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BRIEF REPORT
From the Cancer Genetics Laboratory, Department of
Biochemistry, University of Otago, Dunedin, New Zealand; and Paediatric
Haematology/Oncology, Starship Children's Health, Auckland, New
Zealand.
Germ-line events, such as paternal mutation or genomic imprinting,
contribute to the early onset of childhood cancers such as
retinoblastoma, Wilms tumors, and neuroblastoma. Given the high
frequency of deletion involving chromosome 9p in childhood acute
lymphoblastic leukemia (ALL), this study investigated whether 9p
deletion might reflect preexisting germ-line gene inactivation. To do
this the parental origin of deletion was determined in 10 cases of ALL
with 9p21 loss of heterozygosity. Of these cases, 9 showed loss of the
maternally derived allele, suggesting that a germ-line event involving
a 9p gene may play a role in the onset of childhood ALL.
(Blood. 2002;99:375-377) The young age of onset of childhood cancers can
often be attributed to a constitutional genetic predisposition. In some
cases the predisposition results from the inheritance of a familial mutation, whereas in other cases it reflects the occurrence of de novo
germ-line mutation, as observed in some cases of
retinoblastoma,1 or the involvement of an imprinted gene,
as seen in Wilms tumors and some neuroblastomas.2 Although
childhood acute lymphoblastic leukemia (ALL) is seldom familial, we
hypothesized that a constitutional predisposition might explain its
early age of onset. The chromosome 9p21 region, which contains
CDKN2A and CDKN2B, is a candidate region for a
predisposing genetic event because deletion of this area is the most
common known molecular abnormality in childhood ALL, occurring in 35%
to 60% of cases.3,4 To determine whether constitutional
modification of a chromosome 9p21 gene might be involved in childhood
ALL, we determined the parental origin of the deleted 9p21 allele in a
series of 48 cases of childhood ALL.
A total of 48 children with ALL were recruited at pediatric
oncology clinics throughout New Zealand. Archived slides of bone marrow
aspirate previously obtained at presentation and at remission were
collected after obtaining informed consent in accord with the
instructions of regional ethics committees. The age, diagnosis, proportion of blasts, and the reported karyotype are shown in Table
1. Given that children were recruited at
follow-up clinics up to 3 years after diagnosis, there may have been a
bias toward cases with longer survival.
DNA was extracted from unfixed archived slides of bone marrow aspirate
with the use of a previously published method5 except that
20 mM EDTA was substituted for the 2.5 mM MgCl2 in the
extraction buffer, and the proteinase K digestion was performed at
56°C overnight, followed by an additional 2 hours at 56°C with
fresh proteinase K. Parental DNA was extracted from peripheral blood leukocytes.
Loss of heterozygosity
Sequencing Exon 1- of CDKN2A was amplified by PCR, using
primers as previously described,6 with the addition of a
T3 sequence 5' of the forward primer. PCR products were sequenced by
using Amersham Thermosequenase cycle sequencing kit with IRD-labeled T3
primer. Sequencing products were visualized on a LI-COR DNA sequencer model 4000L.
Of the 48 cases of ALL, 10 showed LOH at one or more of the 3 polymorphic markers on chromosome 9p21 (Table 1, Figure
1). In 9 of these 10 cases the percentage
of leukemic blasts in the bone marrow had been reported as more than
90% (for case 21, 81% blasts were reported). Apart from the results
for case 53 at D9S1679, the amount of the deleted allele present was
consistent with the proportion of normal cells in the bone marrow
aspirate biopsy. The parental origin of the lost allele was determined,
and in 9 of the 10 cases with LOH the lost allele was maternally
derived (P = .02). In 8 of the 10 cases the parental
origin of the lost allele was confirmed at more than one
locus.
