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Journal of Clinical Oncology, Vol 19, Issue 7 (April), 2001: 2033-2040
© 2001 American Society for Clinical Oncology

Methylation of p15 and p16 Genes in Acute Promyelocytic Leukemia: Potential Diagnostic and Prognostic Significance

By C.S. Chim, R. Liang, C.Y.Y. Tam, Y.L. Kwong

From the University Department of Medicine, Queen Mary Hospital, University of Hong Kong, Hong Kong.

Address reprint requests to James C.S. Chim, MD, Senior Medical Officer, Department of Medicine, Queen Mary Hospital, Hong Kong; email: jcschim{at}hkucc.hku.hk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PURPOSE: To investigate the frequency of p15 and p16 gene promoter methylation in acute promyelocytic leukemia (APL), and to define its value in the detection of minimal residual disease (MRD) and treatment prognostication.

PATIENTS AND METHODS: Bone marrow DNA obtained from 26 patients with APL at diagnosis and during follow-up was studied with the methylation-specific polymerase chain reaction (MS-PCR). Serial marrow DNA was studied by MS-PCR for MRD, and disease-free and overall survival were correlated with p15 methylation status at diagnosis.

RESULTS: MS-PCR for p16 and p15 gene methylation has a maximum sensitivity of 10-4 and 10-5. At diagnosis, 19 patients (73.1%) exhibited p15 methylation, whereas only three patients (11.5%) exhibited p16 methylation, all of whom had concomitant p15 methylation. During follow-up, p16 methylation was acquired in two patients, one during the third hematologic relapse, and the other during transformation into therapy-related myelodysplastic syndrome. Six patients were evaluated serially with MS-PCR for p15 methylation at diagnosis and at follow-up examinations. Persistent p15 methylation preceded subsequent hematologic relapses in two patients, and conversion to negative MS-PCR for p15 methylation correlated with prolonged survival in another four patients. The 5-year disease-free survival of patients with p15 methylation was significantly inferior to that of patients without p15 methylation (15% v 62.5%; P = .02), and this remained significant in multivariate analysis.

CONCLUSION: In APL, p15 but not p16 gene methylation is frequent. It is possible that p16 methylation is acquired during clonal evolution. p15 methylation is a potential marker of MRD and might be of prognostic significance.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ACUTE PROMYELOCYTIC leukemia (APL) is characterized by t(15;17) that involves the promyelocytic leukemia (PML) gene on chromosome 15q and the retinoic acid receptor alpha (RAR{alpha}) gene on chromosome 17q.1,2 The translocation results in the generation of a chimeric PML-RAR{alpha} gene that is thought to be responsible for leukemogenesis. Clinically, APL patients usually present at a relatively young age, with a low leukocyte count, and with coagulopathy, which accounts for the high induction mortality associated with conventional chemotherapy.1,2 All-trans retinoic acid (ATRA)3-5 and arsenic trioxide6,7 recently have been demonstrated to induce differentiation and apoptosis6,7 of the abnormal promyelocytes. In addition, the use of ATRA for induction therapy has been demonstrated to reduce the hemorrhagic complications associated with chemotherapy.4,5 Patients also can be monitored for minimal residual disease (MRD) in the bone marrow or peripheral blood by reverse-transcriptase polymerase chain reaction (RT-PCR) for the PML-RAR{alpha} transcript.8,9

Apart from various cellular oncogenes, dysregulation of tumor suppressor genes is an important event in the pathogenesis of cancer. Tumor suppressor genes, especially those involved in cell cycle regulation,10 frequently are inactivated in a variety of cancers. CDKN2A (p16INK4A, MTS1) and CDKN2B (p15INK4B, MTS2) are two closely linked tumor suppressor genes located at 9p21, which encode for p16 and p15 cyclin-dependent kinase inhibitors, respectively. They belong to the INK4 kinase family of cyclin-dependent kinase inhibitors (which consists of p15, p16, p19, and p21), and they negatively regulate the cell cycle through competitive inhibition of the cyclin-dependent kinases 4 and 6 involved in Rb-dependent cell cycle regulation. Although most tumor suppressor genes such as Rb and p53 are inactivated by point mutation in one allele and deletion of the homologous allele by chromosomal loss, the major mechanism of p15 gene inactivation in acute myelogenous leukemia (AML) is methylation of the 5' promoter region of the gene, which leads to transcription silencing.11,12 Homozygous deletion or intragenic mutation of p15 in AML is rare.13-16 Similarly, p16 inactivation by homozygous deletion or intragenic mutations also is uncommon in AML, although this occurs in approximately 30% of acute lymphoblastic leukemia cases.16-18

