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Blood, Vol. 95 No. 6 (March 15), 2000:
pp. 1942-1949
CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Department of Anatomical and Cellular Pathology, Prince of
Wales Hospital, Chinese University of Hong Kong, Hong Kong.
We prospectively analyzed p15 and p16 promoter
methylation patterns using methylation-specific polymerase chain
reaction (PCR) in patients with adult and childhood acute leukemias and
studied the association of methylation patterns with chromosomal
abnormalities and prognostic variables. In nearly all
French-American-British leukemia subtypes, we found p15
methylation in bone marrow or peripheral blood cells from 58% (46/79)
of patients with acute myeloid leukemia (AML), acute lymphoblastic
leukemia (ALL), or acute biphenotypic leukemia (ABL). An identical
alteration was detected in blood plasma from 11 of 12 of these patients
(92%). We also demonstrated for the first time concomitant p16
and p15 methylation in 22% (8/37) of adults with AML or ALL,
exclusively in those with M2, M4, or L2 subtypes. According to
cytogenetic data from 35 patients with ALL, AML, or ABL, 82% (14/17)
of those with unmethylated p15 alleles had normal karyotypes or
hyperdiploidies associated with a favorable prognosis. Conversely, 44%
(8/18) of patients with p15 methylation had chromosomal
translocations, inversions, or deletions, suggesting an interplay of
these abnormalities with p15 methylation. As a prognostic
marker for disease monitoring, p15 methylation appears to be
more widely applicable than BCR-ABL, AF4-MLL, and
AML1-ETO transcripts, which were detectable in only 8% (4/48)
of patients by reverse transcriptase-PCR. Thirty-nine of 43 blood
samples (91%) sequentially collected from 12 patients with AML, ALL,
or ABL showed p15 methylation status in excellent concordance
with morphologic disease stage. Early detection of p15
methylation at apparent remission or its acquisition during follow-up
may prove valuable for predicting relapse. Overall survival of patients
with p15 methylation was notably shortened among 38 adults with
AML and 12 adults with ALL. Aberrant p15 methylation may have
important prognostic implications for clinical monitoring and
risk assessment.
(Blood. 2000;95:1942-1949)
Loss of cell-cycle regulation through changes in the
cyclin D-retinoblastoma (Rb) pathway, is common in human
neoplasia,1 although inactivation of the Rb gene
infrequently occurs in patients with hematologic malignant
disease.2 The p16 and p15 genes, which encode
cyclin-dependent kinase inhibitors, have been recognized as tumor
suppressor genes in solid tumors and hematologic
neoplasms.3-8 Deletions of p16, p15, or
both are commonly found in acute lymphoblastic leukemia (ALL) but
infrequently observed in acute myeloid leukemia (AML).3-5,9-11 Intragenic mutations in p16 and
p15 are only rarely detected in ALL or AML.12,13
The p16/p15 deletion correlates with a high risk of relapse or
death in patients with ALL.14,15
Methylation of the p15/p16 promoter is associated with loss of
transcription in neoplasia.3,7,8,16 Frequent p15
methylation was demonstrated in leukemia cell lines and primary acute
leukemias with use of Southern blot analysis.3 However,
p16 methylation has only rarely been observed in
primary acute leukemias.10,11,17 In acute
leukemias, p16 is predominantly inactivated by homozygous deletion and p15 is mainly inactivated by methylation.
Differential use of p16 or p15 protein in a selective manner has been
implicated, and this is supported by the fact that p16 and
p15 genes are regulated by means of distinct
pathways.17 p16 expression is regulated partly by pRb protein, while Rb expression is in turn
negatively controlled by p53 protein.18-20 There is
evidence that p16 inactivation is closely associated with
aberrant p53 expression, suggesting a collaborating role for
p16 in apoptosis.21,22 Conversely, p15 expression
appears to be independent of pRb but is regulated by the extracellular
growth inhibitors interferon- Multiple chromosomal translocations that aberrantly activate or
deregulate tyrosine kinase or transcription factor genes are remarkably
specific for hematopoietic cells arrested at definitive differentiation
stages in acute leukemias.25-27 The oncogenic fusion genes
may work synergistically with altered tumor suppressor genes in the
multistep process of leukemogenesis. Although the fusion transcripts
may serve as diagnostic and prognostic markers for monitoring minimal
residual disease (MRD), early relapse, and response to therapy, they
are detectable at only low rates in specific morphologic subtypes of
ALL and AML.25-27 In contrast, aberrant p15
methylation is frequently found in adult and childhood ALL and
AML.3,10,11,17 Thus, p15 methylation has potential value as a molecular prognostic marker in acute leukemias. For this
application, methylation-specific polymerase chain reaction (MSP) is
sensitive and specific in detecting epigenetic changes within CpG
dinucleotides that are critical sites for gene silencing.28
In this prospective investigation, we used MSP to analyze p15
and p16 promoter methylation in patients with adult and
childhood acute leukemias of multiple French-American-British (FAB)
classification subtypes and studied the association of p15
methylation with several prognostic variables, including chromosomal
abnormalities, fusion transcripts, and other known risk factors. Also,
we examined cell-free p15 methylation in blood plasma to
investigate its possible biologic implications in relation to the
behavior of leukemic blasts. To assess whether p15 methylation
could be applied as a molecular prognostic marker for detecting MRD or
early relapse, we sequentially monitored p15 methylation status
in peripheral blood from patients with acute leukemias at diagnosis and
during follow-up. The prognostic relevance of p15 methylation
was also evaluated by correlating p15 methylation status with
morphologic disease stage and overall survival. The MSP analysis for
p15 may create exciting possibilities for risk assessment,
early detection of MRD and relapse, and disease monitoring.