The preferential loss of the maternally derived allele in leukemic cells suggests that a parent-specific process modifies chromosome 9p and that germ-line events may play a role in the leukemogenic pathway. This strong bias toward loss of the maternally derived 9p allele is supported by a previous small study by Heyman et al7 in which 4 of 5 cases of childhood ALL showed loss of the maternal allele (combined probability = 0.007). The identity of the leukemogenic gene or genes in the 9p21 region is not yet certain, although good candidates exist. The region contains genes for at least 3 cell cycle regulatory proteins p15, p16, and p14ARF and there is good evidence for the existence of another tumor suppressor gene (TSG) nearby.8 An additional complexity with respect to this region is that some cases of ALL show hemizygous deletion, whereas others show homozygous deletion. Acquisition of homozygous loss during tumor evolution suggests that nullisomy for 9p21 may not be necessary for leukemia initiation, but that progressive loss of genes may enhance malignant growth.9-11 Because analysis of leukemic cells at clinical presentation will reflect the summation of all previous genomic events, the interpretation of observations from 9p21 requires caution. However, we propose 3 models to explain the parental bias of LOH, each of which is consistent with the involvement of a TSG on chromosome 9p and in each the maternal deletion provides the "second hit," unmasking prior changes on the paternal allele. In the first model, maternal LOH unmasks genetic mutation of a 9p TSG
on the paternal allele. A precedent for this model is provided by
bilateral retinoblastoma in which paternal de novo RB
mutations are followed by somatic loss of the maternally derived allele.1 For childhood ALL, mutations of the candidate
genes CDKN2A and CDKN2B are rare, although exon
1- A second model that could account for the apparent germ-line origin but the lack of heritability assumes that the paternal mutation is epigenetic. A pathologic epigenetic event, in which a gene is inactivated by inappropriate methylation during spermatogenesis, provides a plausible explanation because epigenetic inactivation CDKN2B, through promoter methylation, has been observed in some cases of ALL. In these cases, about half of the leukemic DNA samples from leukemias with LOH of 9p genes showed methylation of the remaining allele.16 The third model proposes that the paternally derived gene is not inactivated by mutation but by the normal physiologic process of genomic imprinting. A precedent for this model is the preferential deletion of the maternal copy of p73, an imprinted TSG involved in childhood neuroblastoma.2 Although studies in mice provide no evidence to support genomic imprinting of Cdkn2a or Cdkn2b, it is interesting that imbalances between the relative expression levels of CDKN2A alleles, which might reflect genomic imprinting, have been reported.17 Our data together with previous data from Heyman et al7 provide strong evidence for the involvement of a paternal germ-line factor that predisposes to a proportion of cases of childhood ALL. If this factor involves a pathologic process, such as genetic or epigenetic mutation, then targeted epidemiologic studies of the paternal environment in the subgroup of children with 9p LOH may provide a useful way forward to understanding the environmental factors involved in childhood ALL.
Submitted April 16, 2001; accepted August 23, 2001.
Supported by the Cancer Society of New Zealand, the New Zealand Lottery Grants Board, and the Child Cancer Foundation.
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.
Reprints: Ian M. Morison, Cancer Genetics Laboratory, Department of Biochemistry, University of Otago, PO Box 56, Dunedin, New Zealand; e-mail: ian.morison{at}otago.ac.nz.
1. Kato MV, Ishizaki K, Shimizu T, et al. Parental origin of germ-line and somatic mutations in the retinoblastoma gene. Hum Genet. 1994;94:31-38[CrossRef][Medline] [Order article via Infotrieve]. 2. Kaghad M, Bonnet H, Yang A, et al. Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers. Cell. 1997;90:809-819[CrossRef][Medline] [Order article via Infotrieve]. 3. Takeuchi S, Koike M, Seriu T, et al. Homozygous deletions at 9p21 in childhood acute lymphoblastic leukemia detected by microsatellite analysis. Leukemia. 1997;11:1636-1640[CrossRef][Medline] [Order article via Infotrieve]. 4. Chambon-Pautas C, Cavé H, Gérard B, et al. High-resolution allelotype analysis of childhood B-lineage acute lymphoblastic leukemia. Leukemia. 1998;12:1107-1113[CrossRef][Medline] [Order article via Infotrieve]. 5. Brisco MJ, Condon J, Hughes E, et al. Prognostic significance of detection of monoclonality in remission marrow in acute lymphoblastic leukemia in childhood. Leukemia. 1993;7:1514-1520[Medline] [Order article via Infotrieve].
6.
FitzGerald MG, Harkin DP, Silva-Arrieta S, et al.
