 or HLA-DR, consistent with APL cells. Karyotype analysis of the bone marrow cells revealed 47, XY,del(9)(q?),add(17)(q12),+mar1[3]/48, XY,idem,+mar1[17]. Activity of α2 plasmin inhibitor was 16%, and FDP was 344.0 μg/mL (〈 5.0 μg/mL). The patient was diagnosed to have APL and DIC with fibrinolysis. He achieved a complete remission after one course of chemotherapy and all-trans retinoic acid (ATRA) although the sign of ATRA-induced granulocytic differentiation was not observed. Sequence analysis of the STAT5b-RARα transcript disclosed that the STAT5-RARα fusion gene corresponded to those of previously reported case of STAT5b-RARα-positive APL. Abnormalities of chromosome 17 and resistance to ATRA were commonly observed in 3 patients harboring STAT5b-RARα. Interestingly, there was an additional missense mutation (G596V) within the STAT5b SH2 domain of the STAT5b-RARα gene. Neither STAT5b-RARα fusion gene nor G596V was detected in marrow cells during remission. As the SH2 domain plays an important role in the activation of STAT proteins and the G596 substituted amino acid is highly conserved in the SH2 domain-containing proteins, it would be interesting to analyze the STAT5b and STAT5b-RARα protein harboring G596V mutation. In addition, G596V mutant clone dominate before treatment and wild type subclone was not detected. G596V mutation may occur at an early phase of leukemogenesis or may acquire dominant proliferation during development of APL. STAT5 appears to upregulate DNA repair protein RAD51. One can assume that activated STAT5b-RARα disrupts DNA repair process and results in genomic instability. Further investigations are needed to understand the molecular pathogenesis and clinical features of STAT5b-RARα-positive APL.

Description: ARTS (pro-apoptosis-related protein in the TGF-b signaling pathway) is a septin-like protein that is localized in mitochondria and was reported to promote apoptosis by antagonizing the Inhibitor-of-Apoptosis protein XIAP (Nat Cell Biol 2:915, 2000, EMBO Journal 23:1627, 2004, Oncogene 1:2004). Here, we analyzed the expression and function of ARTS in leukemic and normal cells. We determined the expression levels in leukemic cell lines and in primary leukemia samples by Western blot analysis using an antibody against the C-terminal region of ARTS that identifies 27 unique amino-acids. ARTS was expressed in all AML cell lines studied (OCI-AML3, HL-60, Kasumi-1, NB4, U937, K562), but expression was undetectable in two ALL cell lines (ALL1 and Jurkat). We extended expression studies to primary samples from 34 AML and 14 ALL samples. ARTS was absent or expressed at very low levels in all ALL samples analyzed. In contrast, ARTS was found to be expressed in 50% of primary AML samples. Interestingly, FAB M0 and M1 AML infrequently expressed ARTS (n=1/11), while expression was higher in FAB M2-M5 samples (13/23). These data suggest that ARTS expression is repressed in ALL and in immature AML cells. There was no apparent correlation with cytogenetics. In normal bone marrow and blood cells, ARTS was expressed at low levels. The ARTS gene has a CpG island near its promoter and was reported to be regulated by methylation. We therefore examined ARTS methylation in leukemic cells by methylation specific PCR (MSP). In U937 cells, MSP analysis demonstrated only a methylated product, but ARTS was expressed, while in Jurkat cells the promoter was unmethylated, but ARTS was not expressed. NB4 and Raji cell lines had both, unmethylated and methylated ARTS promoters. In contrast, ARTS mRNA was detected in all cell lines, normal cells and leukemic samples. Our data demonstrate that the methylation status of the ARTS gene is not related to the expression of ARTS. It has been reported that Etoposide induces overexpression of ARTS and binding to XIAP. We therefore treated leukemic cell lines (OCI-AML3 and ALL1), and MCF7 breast cancer cells with Etoposide, Ara-C and Doxorubicin. ARTS expression was increased in MCF7, but not in leukemic cell lines. In MCF7, ARTS was localized in mitochondria before treatment and after 16 hours was partially translocated to the nucleus. On the other hand, Etoposide, Ara-C, and Doxorubicin did not induce ARTS expression in leukemic cells. Co-culture of leukemic cell lines and mesenchymal stem cells increased ARTS expression not only in OCI-AML3, but also in ALL1 cells in 3/3 experiments. Conclusions: This study suggests that ARTS expression is absent in ALL and immature AML cells, but is variably expressed in more differentiated AML and in normal hematopoietic cells. Its expression is not methylation dependent and it is not upregulated by Etoposide, Ara-C and Doxorubicin. However, co-culture with MSC increased ARTS levels. Results, so far, are not consistent with the proposed pro-apoptotic function of ARTS.

