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Blood, Vol. 95 No. 6 (March 15), 2000:
pp. 2138-2143
NEOPLASIA
From the Department of Molecular Genetics, Division of Pathology and
Laboratory Medicine, University of Texas M. D. Anderson Cancer Center,
Houston, TX.
Nonrandom interstitial deletions and monosomy of chromosomes 5, 7, and 17 in refractory myelodysplasia (MDS) and acute myelogenous leukemia (AML) suggest a multistep pathway that culminates in aggressive clinical course. Because cytogenetic studies frequently identify chromosome 5 and 17 deletions within a single clone, we
searched for allele loss for 5q loci and TP53 gene mutations in the
same leukemic samples. Cosegregating deletions of chromosomes 5 and 17 were found to specifically include the 5q13.3 interval between the loci
D5S672 and D5S620/D5S626, a locus hypothesized to
harbor a tumor suppressor gene1 and the TP53 gene
on 17p. A rare patient with secondary refractory MDS and an unbalanced
translocation [der(5;17)], which resulted in deletions of the
5q13.3-qter and 17p loci, provided clues on the sequence of genetic
alterations. Serial molecular analysis of this patient revealed a
dysplastic clone with der(5;17), which gave rise to a leukemic clone on
acquiring an inactivating mutation of TP53. Our findings are
consistent with functional cooperation between a putative tumor
suppressor gene at 5q13.3 that contributes toward the progression of
early stages of MDS, and the TP53 gene when mutated, causes
transformation to AML.
(Blood. 2000;95:2138-2143)
The significance of cytogenetic alterations in
predicting therapeutic outcome was recognized more than a decade
ago.2,3 In refractory anemia with excess blasts (MDS-RAEB),
refractory anemia with excess blasts in transformation (MDS-RAEB-t),
and acute myelogenous leukemia (AML), nonrandom interstitial deletions 5q,7q or monosomy of 5 and 7 are markers for poor prognosis and short
remission periods. Similarly, mutations of the tumor suppressor gene,
TP53, are associated with resistence to therapy and poor prognosis in hematologic malignancies.4 This is in stark
contrast to simple reciprocal translocations such as inv(16) in AML,
for which the outcome is more favorable.
Typically, RAEB, RAEB-t, and AML patients with deletions or monosomy of
either 5q, 7q, or 17p have numerous additional karyotypic changes, the
significance of which has not been extensively investigated. Preferential combination of 1 or more abnormalities may provide clues
on the genetic alterations leading to the progression of AML.
Deletions of 5q result in loss of large segments of the chromosome,
which translates into physical loss of approximately 2% of the haploid
genome. In a previous report, we identified a patient with de novo AML,
who harbored a deletion at 5q13, and allowed the characterization of a
critical locus between proximal markers, AFMb347yf9/D5S672, and
distal markers D5S620/D5S626.1 The importance of
this interval was further confirmed in an AML cell line, ML3, in which
the entire chromosome 5q sequences are grossly intact, except at
5q13.3 and qter.