Recent studies have demonstrated that p15 but not p16 gene inactivation by promoter region methylation occurs frequently in AML.13-15,19-21 Few studies, however, have investigated the role of p15 and p16 methylation in different types of AML. In general, AMLs are heterogeneous, and the many different subtypes each have peculiar molecular mechanisms, so the contributory role of p15 and p16 gene methylation might be different. APL is a well-defined clinical and molecular subtype of AML, and it is the only AML subtype that responds to differentiation therapy. Furthermore, as yet there is no reliable molecular prognostic indicator for APL. Therefore, we examined the frequency of p15 and p16 gene methylation in this leukemia in the largest series to date. We further addressed the hypothesis that p15 and p16 gene methylation might be of value in the detection of MRD as well as in the prognostication of APL.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
The diagnosis of APL was based on typical morphologic characteristics22 and confirmed by the presence of t(15;17) cytogenetically and/or PML-RAR{alpha} molecularly.8,9 From 1985 to 1992, APL patients received induction therapy with cytarabine 100 mg/m2/d for 7 days and daunorubicin 50 mg/m2/d for 3 days. From 1992 onward, patients received ATRA 45mg/m2/d orally in two divided doses until complete remission (CR) or for a total of 6 weeks.4 After CR was attained, all patients (in both groups of patients induced with cytarabine and daunorubicin or ATRA) received two courses of consolidation therapy with cytarabine 100 mg/m2/d for 5 days, daunorubicin 50 mg/m2/d for 2 days, and either etoposide 75 mg/m2/d or thioguanine 100 mg/m2/d for 5 days. Relapsed patients received ATRA, arsenic trioxide, or chemotherapy depending on their initial therapy.4

Methylation-Specific Polymerase Chain Reaction (MS-PCR)
At diagnosis and/or follow-up examinations, high molecular weight genomic DNA was isolated from bone marrow aspirates by use of standard protocols. The MS-PCR for p15 and p16 promoter methylation was performed as described previously.23 Each sample was amplified with two sets of primers, one set for methylated DNA (methylated MS-PCR) and one set for unmethylated DNA (unmethylated MS-PCR). Treatment of DNA with bisulphite for conversion of unmethylated cytosine to uracil (but without an affect on methylated cytosine) was performed with a commercially available CpGenome DNA modification kit (Intergen, New York, NY) according to the manufacturer’s instructions. The primers used for the methylated and unmethylated p15 and p16 gene promoter regions were as reported previously ( Table 1).23 Bisulphite-treated DNA (1 µg) was amplified in duplicate by MS-PCR, and the PCR products were analyzed by polyacrylamide gel electrophoresis and ethidium bromide staining. The specificity of the MS-PCR was verified by direct DNA sequencing in selected cases. DNA from 10 normal donors was used as the negative control, and CpGenome Universal methylated control DNA (Intergen) was used as the positive control in all the experiments. PCR conditions for the four sets of reactions were as follows: 95°C for 10 minutes; then 40 cycles at 95°C for 1 minute, 61°C (p15methylated, p15unmethylated, and p16unmethylated) or 66°C (p16methylated) for 1 minute, and 72°C for 1 minute; and a final extension of 10 minutes at 72°C. The PCR mixture contained 100 ng of bisulfite-treated DNA, 0.25 mmol/L of deoxynucleoside triphosphate, 1.5 mmol/L of MgCl2, 50 pmol of each primer, 1 x PCR Buffer II, and 2.5 units of AmpliTaq Gold (Perkin-Elmer Corp, Foster City, CA) in a final volume of 50 µL. PCR was performed in a GeneAmp PCR System 9600 (Perkin-Elmer Corp). Finally, 10 µL of PCR products were loaded onto 6% nondenaturing polyacrylamide gels, stained with ethidium bromide, and visualized under ultraviolet.


