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Keywords:

  • APL;
  • aberrant methylation;
  • prognosis

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Patients
  6. MSP results
  7. Clinicopathological correlations of aberrant gene methylations
  8. Survival and prognostic analysis
  9. Detection of minimal residual leukaemia
  10. Discussion
  11. Acknowledgments
  12. Note added in proof
  13. References

Summary. Acute promyelocytic leukaemia (APL) has distinct clinicopathological and molecular features. However, the profile of aberrant gene promoter methylation is undefined. In this study, methylation-specific polymerase chain reaction (MSP) was used to define the methylation status of a panel of nine genes, comprising p15, p16, RARβ, oestrogen receptor (ER), E-cadherin (E-CAD), p73, caspase 8 (CASP8), VHL and MGMT, in 29 patients with APL. Aberrant methylation of p15, ER, RARβ, p16 and E-CAD occurred, respectively, in 23 (79%), 14 (48%), six (21%), six (21%) and two (7%) patients at diagnosis, but p73, VHL, CASP8 and MGMT were not methylated in any patients. There was methylation of one gene in 13 patients (45%), two genes in four patients (14%), three genes in six patients (21%) and four genes in three patients (10%). Concurrent methylation of two or more genes occurred in 13 patients (45%). No association was identified between gene methylation and presenting clinicopathological features. However, p15 methylation was significantly associated with an inferior disease-free survival (DFS, P = 0·008), and remained the only poor prognostic factor in multivariate analysis (P = 0·019). In APL, p15, p16, ER and RARβ were most frequently methylated. This profile is distinct from other types of myeloid leukaemias. p15 methylation has a poor prognostic impact on DFS.

DNA methylation, catalysed by DNA methyltransferases, involves the addition of a methyl group to the carbon-5 position of the cytosine ring in a CpG dinucleotide to become methylcytosine (Singal & Ginder, 1999; Baylin & Herman, 2000; Robertson & Wolffe, 2000; Chim et al, 2002). In the normal mammalian genome, CpG dinucleotides have been progressively depleted during evolution and thus are under-represented. In contrast, scattered stretches of CpG-rich DNA, referred to as CpG islands, exist within the promoters of genes. Non-promoter CpG dinucleotides, which constitute more than 80% of all CpGs in the genome, are found in repeat regions and are often methylated. On the other hand, CpG islands located at gene promoters are protected from methylation, so that these genes are in a transcription-ready state. It has been observed that CpG islands of gene promoters may be aberrantly methylated (hypermethylated) in tumours, resulting in repression of gene transcription. This serves as an alternative mechanism of gene inactivation. There is increasing evidence that many genes are hypermethylated in leukaemias (Issa et al, 1997; Baylin et al, 1998; Melki et al, 1999; Chim et al, 2001a,b). However, most studies have focused on methylation studies of individual genes in separate patient groups, so that data on the methylation profiles of leukaemia are scanty.

Acute promyelocytic leukaemia (APL) is characterized by t(15;17) (De The et al, 1990; Kakizuka et al, 1991), which results in the fusion gene PML/RARα that is leukaemogenic (Brown et al, 1997; He et al, 1997). It is also the only AML subtype that responds to differentiation therapy with all-trans retinoic acid (ATRA) (Warrell et al, 1993; Chim et al, 1996), and to arsenic trioxide (Kwong et al, 2001; Au et al, 2002), which induces both differentiation and apoptosis of the neoplastic promyelocytes. Therefore, APL is a unique clinical and molecular subtype of AML.

In this study, we investigated whether aberrant methylation is confined to a single target gene to provide a growth advantage, or if multiple genes are concurrently methylated. The latter might imply a general deregulation of CpG island demethylation. A series of APL patients were examined for the methylation status of a panel of nine genes, in which inactivation by aberrant methylation might confer a growth advantage that contributed to leukaemogenesis. The panel contained genes involved in tumour suppression [p15, p16, p73, oestrogen receptor (ER), RARβ and VHL] (Singal & Ginder, 1999; Baylin & Herman, 2000; Robertson & Wolffe, 2000; Chim et al, 2001a,b, 2002), apoptosis [Caspase 8 (CASP8)] (Teitz et al, 2000) and cell adhesion [E-cadherin (E-CAD)] (Melki et al, 2000), and response to chemotherapy (MGMT, a DNA repair gene) (Esteller et al, 2000). The clinicopathological and prognostic impacts of aberrant gene methylation in APL were also examined.