Patients and controls
Cytogenetic studies
DNA extraction Peripheral blood was centrifuged at 3000g and plasma was collected from a tube containing EDTA. DNA was extracted from 400 µL of plasma by using the QIAamp blood kit (Qiagen, Hilden, Germany). Buffy coats were isolated from peripheral blood or bone marrow, and genomic DNA was extracted by using standard sodium dodecyl sulfate-proteinase K treatment and phenol-chloroform-isoamylalcohol extraction.MSP and Southern blot analysis Bisulfite treatment of DNA converts unmethylated cytosine residues into uracil,28,30 but methylated cytosine residues remain unmodified. Therefore, methylated and unmethylated DNA sequences can be distinguished by using sequence-specific polymerase chain reaction (PCR) primers. We conducted bisulfite conversion by using the CpGenome DNA modification kit (Intergen, New York, NY). Bisulfite-treated buffy coat DNA (1 µg) or extracted plasma DNA was amplified by using p15MF/p15MR and p16MF/p16MR primer sets specific for the methylated p15 and p16 sequences, respectively (Table 1).28 All bisulfite-treated DNA samples were also amplified by using p15UF/p15UR and p16UF/p16UR primer sets specific for the unmethylated p15 and p16 sequences, respectively. Any unconverted DNA was amplified by using p15WF/p15WR and p16WF/p16WR primer sets specific for the wild-type p15 and p16 sequences, respectively.
Sensitivity of MSP HS-Sultan, which was previously shown to have p15 and p16 methylation on Southern blot analysis, served as a methylated control for MSP.7,30 To determine the sensitivity of the MSP, HS-Sultan DNA was serially diluted in water, mixed with normal blood cell DNA, converted with bisulfite, and then amplified with MSP. For the methylated p15 sequence, the lower detection limit was 0.25 ng or 1 ng of HS-Sultan DNA in 1000 ng of normal blood cell DNA with use of 55 or 35 PCR cycles. For the methylated p16 sequence, the lower detection limit was 0.05 ng or 0.2 ng of HS-Sultan DNA in 1000 ng of normal blood cell DNA with use of 55 or 35 PCR cycles. The sensitivity of MSP for p15/p16 reached 2.5 × 10 4 to
5 × 105.
RNA extraction After the cell pellet was washed in phosphate-buffered saline and subjected to centrifugation, it was resuspended in 0.5 mL of guanidinium thiocyanate solution.32 Total RNA was extracted from peripheral blood, bone marrow, or cell line by using a single-step method.32Reverse transcriptase-PCR (RT-PCR) and Southern blot analysis Total RNA (1-2 µg) was denatured at 65°C for 2 minutes and annealed at 37°C for 10 minutes with 1 µg of random primers (GIBCO-BRL, Gaithersburg, MD).31 Complementary DNA (cDNA) was synthesized at 37°C for 1 hour by using Moloney murine leukemia virus reverse transcriptase (GIBCO-BRL). PCR was conducted by using primers specific for mBCR-ABL or MBCR-ABL (fused at minor or major breakpoints), AF4-MLL, or AML1-ETO fusion transcripts (Table 1). 2-microglobulin cDNA was
amplified as a control to ensure that high-integrity RNA was reverse
transcribed in each reaction. The thermal profile included initial
denaturation at 95°C for 12 minutes, followed by 40 cycles at
95°C for 1 minute, 58°C for 1 minute, and 72°C for 1 minute
and final extension at 72°C for 10 minutes. Each sample was
analyzed in duplicate. The identity of the PCR product was verified by
nonradioactive Southern blot analysis using a BCRABLP, AF4MLLP, or
AML1ETOP probe.31
Statistical analysis The correlation between p15 methylation status and known risk factors and the association of p15 methylation status in buffy coats from bone marrow or peripheral blood with methylation positivity in blood plasma were assessed by the Fisher exact test or 2 test. The association between p15 methylation
status and morphologic disease stage was analyzed by using the McNemar
test. Overall survival durations in different subsets of adults with
acute leukemia with or without p15 methylation at diagnosis
were compared by using the Kaplan-Meier method and log-rank test.