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  		Table 1. Primer Sequences for Methylation-Specific PCR and RT-PCR for PML-RAR{alpha}
 
Sequencing
The identity of the methylated and unmethylated sequences was confirmed by automated DNA sequencing. PCR products were electrophoresed in a 2% agarose gel. DNA was recovered from the agarose with the Qiaex II Gel Extraction Kit (Qiagen, Inc, Valencia, CA). Sequencing reactions containing 50 ng of PCR products, 5 pmol of sequencing primer (the same as mentioned above), and 8 µL of ABI Prism dRhodamine Terminator Cycle Sequencing Ready Reaction Mix (Perkin-Elmer Corp) were performed according to manufacturer’s instructions. The sequencing reaction products were purified by the DyeEX Spin Kit (Qiagen, Inc), dried by vacuum, and resuspended in 3 µL of loading buffer that contained blue dextran and formamide in 1:5 ratio. A 377 ABI prism automatic sequencer (Perkin-Elmer Corp) was used according to the manufacturer’s instructions to analyze 1.5 µL of sequencing products. Methylated and unmethylated DNA samples were sequenced in both directions.

Sensitivity of MS-PCR
One microgram of DNA of the methylation positive control (Intergen) was diluted serially (in serial 10-fold dilutions) in normal DNA, modified by bisulphite, and amplified with primers for methylated and unmethylated p15 and p16 sequences. Similarly, p15 MS-PCR sensitivity also was assessed in three patients’ samples that tested positive for p15 methylation. In these cases, the blast and promyelocytes constituted 64% to 78% of the nucleated cells in the marrow.

Nested RT-PCR for PML-RAR{alpha}
Total RNA was extracted from the buffy coat of peripheral blood or bone marrow by the standard guanidine thiocyanate and phenol/chloroform method.24 In brief, total RNA (1 µg) was denatured at 65°C and annealed at 42°C with 5 pmol of the 3' primer (of the first-round PCR). cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase (Gibco BRL, Gaithersburg, MD). The PCR was conducted using primers specific for PML-RAR{alpha} (Table 1). Integrity of RNA was demonstrated by successful amplification of the ß2-microglobulin in all cases.

Statistical Analysis
CR was defined as the complete disappearance of abnormal promyelocytes in normocellular bone marrow, with recovery of peripheral blood counts (hemoglobin > 10g/dL, platelet > 100 x 109/L, and neutrophil > 1.5 x 109/L), and normalization of coagulation parameters. Disease-free survival (DFS) was measured from the date of CR to the date of last follow-up examination, death, or relapse. Overall survival (OS) was measured from the date of diagnosis to the date of last follow-up examination or death. Survival was censored at the time of bone marrow transplantation. Survival was plotted by the Kaplan-Meier method and compared by the log-rank test.25 Prognostic factors (p15 methylation, age, sex, initial leukocyte count, and morphologic subtypes) predictive of DFS were analyzed forward stepwise by the Cox proportional hazard model26 using SPSS version 10.0 software (SPSS, Inc, Chicago, IL). To confirm that there was no difference in the distribution of risk factors for patients with and without p15 methylation, correlation between p15 methylation status and other clinical parameters (age, sex, presenting leukocyte count, use of ATRA) were analyzed by Fisher’s exact test. Because the use of ATRA for induction therapy might influence survival, the log-rank test was used to compare DFS and OS for patients who did and did not receive ATRA as induction therapy. All P values were two sided.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patients and Controls
Twenty-six APL patients were analyzed at the time of diagnosis. Their median age was 28 years (range, 18 to 70 years), and the median leukocyte count was 6.8 (range, 1 to 22) x 109/l. The demographic data of patients with and without p15 gene methylation are detailed in Table 2. There was no significant difference in the clinical characteristics and the treatment regimen between patients with and without p15 methylation.