Patients. Twenty-nine patients with newly diagnosed APL were analysed for gene methylation profiling. The diagnosis of APL was based on typical morphological characteristics, and confirmed by the presence of t(15;17) cytogenetically and/or PML/RARA molecularly. APL patients diagnosed between 1985 and 1992 received induction therapy with cytosine arabinoside (Ara-C; 100 mg/m2/d × 7) and daunorubicin (50 mg/m2/d × 3). From 1992 onwards, patients received ATRA at 45 mg/m2/d orally in two divided doses until complete remission (Chim et al, 1996). After complete remission (CR) was attained, all patients (in both groups of patients induced with cytarabine/daunorubicin or ATRA) received two courses of consolidation therapy with Ara-C (100 mg/m2/d × 5 d), daunorubicin (50 mg/m2/d × 2 d), and etoposide (75 mg/m2/d × 5 d) or thioguanine (100 mg/m2/d × 5 d). Relapsed patients received ATRA, arsenic trioxide or chemotherapy depending on their prior therapies (Chim et al, 1996; Kwong et al, 2001; Au et al, 2002).

Methylation-specific polymerase chain reaction (MSP). High-molecular-weight genomic DNA was isolated from the bone marrow aspirates of APL patients at diagnosis. The methylation-specific polymerase chain reaction (MSP) for gene promoter methylation was performed as previously described (Chim et al, 2001a,b). Briefly, treatment of DNA with bisulphite for conversion of unmethylated cytosine to uracil (but not affecting methylated cytosine) was performed with a commercially available kit (CpGenome DNA modification kit; Intergen, New York, NY, USA), according to the manufacturer's instructions. The primers for the methylated (M-MSP) and unmethylated (U-MSP) promoters of the target genes are shown in Table I. The polymerase chain reaction (PCR) mixture contained 50 ng of bisulphite-treated DNA, 0·2 mmol/l dNTPs, 2 mmol/l MgCl2, 10 pmol/l of each primer, 1 × PCR buffer II and 2·5 units of AmpliTaq Gold (PE Biosystems, Foster City, CA, USA) in a final volume of 50 µl. PCR was performed in an automated thermal cycler (9700; PE Biosystems), using the cycling conditions shown in Table I. All experiments contained a positive control with methylated DNA (CpGenome Universal Methylated DNA; Intergen), a negative control with DNA from normal donors, and blanks. Each patient sample was tested in duplicate.

Table I.  MSP: primer sequences and reaction conditions.
GeneForward primerReverse primerTm/ no. of cyclesSize
  1. Tm, annealing temperature; M-MSP, methylation-specific polymerase chain reaction for the methylated allele; U-MSP, MSP for the unmethylated allele.