Subsequently, several studies on solid tumors support a critical locus
at 5q13, which undergoes loss of heterozygosity (LOH) or allelic
imbalance. These include malignancies of the lung,5 prostate,6 ovary,7 stomach,8
pancreas,9 and bladder.10 More importantly, in
each of these studies, LOH of 5q13 loci is evident but not 5q23 (APC
locus) or 5q31.1 (AML/MDS locus), indicating a distinct tumor
suppressor locus at chromosome 5q13.3. The deletions of 5q13
in ovarian cancer are particularly striking in that the 5q13
deletions correlate with mutations of the TP53 gene in 78% of tumors.7
In AML and MDS, mutations of the TP53 gene are infrequent, less
than 10%. However, in a subset of patients with "17p- syndrome," the TP53 gene is frequently mutated.11 A recent
report on this distinct clinical entity demonstrated loss of the
TP53 gene by 17p deletion in 14 of the 16 cases analyzed by
fluorescent in situ hybridization (FISH). Enhanced expression or
mutation of the remaining allele was observed in 11 of 14 cases.12 Interestingly, 15 of 17 patients in this study
also had karyotypic deletions of chromosome 5. The primary mechanism of
loss of chromosome 5 and 17 sequences was by an unbalanced
translocation between the 2 chromosomes (9/15 cases). Unbalanced
translocations or dicentrics involving chromosomes 5 and 17 have been
recognized as recurring abnormalities in myeloid neoplasms by other
investigators as well.13 The nonrandom participation of
chromosome 5 raises the possibility of an unidentified gene on
chromosome 5 whose loss synergizes with the inactivation of the
TP53 gene. Because most of these patients have numerous
cytogenetic abnormalities at presentation, it is difficult to determine
whether the mutation in the TP53 gene occurs first and the
unbalanced 5;17 translocation results from the ensuing genomic
instability, or whether the der(5;17) is an initial, critical step.
In this report, we show that a putative tumor suppressor locus at
5q13.3 may be the target of a critical gene because patient samples
with a mutated TP53 allele also have invariant loss or disruption of 5q13.3. Thus, loss of chromosome 5q13.3 sequences confers
a proliferative advantage to a dysplastic clone that can be fully
transformed upon acquiring a mutation in the TP53 gene.
Patient samples
Patient database
Loss of heterozygosity (LOH) analysis Information for the polymorphic markers that were used in this study was obtained from the Whitehead Institute (http://www.genome.wi.mit.edu) or the Stanford Human Genome Center (SHGC) (http://www-shgc.stanford.edu). Primers and polymerase chain reaction (PCR) amplification conditions for chromosome 5 and 17 markers were obtained from the Genome Database (http://www.gdb.org), the Whitehead institute, or the SHGC. The technique for determining LOH is detailed elsewhere.14Physical map of 5q13.3 loci Our previous report on the delineation of the critical 5q13.3 locus relied solely on the radiation hybrid mapping data available at that time from the Whitehead Institute. The current study was facilitated by second generation radiation hybrid maps from the SHGC, Sanger Center. UK, and a Yeast Artificial Chromosome (YAC) contig from the Whitehead Institute. All the markers were verified independently against the YACs. Only those markers that tested positive in 2 or more screens were considered truly positive. Orientation and overlap of the YACs were further confirmed by screening for ends of YACs rescued by inverse PCR.15 The current order is most consistent with centromere-AFMb347yf9-D5S672-D5S620/D5S626-D5S641-GATAP18 104-D5S401-telomere, rather than the previous order of centromere-D5S672-AFMb347yf9-GATAP18 104-D5S620/D5S626-D5S641-D5S401-telomere. Inclusion of additional sequence tagged site (STS) markers D5S1464, D5S806, D5S2029 enhances the resolution of the map.Somatic cell hybrids Somatic cell hybrids were generated by fusing the peripheral blood leukemic blasts from patient 1 and LMTK- murine fibroblasts. Ficoll Hypaque separated mononuclear cells in suspension were fused with polyethylene glycol (PEG 1000) and selected in hypoxanthine, aminopterin, and thymidine (HAT) containing medium.16 Colonies were screened by PCR to identify those with human chromosome 5 and 17 sequences.Cell lines The AML cell line ML3 was cultured in RPMI 1640 medium (Life Technologies, Rockville, MD) containing 10% fetal calf serum. LMTK- mouse fibroblasts were cultured in Dulbecco's Modified Eagle medium (DMEM) (Life Technologies, Rockville, MD) containing 10% fetal calf serum.Fluorescence in situ hybridization Alu PCR probes were made by PCR amplifying against YAC DNA. Primers and amplification conditions are described by Liu et al.17 Probes were biotinylated using Bionick Labeling System (Life Technologies, Rockville, MD) according to the manufacturer's instructions. Painting probes were purchased from Oncor (Gaithersberg, MD). Hybridization procedures are detailed elsewhere.18Mutation screening for the TP53 gene The TP53 exons 4-8 were amplified with intronic primers and the products were sequenced in an ABI 377 automated DNA sequencer using standard dye terminator chemistry. Mutations were verified by at least 2 independent PCR amplifications.