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  		Table 2. P15 Gene Promoter Methylation
 
p15 and p16 Methylation
Ten normal donors were tested and the results all were negative for p15 and p16 methylation ( Fig 1). The specificity of the MS-PCR was demonstrated by DNA sequencing ( Fig 2). DNA sequence of methylated control DNA (p15 methylated) was aligned and compared with germline sequence of wild-type p15 DNA. Methylated cytosine residues in CpG dinucleotide remained as "C" whereas unmethylated cytosine read as "T" after bisulphite conversion. The last nucleotides "TGT" demonstrated in the methylated DNA sequence were at position -170 from the p15 initiation methionine (ATG) codon (Genbank accession number, S75756).



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  		Fig 1. MS-PCR for methylated control DNA and normal DNA with primer sets for methylated (methylated MS-PCR) and unmethylated (unmethylated MS-PCR) DNA. Lanes: M, molecular weight marker; P, methylated MS-PCR for methylated control DNA showing positive result; NU, unmethylated MS-PCR with normal DNA demonstrating positive result; NM, methylated MS-PCR with normal DNA showing no amplification; B, regent blank.

 


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  		Fig 2. Sequencing of bisulphite converted methylated control DNA by forward primer for methylated MS-PCR. The DNA sequence of methylated control DNA (p15M) is aligned and compared with germline sequence of wild-type p15 DNA. Methylated cytosine residues in CpG dinucleotide are underlined; the reverse primer is indicated by bold italics.

 
Of 26 patients evaluated at diagnosis, 19 (73.1%) tested positive for p15 methylation ( Fig 3). Methylated and unmethylated MS-PCR for p15 yielded PCR products with 148 and 154 base pairs (Fig 3), and methylated and unmethylated MS-PCR for p16 produced PCR products with 150 and 151 base pairs. Only three cases (11.5%) were positive for p16 methylation, however, and all three demonstrated concomitant p15 methylation. One patient who tested negative for p16 methylation at diagnosis and at first and second relapses demonstrated p16 methylation at the third relapse. Another patient developed pancytopenia 7 years afterward; bone marrow exhibited refractory anemia with excess blasts in transformation, and karyotypic analysis revealed deletion of chromosomes 5q and 7.27 These developments were consistent with therapy-related myelodysplastic syndrome (MDS). This patient tested negative for both p15 and p16 methylation at diagnosis but acquired both p15 and p16 methylation at the diagnosis of therapy-related MDS.



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  		Fig 3. Methylated MS-PCR for p15. Lanes: M, molecular weight marker; P, methylated control DNA showing positive amplification; N, normal DNA showing no amplification; 1 to 5, primary APL marrow DNA; B, reagent blank.

 
Sensitivity of Methylated MS-PCR
For the positive control, the sensitivity of MS-PCR for p15 methylation was 1 x 10-5 ( Fig 4A), and that of the p16 gene was 1 x 10-4. For the three patients’ samples, the sensitivity of p15 MS-PCR ranged from 1 x 10-3 to 1 x 10-4 (Fig 4B).



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  		Fig 4. (A) Sensitivity of methylated MS-PCR for p15. Lanes: M, molecular weight marker; N, normal DNA showing no amplification; 1 to 6, serial dilution of methylated control DNA from 100 to 10-5; B, reagent blank. (B) Serial dilutions of test sample DNA with marrow blast and promyelocytes accounting for 64% and 78% of all nucleated marrow cells, respectively. Lanes: 1 to 5, (100 to 10-4); 7 to 12, (100 to 10-5), M, molecular weight marker; N, normal DNA showing no amplification; B, reagent blank.