p15
M-MSPGCG TTC GTA TTT TGC GGT TCGT ACA ATA ACC GAA CGA CCG A63°C/35148 bp
U-MSPTGT GAT GTG TTT GTA TTT TGT GGT TCCA TAC AAT AAC CAA ACA ACC AA 154 bp
p16
M-MSPTTA TTA GAG GGT GGG GCG GAT CGCGAC CCC GAA CCG CGA CCG TAA65°C/35150 bp
U-MSPTTA TTA GAG GGT GGG GTG GAT TGTCAA CCC CAA ACC ACA ACC ATA A 151 bp
RARβ
M-MSPGGA TTG GGA TGT CGA GAA CTAC AAA AAA CCT TCC GAA TAC G64°C/3593 bp
U-MSPAGG ATT GGG ATG TTG AGA ATGTTA CAA AAA ACC TTC CAA ATA CA 95 bp
ER
M-MSPCGA GTT GGA GTT TTT GAA TCG TTCCTA CGC GTT AAC GAC GAC CG66°C/35152 bp
U-MSPATG AGT TGG AGT TTT TGA ATT GTT TATA AAC CTA CAC ATT AAC AAC AAC CA 152 bp
CASP8
M-MSPTAG GGG ATT CGG ACA TTG CGACGT ATA TCT ACA TTC GAA ACG A56°C/45321 bp
U-MSPTAG GGG ATT TGG AGA TTG TGACCA TAT ATA TCT ACA TTC AAA ACA A 322 bp
E-CAD
M-MSPGTG GGC GGG TCG TTA GTT TCCTC ACA AAT ACT TTA CAA TTC CGA CG66°C/35173 bp
U-MSPGGT GGG TGG GTT GTT AGT TTT GTAAC TCA CAA ATC TTT ACA ATT CCA ACA 173 bp
MGMT
M-MSPTTT CGA CGT TCG TAG GTT TTC GCGCA CTC TTC CGA AAA CGA AAC G54°C/3581 bp
U-MSPTTT GTG TTT TGA TGT TTG TAG GTT TTT GTAAC TCC ACA CTC TTC CAA AAA CAA AAC A 93 bp
p73
M-MSPGGA CGT AGC GAA ATC GGG GTT CACC CCG AAC ATC GAC GTC CG68°C/3560 bp
U-MSPAGG GGA TGT AGT GAA ATT GGG GTT TATC ACA ACC CCA AAC ATC AAC ATC CA 69 bp
VHL
M-MSPTGG AGG ATT TTT TTG CGT ACG CGAA CCG AAC GCC GCG AA56°C/35158 bp
U-MSPGTT GGA GGA TTT TTT TGT GTA TGTCCC AAA CCA AAC ACC ACA AA 165 bp

Specificity and sensitivity of MSP. The specificity of the MSP was verified by direct DNA sequencing of the M-MSP and U-MSP PCR products. The sensitivity of MSP was estimated by serial 10-fold dilution of methylated DNA (Intergen) in normal donor DNA, followed by bisulphite modification and amplification by M-MSP.

Clinicopathological correlations of aberrant gene methylation. The presenting clinicopathological features including age, sex, presenting leucocyte count and M3 subtype were correlated with aberrant methylation of the tested genes. Categorical variables such as sex and M3 subtype were analysed by the chi-square or Fisher's Exact test. Comparison of the means of age and presenting leucocyte count with methylation status was performed by Student's t-test. All P-values were two sided, and were considered significant when < 0·05.

Survival and prognostic factor analysis. Only patients receiving induction therapy with ATRA were included for survival and prognostic analysis. Complete remission (CR) was defined as disappearance of abnormal promyelocytes with recovery of peripheral blood counts. Disease-free survival (DFS) was measured from the time of CR to the time of last follow-up, death or relapse. Overall survival (OS) was measured from the time of diagnosis to the time of last follow-up or death. Survival was censored at the time of bone marrow transplantation. Survival was calculated by the Kaplan–Meier method and compared by the log-rank test. The impact of p15 and p16 methylation, age, sex, and diagnostic leucocyte count on DFS was assessed by multivariate analysis with Cox regression [Forward conditional, statistical package for the social sciences (spss) software, version 11·0] (Cox, 1972).

Patients

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Patients
  6. MSP results
  7. Clinicopathological correlations of aberrant gene methylations
  8. Survival and prognostic analysis
  9. Detection of minimal residual leukaemia
  10. Discussion
  11. Acknowledgments
  12. Note added in proof
  13. References

There were 13 male and 16 female patients, at a median age of 35 years (18–76 years). The median presentation leucocyte count was 4·3 × 109/l (1·6–78 × 109/l). Twenty-eight patients were of the classical hypergranular subtype, and one patient had a microgranular subtypes.

MSP results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Patients
  6. MSP results
  7. Clinicopathological correlations of aberrant gene methylations
  8. Survival and prognostic analysis
  9. Detection of minimal residual leukaemia
  10. Discussion
  11. Acknowledgments
  12. Note added in proof
  13. References

Eight normal marrow donors were tested and were negative for methylation in all the nine genes tested (Fig 1). The specificity of the MSP was shown by DNA sequencing (Fig 2). All results from duplicate experiments were concordant. Of the 29 APL patients, aberrant methylation of p15, ER, RARβ, p16 and ECAD occurred, respectively, in 23 patients (79%), 14 patients (48%), six patients (21%), six patients (21%) and two patients (7%) at diagnosis. None of the patients showed methylation of p73, VHL, CASP8 and MGMT. Apart from three who that did not show aberrant methylation, hypermethylation occurred in one gene in 13 patients (45%), two genes in four patients (14%), three genes in six patients (21%) and four genes in three (10%) patients (Table II).