Aberrant p15 methylation in adult and childhood acute leukemias of nearly all morphologic subtypes In this prospective analysis of bone marrow and peripheral blood cells, p15 methylation was detected at diagnosis by MSP in 58% (46/79) of patients with adult or childhood acute leukemias of nearly all the FAB subtypes (Figure 1, panel A). As shown in Table 2, p15 methylation was found in 64% (27/42) of patients with AML, 50% (17/34) of patients with ALL, and 67% (2/3) of patients with ABL. Using MSP, we found higher p15 methylation frequencies in patients with the M3 (3/3 patients), M4 (8/10), M2 (10/13), or M7 (2/3) subtypes than in those with the M1 (2/4 patients), M6 (1/3), or M5 (1/6) subtypes (Figure 2). Comparable frequencies of p15 methylation were found in patients with L1 (8/18 patients) and L2 (8/14) of B lineage and T-ALL of the L2 subtype (1/2). p15 methylation was not detected in blood cells or bone marrow from 12 healthy control subjects.
Cell-free methylated p15 sequences in blood plasma from patients We also examined p15 methylation status in peripheral blood plasma obtained at diagnosis from 16 patients with acute leukemia (9 with AML and 7 with ALL). We detected methylated p15 sequences in plasma from 92% (11/12) of patients who had identical methylation in blood cells or bone marrow (Figure 1, panels B and C). In contrast, p15 methylation was not detected in plasma from 10 healthy subjects or any of the 4 patients without p15 methylation in blood cells or bone marrow. The association of p15 methylation status in blood cells or bone marrow with methylation positivity or negativity in plasma was significant (P = .003 by Fisher exact test).Concomitant p16 and p15 methylation in adult acute leukemias of particular subtypes Aberrant p16 methylation was not detected in bone marrow or peripheral blood cells from 11 patients with childhood AML, ALL, or ABL or 12 healthy control (Table 2). Among 37 adults with acute leukemias, p16 methylation was detected at diagnosis in 7 (M2 or M4 subtype) of 30 patients with AML (23%) and in 1 (L2 of B lineage) of 6 patients with ALL (17%) but not in the patient with ABL (Figure 1, panel D). Notably, p16 methylation occurred concomitantly with p15 methylation in the 8 adults with acute leukemias. Seven of the 8 patients were 50 years old and had WBC counts of < 50 × 109/L (median WBC count,
7.6 × 109/L). The other patient, who was 31 years
old and had the M4 subtype, had a WBC count of
207 × 109/L. Of these 8 patients, 3 (M2 or M4
subtype) died of leukemia within 3 months after diagnosis.
p15 methylation, karyotypes, and fusion transcripts Among 35 patients with adult or childhood AML, ALL, or ABL for whom cytogenetic information was available, 82% (14/17) of patients carrying unmethylated p15 alleles had normal karyotypes or hyperdiploidies associated with good prognosis (Table 3). In contrast, 44% (8/18) of patients with p15 methylation at diagnosis had chromosomal deletions, the inversion inv(16)(p13q22), or one of the following translocations: t(9;22)(q34;q11), t(4;11)(q21;q23), t(7;22)(p15;q23), t(15;17)(q22;q12), or t(8;21)(q22;q22) (P = .088 by2 test). The mBCR-ABL and AF4-MLL
transcripts were detected by RT-PCR in 6% (1/16) and 8% (1/13),
respectively, of patients with ALL (Table 3). AML1-ETO
transcripts were found in 6% (2/32) of patients with AML (Table 3). In
contrast to the low rates of detection of these fusion transcripts,
p15 methylation appeared to be more applicable as a molecular
marker in 56% (27/48) of the same patients. Both methylated
p15 alleles and BCR-ABL transcripts were detected in
blood cells from a patient with ALL in morphologic remission,
suggesting the presence of MRD (Table 3).