 
MRD by p15 MS-PCR
Sequential marrow DNA from six patients who had p15 methylation at diagnosis was used for MRD analysis, which demonstrated results highly concordant with data from clinical and morphologic analyses ( Fig 5). In two patients, persistent p15 methylation during morphologic remission at 2 and 3 months after diagnosis occurred before frank hematologic relapse 2 and 8 months later. In two patients, persistent p15 methylation occurred posttreatment despite marrow morphologic remission. Both patients became MS-PCR negative after allogeneic bone marrow transplantation, however, and they have remained in CR since then. In two patients, p15 methylation was transiently present at 1 and 2 months after induction therapy but disappeared after further treatment. Both patients have remained in CR 12 and 55 months later. Of the 29 samples from these six patients, 13 had corresponding RT-PCRs for PML-RAR{alpha}. Twelve (92%) of the 13 RT-PCR results concurred with the p15 methylated MS-PCR results. In the only discordant result, MS-PCR seemed more accurately predictive of ultimate relapse than RT-PCR.



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  		Fig 5. MRD by methylated MS-PCR for p15 methylation and RT-PCR PML-RAR{alpha}. All tests were performed on marrow DNA samples. (•) presence of p15 methylation DNA during CR; ({circ} absence of p15 methylation in CR; ({star}) presence of p15 methylation at frank hematologic relapse; (+) presence of PML-RAR{alpha}; (–) absence of PML-RAR{alpha}.

 
Survival and Prognostic Factor Analysis
The 5-year DFS was significantly inferior in patients with p15 methylation compared with those without p15 methylation (15% v 62.5%; P = .02)( Fig 6A). Conversely, the 5-year OS was not significantly different (80% v 100%; P = .25). The projected DFS (P = .54) (Fig 6B) and OS (P = .83) of patients who received induction therapy with ATRA or combined cytarabine and daunorubicin were not statistically different. In a multivariate analysis of the prognostic impact of various factors on DFS (p15 methylation status, age, sex, and initial leukocyte count), only p15 methylation remained statistically significant (P = .03)



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  		Fig 6. (A) DFS for patients with and without p15 methylation in the diagnostic marrow DNA (log-rank test; P = .02). (B) DFS for patients who received ATRA or combination cytarabine and daunorubicin as induction therapy (P = .54).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In our study, 73% of patients had methylation of the p15 promoter. This frequency was comparable with previous reports that demonstrated p15 methylation in 61% to 94% of all AML subtypes.13-15,19,20 However, few studies have addressed p15 methylation in APL specifically. One study reported p15 methylation in 100% of APL cases, but only three cases were analyzed.19 Our series, therefore, is the largest to date that examined p15 methylation in APL specifically. The high frequency of p15 methylation found in our study implied that it might play an important role in leukemogenesis. In transgenic models,28,29 PML-RAR{alpha} expression leads to an APL-like syndrome, but frank leukemia develops only after a latent period of 6 to 12 months; this implies that other mutational events might be involved in conjunction with PML-RAR{alpha} that result in the leukemia phenotype. Our results suggest that inactivation of p15 through promoter methylation may be one possible mechanism.

In this series, p16 gene methylation occurred at a low frequency, which was consistent with previous studies that demonstrated rare p16 methylation in other types of AML.14,15 All of our patients with p16 methylation demonstrated concomitant p15 methylation. One other study also reported concomitant p15 methylation in all seven adult AML M2 and M4 patients with p16 methylation, but not in any of three APL patients.19 The results suggest that p16 inactivation through methylation probably is not an important primary event in leukemogenesis, but it may play a contributory role in the further enhancement of cellular proliferation. It is of interest that one of our patients, who tested negative for p16 methylation at diagnosis, acquired methylation at relapse. Thus, p16 methylation seemed to be a marker of clonal progression in this patient. This observation is consistent with previous studies demonstrating that acquisition of p16 methylation is associated with disease progression in adult T-cell leukemia30 and other types of invasive or metastatic malignancies.31-33 This point was further demonstrated in another patient in our series who developed MDS and acquired both p15 and p16 methylations at transformation. This phenomenon also was observed in leukemic transformation of other types of MDS.34