image

Figure 1. MSP of E-CAD, RARβ and ER genes. (A) M- and U-MSP for E-CAD. MW: molecular weight marker; lanes 1–4: M- and U-MSP of four different primary APL samples showing no amplifications by M-MSP despite good DNA quality as shown by positive amplification in U-MSP; N1, N2: normal marrow DNA showing no M-MSP amplification; P: methylated control DNA showing positive M-MSP amplification; B: reagent blank. (B) M-MSP for RARβ. Lanes 1–4: primary APL marrow DNA with positive amplification for the methylated allele; N: normal DNA showing no amplification; P: methylated control DNA showing positive amplification; MW: molecular weight marker; B: blank. (C) M-MSP for ER. Lanes 1–4: primary APL marrow DNA with positive amplification for the methylated allele; N: normal DNA showing no amplification; P: methylated control DNA showing positive amplification; B: blank. (D) Sensitivity of M-MSP for RARβ. Positive amplification was discernable up to 10−3 dilution. M: molecular weight marker; B: blank.

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image

Figure 2. Specificity of MSP for p73 and ER. DNA sequence of the wild-type (WT) allele is aligned and compared with the methylated allele (Me). Methylated cytosine residues in CpG dinucleotides remained as ‘C’ whereas unmethylated cytosine was read as ‘T’ after bisulphite conversion.

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Table II.  Methylation profile of the 29 APL patients at diagnosis.
Patientp15 * ERRARβp16 * E-CADp73VHLCASP8MGMTn
  1. * p15 and p16 methylation status of 20 patients have previously been reported (Chim et al, 2001a).

  2. n, number of genes that were methylated.

1++++4
2++++4
3++++4
4+++3
5+++3
6+++3
7+++3
8+++3
9+++3
10++2
11++2
12++2
13++2
14+1
15+1
16+1
17+1
18+1
19+1
20+1
21+1
22+1
23+1
24+1
25+1
26+1
270
280
290

Clinicopathological correlations of aberrant gene methylations

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Patients
  6. MSP results
  7. Clinicopathological correlations of aberrant gene methylations
  8. Survival and prognostic analysis
  9. Detection of minimal residual leukaemia
  10. Discussion
  11. Acknowledgments
  12. Note added in proof
  13. References

No association was identified between the patterns of aberrant gene methylation and clinicopathological features at presentation, including age, sex and diagnostic leucocyte count.

Survival and prognostic analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Patients
  6. MSP results
  7. Clinicopathological correlations of aberrant gene methylations
  8. Survival and prognostic analysis
  9. Detection of minimal residual leukaemia
  10. Discussion
  11. Acknowledgments
  12. Note added in proof
  13. References

There was no significant impact of RARβ, ER and E-CAD methylation on the median or projected OS and DFS of the 29 APL patients studied. For p15 and p16, the data of six other patients previously reported (Chim et al, 2001a) were combined with the data of the 29 patients currently studied. Aberrant methylation of p15 had a significant impact on DFS (P = 0·008) (Fig 3) but not OS (P = 0·88), and remained the only significant prognostic factor in multivariate analysis together with other variables including age, sex and diagnostic leucocyte count (P = 0·019). The methylation status of p16 had no prognostic effect.

image

Figure 3. DFS of APL patients treated with ATRA, with respect to aberrant p15 gene methylation. Patients with p15 methylation had a significantly inferior survival when compared with those without p15 methylation.

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Detection of minimal residual leukaemia

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Patients
  6. MSP results
  7. Clinicopathological correlations of aberrant gene methylations
  8. Survival and prognostic analysis
  9. Detection of minimal residual leukaemia
  10. Discussion
  11. Acknowledgments
  12. Note added in proof
  13. References

The sensitivity of MSP was 1 × 10−3 for ER and RARβ, and 1 × 10−5 for p15 (Fig 1). Three patients were evaluated serially with MSP for p15, ER and RARβ methylation at diagnosis and follow-up (Table III). The MSP for p15 apparently had a higher sensitivity in detecting minimal residual leukaemia. In patient 2, the MSP for p15 remained positive at 3 months (but not ER and RARβ), heralding an ultimate relapse.