Prevalence of germ-line mutations in p16, p19ARF, and CDK4 in familial melanoma: analysis of a clinic-based population.
Proc Natl Acad Sci U S A.
1996;93:8541-8545
7.
Heyman M, Grandér D, Bröndum-Nielsen K, Liu Y, Söderhäll S, Einhorn S.
Deletions of the short arm of chromosome 9, including the interferon- 8. Hamada K, Kohno T, Takahashi M, et al. Two regions of homozygous deletion clusters at chromosome band 9p21 in human lung cancer. Genes Chromosomes Cancer. 2000;27:308-318[CrossRef][Medline] [Order article via Infotrieve].
9.
Glendening JM, Flores JF, Walker GJ, Stone S, Albino AP, Fountain JW.
Homozygous loss of the p15INK4B gene (and not the p16INK4 gene) during tumor progression in a sporadic melanoma patient.
Cancer Res.
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Maloney KW, McGavran L, Odorn LF, Hunger SP.
Acquisition of p16INK4a and p15INK4b gene abnormalities between initial diagnosis and relapse in children with acute lymphoblastic leukemia.
Blood.
1999;93:2380-2385 11. Carter TL, Reaman GH, Kees UR. INK4A/ARF deletions are acquired at relapse in childhood acute lymphoblastic leukaemia: a paired study on 25 patients using real-time polymerase chain reaction. Br J Haematol. 2001;113:323-328[CrossRef][Medline] [Order article via Infotrieve].
12.
Quelle DW, Cheng M, Ashmun RA, Sherr CJ.
Cancer-associated mutations at the INK4a locus cancel cell cycle arrest by p16INK4a but not by the alternative reading frame protein p19ARF.
Proc Natl Acad Sci U S A.
1997;94:669-673 13. Ruas M, Peters G. The p16INK4a/CDKN2a tumor suppressor and its relatives. Biochim Biophys Acta. 1998;1378:F115-F177[Medline] [Order article via Infotrieve].
14.
Gardie B, Cayuela J-M, Martini S, Sigaux F.
Genomic alterations of the p19ARF encoding exons in T-cell acute lymphoblastic leukemia.
Blood.
1998;91:1016-1020 15. Hawkins MM, Draper GJ, Winter DL. Cancer in the offspring of survivors of childhood leukaemia and non-Hodgkin lymphomas. Br J Cancer. 1995;71:1335-1339[Medline] [Order article via Infotrieve].
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Herman JG, Civin CI, Issa J-P, Collector MI, Sharkis SJ, Baylin SB.
Distinct patterns of inactivation of p15INK4B and p16INK4A characterize the major types of hematological malignancies.
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Differential expression of p16INK4a and p16
© 2002 by The American Society of Hematology.
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  J. L. Wilson, I. M. Morison, K. Paulsson, and B. Johansson No evidence for preferential maternal origin of duplicated chromosome 14 in hyperdiploid ALL Blood, February 15, 2005; 105(4): 1837 - 1838. [Full Text] [PDF]
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 M. Raghavan, D. M. Lillington, S. Skoulakis, S. Debernardi, T. Chaplin, N. J. Foot, T. A. Lister, and B. D. Young Genome-Wide Single Nucleotide Polymorphism Analysis Reveals Frequent Partial Uniparental Disomy Due to Somatic Recombination in Acute Myeloid Leukemias Cancer Res., January 15, 2005; 65(2): 375 - 378. [Abstract] [Full Text] [PDF]
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K. Paulsson, I. Panagopoulos, S. Knuutila, K. J. Jee, S. Garwicz, T. Fioretos, F. Mitelman, and B. Johansson Formation of trisomies and their parental origin in hyperdiploid childhood acute lymphoblastic leukemia Blood, October 15, 2003; 102(8): 3010 - 3015. [Abstract] [Full Text] [PDF]
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C. J. M. van Schooten, L. M. Ellis, I. M. Morison, and J. Webb Reassessment of loss of heterozygosity within MLL in childhood acute lymphoblastic leukemia Blood, May 15, 2003; 101(10): 4222 - 4222. [Full Text] [PDF]
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Abstract
Introduction
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