Sequential monitoring of p15 methylation in patients During a median follow-up period of 8 months, we used MSP for sequential monitoring of p15 methylation status in 43 peripheral blood samples from 12 patients (9 adults and 3 children) with AML, ALL, or ABL (median time to achieve first remission, 38 days). p15 methylation was detected in all 12 blood samples obtained at diagnosis. During follow-up, p15 methylation was not detected in 9 samples from 5 patients (2 with ALL and 3 with AML) and, for 8 samples, the unmethylated status was in concordance with morphologic remission and lack of residual leukemia (Figure 3, panels A and B). Among these 5 patients, 1 with the M6 subtype had p15 methylation during the active leukemia stage (Figure 3, panel B, lane 9). In an additional 7 patients (5 with AML, 1 with ABL, and 1 with ALL), p15 methylation was detected in 9 samples, and the methylation status of 7 samples was in agreement with morphologic relapse or active or residual leukemia (Figure 3, panels C and D). Conversely, p15 methylation was not detected in 12 samples from these 7 patients, and the unmethylated status of 11 samples correlated with morphologic remission and lack of residual leukemia.
Overall survival of adults with acute leukemias Among 38 adults with AML, the median survival time for the 23 patients with p15 methylation at diagnosis was 13 months, which was apparently shorter than the median survival time of 21 months for the 15 patients with unmethylated p15 alleles (P = .145 by Kaplan-Meier method and log-rank test; Figure 4, panel A). There appeared to be a trend toward improved survival after 21 months among the patients with AML who had unmethylated p15 alleles. Among 12 adults with ALL, the median survival time for 8 patients with p15 methylation at diagnosis was also 13 months, which represented a > 2-fold reduction compared with the 29-month survival time for 4 patients with unmethylated p15 alleles (P = .079 by Kaplan-Meier method and log-rank test; Figure 4, panel B).
For nearly all the FAB subtypes, our studies using MSP demonstrated aberrant p15 promoter methylation in a significant proportion of patients with adult or childhood AML, ALL, or ABL. We clearly depicted p15 methylation patterns in many subtypes arising in different lineages and differentiation stages. With MSP, we confirmed that frequent p15 methylation is likely a crucial event in acute leukemias. Our findings suggest that p15 methylation arises universally and de novo during leukemic transformation and progression in hematopoietic progenitors developing in the myeloid/lymphoid pathway (B or T lineage) or in primitive stem cells with multilineage potential. Lack of correlation between p15 methylation and WBC and bone marrow blast counts might provide evidence suggesting that p15 methylation is one of the early events in leukemogenesis.
We thank our colleagues in the hematology/BMT laboratories and the hematology and oncology units for support during this project and Eric Wong for helpful advice on statistical analysis of the data.
Submitted June 9, 1999; accepted November 18, 1999.
Supported by research grants 2040505 and 2040507 from the Chinese University of Hong Kong.
Reprints: Margaret H. L. Ng, Hematology Section, Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR; e-mail: margaretng{at}cuhk.edu.hk.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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S. Agrawal, M. Unterberg, S. Koschmieder, U. zur Stadt, U. Brunnberg, W. Verbeek, T. Buchner, W. E. Berdel, H. Serve, and C. Muller-Tidow DNA Methylation of Tumor Suppressor Genes in Clinical Remission Predicts the Relapse Risk in Acute Myeloid Leukemia Cancer Res., February 1, 2007; 67(3): 1370 - 1377. [Abstract] [Full Text] [PDF]
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S. H. Tang, D. H. Yang, W. Huang, H. K. Zhou, X. H. Lu, and G. Ye Hypomethylated P4 Promoter Induces Expression of the Insulin-Like Growth Factor-II Gene in Hepatocellular Carcinoma in a Chinese Population. Clin. Cancer Res., July 15, 2006; 12(14): 4171 - 4177. [Abstract] [Full Text] [PDF]
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J. Roman-Gomez, A. Jimenez-Velasco, J. A. Castillejo, X. Agirre, M. Barrios, G. Navarro, F. J. Molina, M. J. Calasanz, F. Prosper, A. Heiniger, et al. Promoter hypermethylation of cancer-related genes: a strong independent prognostic factor in acute lymphoblastic leukemia Blood, October 15, 2004; 104(8): 2492 - 2498. [Abstract] [Full Text] [PDF]