Description: Transcription factors involved in myeloid cell differentiation are frequent targets of chromosomal translocations and point mutations in patients with acute myeloblastic leukemia (AML). Familial AML harboring a mutation in a transcription factor should provide an association of clinical features with functions of the transcription factor. Recently, two pedigrees of AML carrying a germ-line mutation in the CEBPA, a gene encoding transcription factor C/EBPα have been reported. As C/EBPα-null mice lack of mature neutrophil and eosinophil granulocytes, C/EBPα is thought to play a central role in regulating the differentiation of granulocytes. We here present clinical and molecular features of a Japanese family in which two individuals developing AML and one healthy individual had an identical CEBPA mutation. Father had received a diagnosis of AML M2 in 1988 at the age of 39 years. Following a relapse 7 years after his first complete remission (CR), he received autologous stem cell transplantation, after which he has been in a lasting CR. His elderly son was diagnosed to have M2Eo in 2004 at the age of 26 years. He has achieved a continuous CR. Bone marrow cells at the time of diagnosis and peripheral blood cells obtained during CR in both patients showed a 4-base pair insertion in the N-terminal region of the CEBPA (350_351insCTAC). The corresponding protein is predicted to terminate prematurely at codon 107 (I68fsX107). Therefore, this heterozygous mutation causes truncation of the 42-kD C/EBPα protein and overproduction of a 30-kD isoform, which lacks a transactivation domain and functions in a dominant negative fashion, causing a decrease in C/EBPα activity. His younger son unaffected by AML aged 21 years also had the same mutation. The N-terminal C/EBPα mutations in sporadic AML patients are associated with FAB M1/M2 subtypes, presence of Auer rods, CD7 expression, normal karyotype, and a favorable prognosis. Familial AMLs with an N-terminal C/EBPα mutation demonstrate links of these unique features to a dominant negative C/EBPα mutation. It is of note that different in-frame insertion mutations in the C-terminal region of C/EBPα were identified in both patients at the time of diagnosis. As these C-terminal CEBPA mutations were not found in peripheral blood cells during CR in both patients, these in-frame insertion mutations in the C/EBPα basic region leucine zipper DNA-binding domain should be somatic mutations. Interestingly, father showed different in-frame insertion mutations in the C-terminal CEBPA at the time of diagnosis and relapse. These N- and C-terminal biallelic mutations in the CEBPA are thought to result in complete loss-of-function of C/EBPα. A recent study using conditional Cebpa knockout mice showed that C/EBPα deficiency in adult mice leads to persistence of immature myeloid blasts in the bone marrow without developing AML. Although the mechanism underlying the development of AML is yet unclear, this study indicates that the N-terminal CEBPA mutation appears to confer an increased risk occurring C-terminal C/EBPα mutations and other mutations as a second and further genetic hits, generating undifferentiated myeloid cells and eventually leading to AML over a long latency.

Description: Aberrant DNA methylation of promoter-associated CpG islands is a frequent event in ALL. DNA methylation of specific subsets of genes is associated with poor prognosis and is stable in a majority of patients (pts) at the time of relapse (Clinical Cancer Res2002;8:1897), and therefore tracking these epigenetic marks during remission may predict for relapse risk. Most commonly used methods to detect DNA methylation exploit the availability of sodium bisulfite that transforms unmethylated C into A/T leaving methylated C as such. This has allowed the development of several techniques that use conventional PCR or sequencing to detect methylated alleles. Although there are significant differences in the sensitivity and specificity of these assays, with bisulfite sequencing considered the gold standard, most of them are labor intensive and require several days to be performed. To circumvent some of these problems, we have developed a real-time bisulfite PCR assay to detect methylation of p57KIP2, p73, and p15 in samples obtained from bone marrows in pts with ALL at remission. Methylation of these genes has been shown to be common in ALL and to predict for poor prognosis at initial presentation (Blood2003;101:4131). This method allows for the simultaneous analysis of multiple samples in less than 24 hours, is quantitative, and requires less than 0.2μg of DNA for each target gene. To amplify bisulfite treated DNA, we designed primer sets and probes for all three genes in regions known to be inversely correlated with gene expression. To quantify methylation density, we used the interferon γ (INFG) gene as an internal control because it has very rare CpG sites, is a single copy gene and has no homology with other known genes. Methylation density is defined as: Methylation (%) = 2CT of target gene / 2 CT of EFM x 100. CT is the number of cycles threshold, and EFM the estimated 100% fully methylation (EFM) of the target gene in control experiments. Using DNA artificially methylated with SssI in dilution experiments with unmethylated DNA, p57, p15 and p73 methylation density could be detected at dilutions of 0.2%, 1.2% and 0.1%, respectively. We then studied the methylation status of 30 cell lines (22 hematopoietic and 8 no-hematopoietic). Methylation of p57, p15 and p73 gene was detected in 17(57%), 19(63%) and 16(53%) cell lines respectively. Methylation of 3 genes, 2 genes and 1 gene was observed in 9(30%), 8(27%) and 7(23%) cell lines respectively. DNA methylation of p15 and p73 was not detected in the marrow from 6 healthy volunteers but p15 was detected (0.1% methylation) in 1 out of 6 of these specimens. Subsequently, we studied 50 pts with ALL at the time of remission. We found the frequencies of p57, p15 and p73 gene to be 2(4%), 26(52%) and 10(20%), respectively. There was a trend towards a better overall survival for pts with methylation of 0 or 1 gene (209 weeks) compared with those with methylation of 2 or more genes (71 weeks), p=0.1. In conclusion, the real-time bisulfite PCR described here allows for the rapid detection of aberrant DNA methylation in samples obtained at the time of remission in pts with ALL and may have a role in the development of techniques to assess minimal residual disease in this group of pts.