Deletions of chromosomes 5 and 17 cosegregate in RAEB, RAEB-t, and AML patients We noted that 6 of the 12 patients who allowed the initial delineation of the critical 5q13.3 locus also had deletions of 17p or monosomy for chromosome 17.1 To determine whether this was due to a selection bias or a nonrandom correlation, we used our institutional database that contains karyotypic data for AML and MDS patients. The overall frequency of loss of 5, 7, and 17 were 11.3%, 13.4%, and 5.97%, respectively. Of those 350 patients with chromosome 5 abnormalities, 134 (38%) had a chromosome 17 abnormality and 161 (46%) had a chromosome 7 abnormality. The correlation between chromosomes 5 and 17 is similar to that seen between chromosomes 5 and 719 (Figure 1). No other chromosomal abnormality showed such a strong cosegregation. For example, anomalies of the long arm of chromosome 16, which is reported to show allelic imbalance in solid tumors,20 are seen only in 55 (16%) of the 350 patients with chromosome 5 anomalies. Additionally, none of the 55 patients with the anomaly inv(16) that is associated with the M4 subtype of AML had loss of chromosome 5. Thus, there is a nonrandom cosegregation of deletions of chromosome 5q and 17p loci in AML and MDS.
Delineation of the critical 5q13.3 locus from patients with der(5;17) Two patients with unbalanced translocations between chromosomes 5q and 17p facilitated the search for the smallest region of loss on 5q. The der(5;17) in both patients resulted in deletions of 5q13.3 loci, but were in opposite orientations. Patient 2, with de novo AML, retained heterozygosity for all the 5q31.1 loci and the distal 5q13.3 markers D5S2029, D5S620, and D5S626, yet showed a minimal deletion from 5q13.1-q13.3, which included the 5q13.3 markers AMFb347yf9 and D5S672 (Figure 2). A similar patient with der(5;17) (patient 1) showed a deletion which apparently overlapped with that of patient 2. As shown in Figure 2B, the informativeness of the AFMb347yf9, D5S672, and D5S2029 loci in both these patients is revealed by the presence of 2 major alleles in the normal sample. The leukemic cells of patient 1 retains 2 alleles of AFMb347yf9 and a single allele of both D5S672 and D5S2029 loci. Patient 2, however, retains 2 alleles of D5S2029 and reveals allele loss for both D5S672 and AFMb347yf9. In addition, the deletion pattern in patient 1 confirms the revised order of markers, namely, centromere AFMb347yf9-D5S672-telomere.
Generation of a YAC contig spanning the D5S1464-D5S641 interval To physically link the markers within the critical 5q13.3 region, we initiated a chromosomal walk from the telomeric end of this locus. The YAC 944d6 served as the starting point and the end rescued fragment from the right arm tested positive by PCR for the YACs 965b11 and 934c2; thus, orienting the D5S641, D5S2094, D5S1959, and GATA-P18104 loci telomeric of the D5S620 and D5S626 loci (Figure 3). Screening of the YACs 729f12, 765a5, 940d1, 934c2, and 744d10 with the additional markers yielded an order that is most consistent with centromere-D5S1464-(D5S2029/D5S806)-(D5S620/D5S626)-(D5S641/GATA-P18104)-telomere.