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T. Takahashi, N. Shivapurkar, J. Reddy, H. Shigematsu, K. Miyajima, M. Suzuki, S. Toyooka, S. Zochbauer-Muller, J. Drach, G. Parikh, et al. DNA Methylation Profiles of Lymphoid and Hematopoietic Malignancies Clin. Cancer Res., May 1, 2004; 10(9): 2928 - 2935. [Abstract] [Full Text] [PDF]
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I. H. N. Wong, J. Chan, J. Wong, and P. K. H. Tam Ubiquitous Aberrant RASSF1A Promoter Methylation in Childhood Neoplasia1 Clin. Cancer Res., February 1, 2004; 10(3): 994 - 1002. [Abstract] [Full Text] [PDF]
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I. H. N. Wong, J. Zhang, P. B. S. Lai, W. Y. Lau, and Y. M. Dennis Lo Quantitative Analysis of Tumor-derived Methylated p16INK4a Sequences in Plasma, Serum, and Blood Cells of Hepatocellular Carcinoma Patients Clin. Cancer Res., March 1, 2003; 9(3): 1047 - 1052. [Abstract] [Full Text] [PDF]
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A. El-Osta, M. Lubbert, P. W. Wijermans, T. Licht, and P. A. Jones On the use of DNA methylation inhibitors and the reversal of transcriptional silencing Blood, February 15, 2003; 101(4): 1656 - 1657. [Full Text] [PDF]
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S. Kusy, M. Cividin, N. Sorel, F. Brizard, F. Guilhot, A. Brizard, C. Larsen, and J. Roche p14ARF, p15INK4b, and p16INK4a methylation status in chronic myelogenous leukemia Blood, January 1, 2003; 101(1): 374 - 374. [Full Text] [PDF]
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G. Garcia-Manero, J. Daniel, T. L. Smith, S. M. Kornblau, M.-S. Lee, H. M. Kantarjian, and J.-P. J. Issa DNA Methylation of Multiple Promoter-associated CpG Islands in Adult Acute Lymphocytic Leukemia Clin. Cancer Res., July 1, 2002; 8(7): 2217 - 2224. [Abstract] [Full Text] [PDF]
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G. Garcia-Manero, C. Bueso-Ramos, J. Daniel, J. Williamson, H. M. Kantarjian, and J.-P. J. Issa DNA Methylation Patterns at Relapse in Adult Acute Lymphocytic Leukemia Clin. Cancer Res., June 1, 2002; 8(6): 1897 - 1903. [Abstract] [Full Text] [PDF]
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J. H. Dalle, M. Fournier, B. Nelken, F. Mazingue, J.-L. Lai, F. Bauters, P. Fenaux, and B. Quesnel p16INK4a immunocytochemical analysis is an independent prognostic factor in childhood acute lymphoblastic leukemia Blood, April 1, 2002; 99(7): 2620 - 2623. [Abstract] [Full Text] [PDF]
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D. A. Sweetser, C.-S. Chen, A. A. Blomberg, D. A. Flowers, P. C. Galipeau, M. T. Barrett, N. A. Heerema, J. Buckley, W. G. Woods, I. D. Bernstein, et al. Loss of heterozygosity in childhood de novo acute myelogenous leukemia Blood, August 15, 2001; 98(4): 1188 - 1194. [Abstract] [Full Text] [PDF]
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C.S. Chim, R. Liang, C.Y.Y. Tam, and Y.L. Kwong Methylation of p15 and p16 Genes in Acute Promyelocytic Leukemia: Potential Diagnostic and Prognostic Significance J. Clin. Oncol., April 1, 2001; 19(7): 2033 - 2040. [Abstract] [Full Text] [PDF]
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M. Esteller, P. G. Corn, S. B. Baylin, and J. G. Herman A Gene Hypermethylation Profile of Human Cancer Cancer Res., April 1, 2001; 61(8): 3225 - 3229. [Abstract] [Full Text]
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I. H. N. Wong, Y. M. Dennis Lo, W. Yeo, W. Y. Lau, and P. J. Johnson Frequent p15 Promoter Methylation in Tumor and Peripheral Blood from Hepatocellular Carcinoma Patients Clin. Cancer Res., September 1, 2000; 6(9): 3516 - 3521. [Abstract] [Full Text]
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E. P. Das-Gupta and N. H. Russell Anticorresponding p15 promoter methylation and microsatellite instability in acute myeloblastic leukemia Blood, September 1, 2000; 96(5): 2002 - 2002. [Full Text] [PDF]
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I. H. N. Wong, A. T. Chan, and P. J. Johnson Quantitative Analysis of Circulating Tumor Cells in Peripheral Blood of Osteosarcoma Patients Using Osteoblast-specific Messenger RNA Markers: A Pilot Study Clin. Cancer Res., June 1, 2000; 6(6): 2183 - 2188. [Abstract] [Full Text]
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 O. Galm, M. R. Rountree, K. E. Bachman, K.-W. Jair, S. B. Baylin, and J. G. Herman Enzymatic Regional Methylation Assay: A Novel Method to Quantify Regional CpG Methylation Density Genome Res., January 1, 2002; 12(1): 153 - 157. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
and transforming growth factor-