Table III.  Minimal residual disease study by M-MSP of p15, ER and RARβ.
PatientAberrantly methylated genesPML/RARα
p15ERRARβ
  1. CR, complete remission; NA, not available.

Patient 1
Diagnosis++++
1 month (CR)+++NA
2 months (CR)+NA
11 months (CR)
Patient 2
Diagnosis++++
1 month (CR)NA
3 months (CR)+
11 months (relapse)++++
Patient 3
Diagnosis++++
1 months (CR)++NA
2 months (CR)
5 months (CR)

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Patients
  6. MSP results
  7. Clinicopathological correlations of aberrant gene methylations
  8. Survival and prognostic analysis
  9. Detection of minimal residual leukaemia
  10. Discussion
  11. Acknowledgments
  12. Note added in proof
  13. References

Although aberrant gene promoter methylation is increasingly recognized to be important in carcinogenesis, the mechanism of how this is mediated remains unclear. Increased levels of DNA methyltransferases (DNMT) in leukaemias (Mizuno et al, 2001) and colon cancers (Issa et al, 1993) have been found. However, if this were the only mechanism of aberrant methylation, gene methylation would very probably be non-specific, meaning that most of the genes possessing promoters with CpG dinucleotides would be methylated. Instead, our observations and those of other studies showed that a specific methylation profile existed for a particular tumour (Costello et al, 2000; Esteller et al, 2001), which argued against a general increase in gene methylation. Indeed, global hypomethylation has been found in tumours, together with specific hypermethylation of selected genes (Leonhardt & Cardoso, 2000).

In this study, we showed that p15, ER and RARβ were frequently methylated in APL, whereas the other genes (p16, p73, VHL, MGMT, CASP8) were rarely or not methylated. None of the normal control marrow samples showed methylation of any of the genes tested. Although the normal counterpart of the APL cells, i.e. blasts/promyelocytes, or CD34+ cells might make better normal controls, these cells account for about 1% of cells in the marrow. If gene methylation had occurred in these cells, it would have been detected by MSP, which had a sensitivity of at least 10−3. Therefore, it might be concluded that aberrant gene methylation in the APL samples had actually occurred during leukaemogenesis.

The observed pattern of aberrant gene methylation in APL was in contrast to the results found in other solid tumours, where aberrant methylations of VHL (Brauch et al, 2000), MGMT (Esteller et al, 2001) and CASP8 (Teitz et al, 2000) are frequent. The preferential methylation of specific genes has also been reported in a previous study of AML, which showed that while concurrent methylation of multiple genes, including calcitonin, p15, ER, HIC1 and E-CAD, might occur, Rb and GSTP1 were protected from methylation (Baylin et al, 1998). Therefore, the methylation patterns of myeloid malignancies appear to be different from those of many solid tumours, where frequent methylation of p16, MGMT (colon cancer and glioma) and GSTP1 occurs, while p15 is infrequently methylated (Esteller et al, 2001).

APL is a distinct pathological subtype of AML, both pathologically in its possession of the t(15;17) and the fusion gene PML/RARα, and clinically in its unique response to ATRA and arsenic trioxide. Our results showed that the methylation profile of APL also appeared to be different from other AML subtypes. Although the frequencies of methylation of p15, p16 and ER methylation are comparable to AML in general (Melki et al, 1999; Chim et al, 2001b;Toyota et al, 2001), E-CAD methylation was found in only 7% of APL, as compared with 32–78% in other subtypes of AML (Melki et al, 1999; Corn et al, 2000) (Table IV). Furthermore, RARβ methylation has not been described previously in AML (Melki et al, 1999; Chim et al, 2001b;Toyota et al, 2001), but was found in nearly a quarter of the APL patients in this study. The clinical and biological implications of a different methylation profile in APL compared with other subtypes of AML will need to be investigated.

Table IV.  Methylation frequencies of p15, p16, ER, E-CAD and RARβ in acute myeloid leukaemia (AML) and acute promyelocytic leukaemia (APL).
 Subtypesp15p16ERE-CADRARβ
  1. NS, not specified.