Delineation of the 5q13.3 chromosomal breakpoint in the AML cell line, ML3 The AML cell line, ML3, has a der(3;5) and retains heterozygosity for most of the chromosome 5q loci. The translocation disrupts the 5q13.3 region and so facilitated further delineation of the critical region. Inter-alu PCR products from 5q13.3 YACs (Figure 3) were used as FISH probes on metaphases from these cells. YACs 745a9, containing the marker AFMb347yf9, hybridized to the der(5) and YAC 965b11, containing markers D5S620 and D5S626, hybridized to the der(3).1 YACs 848c4 and 765a5, containing markers D5S672 and D5S1464 respectively, hybridized to the telomeric end of the der(5) chromosome. YAC 940d1, containing the markers D5S1464 as well as D5S620, also hybridized to the der(5) chromosome. YAC932c2, with markers D5S2029/D5S806 and D5S620/D5S626 hybridized to the der (3) chromosome. Figure 3 demonstrates that the 5q13.3 locus disrupted in the ML3 cell line resides between D5S1464 and D5S620/D5S626, a breakpoint at the telomeric end of the critical region of loss (Figure 2).The TP53 gene is deleted in patients with 5q13.3 translocations and deletions Microsatellite analysis was used to determine whether patients who show allele loss for 5q loci and anomalies of chromosome 17p also show a region of loss that included the TP53 gene. Two pairs of markers that are centromeric (D17S954 and D17S786) and telomeric (D17S938 and D17S796) of the TP53 gene were selected. The microsatellite marker D17S1353 maps within the same YAC 728b11 as the TP53 gene, but its orientation with respect to the TP53 gene is unclear. In total, 15 patients with chromosome 5 deletions, including 2 patients whose interstitial deletions were entirely telomeric of the 5q13.3 locus, were analyzed. Surprisingly, we found that the 4 patients who had allele loss for 17p loci had the 5q13.3 deletion in common (ie, contiguous loss of loci within the D5S672-D5S626 interval). The karyotypes and diagnoses of these 4 patients are shown in Table 1.
TP53 gene is mutated in patients with allele loss
for 17p and the cell line, ML3
The der(5;17) precedes mutation of TP53 that is involved in
leukemic progression
This study is a combinatorial analyses of 2 distinct cytogenetic
alterations, namely, deletions of 5q and 17p. The investigations allow
us to conclude that unbalanced translocations between chromosomes 5 and
17 target a critical locus at 5q13.3 and the TP53 gene
(patients 1 and 2). Both of these loci may also be deleted by separate
events as seen in ML3 and patients 3 and 4.
Cosegregation of deletions of chromosomes 5 and 17
Deletions of chromosome 5 occur in a broad range of clonal myeloid anomalies An eclectic group that includes the indolent, transfusion-dependant refractory anemia (RA), the highly refractory and aggressive RAEB-t, and AML harbor large deletions of chromosome 5. Very little is known about the relationship between the basis of these diverse genetic entities besides the 2 distinct critical regions of loss identified at 5q31.22,23 A rare case of RAEB may show noncontiguous deletions of both 5q31 loci,24 whereas other cases of RA have also been reported with del(5)(q11q13).25 The evidence provided here identifies a distinct refractory subset in which the D5S2634-D5S2029/D5S806 interval is consistently lost and the TP53 gene is mutated.Nature of TP53 mutations All the mutations we identified were in the DNA binding domain of TP53. Of the 5 TP53 mutations that we identified, 2 were not typical. Codon 220 is a common site for mutation, but is usually a change from Tyr to Cys. Codon 294 is rarely mutated.26 Only 3 of the 5 mutations that we identified were transition mutations indicating that the common C to T transition mutation seen in solid tumors may not be the only mechanism of mutation in leukemia. A larger survey of TP53 mutations in secondary AML and MDS will be useful in understanding the mechanism as the mutational spectra in MDS and AML may be different from other cancers.27Loss of 5q13.3 locus precedes mutations in the TP53 gene Patient 1 allows us to conclude that deletions of 5q and 17p loci precede the TP53 inactivation although clonal expansion of cells harboring der(5;17) is clearly evident, as