We have evaluated further whether p15 gene methylation might be a target for detection of MRD. In this study, the sensitivity of p15 methylated MS-PCR from serial dilutions of the control DNA was 1 x 10-5. Use of the patients’ marrow samples obtained at diagnosis resulted in lower sensitivity (1 x 10-3 to 10-4). As a test for MRD in leukemia, the sensitivity of MS-PCR is intermediate between two other strategies, on the basis of PCR of a rearranged immunoglobulin/T-cell receptor gene (1 x 10-3 to 10-5)35-37 and RT-PCR for leukemia-specific fusion transcripts (1 x 10-5 to 10-6).8,9,37 In this study, results of MS-PCR correlated well with clinical and pathologic features of disease status. Furthermore, 92% of the samples with concurrent p15 MS-PCR demonstrated concordant results with RT-PCR for PML-RAR{alpha}. This was expected, as both p15 methylated MS-PCR (10-3 to 10-5) and RT-PCR (10-3 to 10-6) had comparable sensitivity.9 In leukemias for which no leukemia-specific molecular markers are available, MS-PCR for p15 methylation may provide an alternative strategy to monitor MRD. In leukemias in which specific markers such as fusion transcripts are present, serial MS-PCR for p15 methylation may serve as a marker of clonal progression if the methylation is acquired during the course of the disease. Furthermore, the requirement of DNA instead of RNA for RT-PCR of fusion transcript makes for easier specimen handling and logistics. Therefore, the potential role of MS-PCR for p15 methylation warrants verification in future prospective studies and should be extended to other AMLs in which translocation and fusion transcript is less frequent.

Finally, we have demonstrated that APL patients with p15 methylation have an inferior DFS. OS was not significantly different because of the highly effective salvage regimen in APL (ATRA, arsenic trioxide, and chemotherapy). Although we did not examine other mechanisms of gene inactivation, such as homozygous deletion and intragenic mutation, these are virtually nonexistent in AML; we think that p15 gene methylation is sufficient to inactivate the p15 gene and it confers prognostic value. The negative prognostic impact of p15 methylation has been demonstrated in solid tumors such as sarcomas38 as well as in childhood acute lymphoblastic leukemia39 and other types of adult leukemia.19 Although the primary objective of this study was not to evaluate the efficacy of ATRA for APL, we noticed that the 28% DFS at 97 months in ATRA-treated patients was inferior to that reported in other clinical trials. The projected DFS for patients who received ATRA as induction therapy, followed by chemotherapy consolidation, has been reported to range from 62% at 4 years to 92% at 2 years.40-43 There may be several reasons for the inferior DFS observed in our patients. First, we studied only APL patients with diagnostic DNA samples, and not all consecutive patients were included. Second, the follow-up time for our patients was long, and some relatively late relapses occurred that might not have been included in previous studies. However, the standard APL risk factors in our study patients, such as median age, presenting leukocyte count, and M3 variant, seemed similar to the patterns observed in other reported APL series.40,42,43 Because p15 methylation was found to be an independent adverse prognostic factor in our study and was observed in a high proportion (73%) of our APL patients, it might account for the inferior clinical outcome of our patients. Therefore, the potential prognostic impact of p15 methylation warrants further evaluation in larger prospective studies.

In conclusion, p15 gene methylation might play an important role in leukemogenesis and might potentially serve as a marker of residual leukemia, particularly when RNA is not available to perform RT-PCR for PML-RAR{alpha}. Our findings are the first to document a poorer prognostic impact of p15 methylation in APL in a multivariate analysis. Because the number of patients in our study was small, this observation must be validated in future prospective studies with larger numbers of patients and other prognostic markers, including additional karyotypic aberrations, CD2 expression, and M3 variant. The prognostic significance of p15 methylation also might warrant consideration in the design of an optimal therapeutic strategy for APL that will maximize the curative potential of this leukemia.


   ACKNOWLEDGMENTS
 
Supported by a Conference and Research Grant Committee grant, University of Hong Kong.

We thank Profs David Todd and T.K. Chan for their kind advice and Prof L.C. Chan, Dr Edmond Ma, and Dr Clarence Lam for helping with diagnosis.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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Submitted May 3, 2000; accepted December 14, 2000.


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