Melki et al (2000)NS68%30%54%69%
Corn et al (2000)NS32%
Toyota et al (2001)NS31%17%56%
Chim et al (2001b)AML except APL93%
Present studyAPL79%21%48%7%21%

The high frequency of aberrant methylation of multiple genes in APL suggests that this might be important in leukaemogenesis. In transgenic mouse models of APL, PML/RARα expression leads to an APL-like syndrome, but frank leukaemia develops only after a latent period of 6 to 12 months, implying that other mutational events might be involved. Interestingly, a recent study showed that the expression of PML/RARα resulted in methylation of RARβ by the recruitment of DNA methyltransferase (Di Croce et al, 2002). RARβ methylation was responsible for a block of differentiation of the neoplastic promyelocytes. Importantly, retinoic acid treatment resulted in RARβ promoter demethylation, gene re-expression and reversal of the transformed phenotype. These results showed a direct mechanistic link between genetic and epigenetic changes in the pathogenesis of APL, which might explain the observed methylation of selected genes. Accordingly, the genes that are aberrantly methylated would be those that contribute directly to leukaemogenesis, implying that the definition of the methylation profile might disclose genes critically involved in the disease evolution of APL. Such a hypothesis would need to be tested in the future.

Data on prognostic factor in APL are scanty. Previous studies of prognostic factors in APL patients receiving ATRA as primary induction therapy have shown that a high diagnostic leucocyte count (Asou et al, 1998; Sanz et al, 2000; Tallman et al, 2002), low diagnostic platelet count (Sanz et al, 2000), advanced age (> 30 years) (Asou et al, 1998), male sex (Tallman et al, 2002) and the microgranular variant (Tallman et al, 2002) are poor prognostic factors for relapse- or disease-free survivals. Moreover, our previous study showed that p15 methylation was a potential negative prognostic marker in a group of APL patients treated with chemotherapy or ATRA for induction of remission (Chim et al, 2001a). In the current study, we have restricted prognostic analysis only to patients receiving ATRA, which has now superseded combination chemotherapy as the induction therapy (Chim et al, 1996; Tallman et al, 1997). Our results showed that aberrant p15 methylation was significantly associated with an inferior DFS. However, the OS was not affected, owing to the effective salvage of relapsed patients with arsenic trioxide. This observation, if validated by prospective studies of larger numbers of APL patients treated with ATRA, will be of importance in the planning of postremission therapy.

Finally, the MSP sensitivity of the different genes ranged from 10−3 to 10−5, suggesting that this might be a useful surrogate marker for the detection of minimal residual leukaemia (Melki et al, 1999; Wong et al, 2000). The clinical utility of MSP in comparison with detection of PML/RARα for monitoring of APL will need to be further investigated.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Patients
  6. MSP results
  7. Clinicopathological correlations of aberrant gene methylations
  8. Survival and prognostic analysis
  9. Detection of minimal residual leukaemia
  10. Discussion
  11. Acknowledgments
  12. Note added in proof
  13. References

We thank the members of the Department of Pathology for the diagnosis, and the Kadoorie Charitable Foundation and the University Department of Medicine for funding support.

Note added in proof

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Patients
  6. MSP results
  7. Clinicopathological correlations of aberrant gene methylations
  8. Survival and prognostic analysis
  9. Detection of minimal residual leukaemia
  10. Discussion
  11. Acknowledgments
  12. Note added in proof
  13. References

A recent study also reported a negative impact of p15 methylation on DFS in APL. [Teofili, L., Martini, M., Luongo, M., Diverio, D., Capelli, G., Breccia, M., Lo Coco, F., Leone, G. & Larocca, L.M. (2003). Hypermethylation of GpG islands in the promoter region of p15(INK4b) in acute promyelocytic leukemia represses p15 (INK4b) expression and correlates with poor prognosis. Leukemia, 17, 919–924.]

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  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Patients
  6. MSP results
  7. Clinicopathological correlations of aberrant gene methylations
  8. Survival and prognostic analysis
  9. Detection of minimal residual leukaemia
  10. Discussion
  11. Acknowledgments
  12. Note added in proof
  13. References
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