shown in Table 2. She was asymptomatic during the evolution of the der(5;17) clone suggesting that the unbalanced translocation conferred a proliferative advantage and did not affect the differentiation potential. Deletion of chromosome 5q is also seen as an acquired anomaly in familial AML and MDS without genetic linkage to chromosome 5q.28 Taken together, the early events leading to the initiation of a dysplastic clone may be independent of loss of chromosome 5q; whereas, deletions in 5q may allow an expansion of the clone that can then be transformed by mutations on TP53 or other genetic alterations. Figure 6 illustrates a proposed order and functional consequences of the somatic mutations during the progression of MDS and AML. As TP53 mutations are infrequent in refractory MDS and AML, there are likely to be other genetic alterations that would also facilitate transformation in cells with deletions of chromosome 5. To support this model, additional patients with chromosome 5 and 17 abnormalities will be studied in the future. Furthermore, the placement of TP53 mutation late in the sequence of events is reminiscent of the colon cancer model of transformation.29
Our special thanks are due to Dr Elihu Estey for helpful discussions on patient 1. We thank Drs Miloslav Beran, David Claxton, Steven Kornblau, and Susan Roberts for patient material and Dr Hong Liang for helpful discussions. We also thank Rui Yu Wang, Xiuying Lina Wu, and Lisa Chu for assistance with FISH analysis and Rashmi Pershad for the automated DNA sequence analysis. The enthusiastic assistance of Gisela Sanchez William and Ellen Jackson with the patient database is gratefully acknowledged. We thank Dr Walter Hittelman for critical reading of the manuscript.
Submitted July 12, 1999; accepted November 30, 1999.
Supported by grants from Ladies Leukemia League, Metairie, LA, CA66982 and CA55164 from the National Institutes of Health.
Reprints: Lalitha Nagarajan, Department of Molecular Genetics, Box 45, M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030; e-mail: lalitha{at}odin.mdacc.tmc.edu.
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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 A. G. Smith, L. J. Worrillow, and J. M. Allan A common genetic variant in XPD associates with risk of 5q- and 7q-deleted acute myeloid leukemia Blood, February 1, 2007; 109(3): 1233 - 1236. [Abstract] [Full Text] [PDF]
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 M. Osanai, M. Murata, N. Nishikiori, H. Chiba, T. Kojima, and N. Sawada Epigenetic Silencing of Occludin Promotes Tumorigenic and Metastatic Properties of Cancer Cells via Modulations of Unique Sets of Apoptosis-Associated Genes. Cancer Res., September 15, 2006; 66(18): 9125 - 9133. [Abstract] [Full Text] [PDF]
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D. H. Christiansen, M. K. Andersen, and J. Pedersen-Bjergaard Mutations of AML1 are common in therapy-related myelodysplasia following therapy with alkylating agents and are significantly associated with deletion or loss of chromosome arm 7q and with subsequent leukemic transformation Blood, September 1, 2004; 104(5): 1474 - 1481. [Abstract] [Full Text] [PDF]
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 D. H. Christiansen, M. K. Andersen, and J. Pedersen-Bjergaard Mutations With Loss of Heterozygosity of p53 Are Common in Therapy-Related Myelodysplasia and Acute Myeloid Leukemia After Exposure to Alkylating Agents and Significantly Associated With Deletion or Loss of 5q, a Complex Karyotype, and a Poor Prognosis J. Clin. Oncol., March 1, 2001; 19(5): 1405 - 1413. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
murine fibroblasts. A
hybrid clone that contained the der(5;17) chromosome but not the normal
chromosome 5 was identified and characterized. STS markers localized to
this interval by the SHGC were tested against the hybrid by PCR
amplification. The der(5;17) chromosome contained markers
AFMb347yf9 and D5S2634 but not D5S672. D5S2634, which is retained in the hybrid is within 30 kb (less than 1 cR) of D5S672, according to the SHGC radiation hybrid
map. Therefore, the proximal breakpoint in patient 1 resides between the loci D5S672 and D5S2634 (Figure 
