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Chromosomal Loss and Deletion Are the Most Common Mechanisms for Loss of
Heterozygosity From Chromosomes 5 and 7 in Malignant Myeloid Disorders
By Wilma L. Neuman, Charles M. Rubin, Rachel B. Rios, Richard A. Larson, Michelle M.Le Beau, Janet D. Rowley,
James W. Vardiman, Jeffrey L. Schwartz, and Rosann A. Farber
We have examined a population of patients with acute
myeloid leukemia (AML) or myelodysplastic syndrome (MDS)
for loss of heterozygosity of polymorphic markers on chromosomes 5 and 7. The rationale for this study was the observation that the majority of patients with therapy-related leukemia (t-AML or t-MDS), resulting from cytotoxic treatment for
prior malignancies, have loss of chromosome 5 and/or 7 or
deletions involving the long arms of one or both of these
chromosomes.This cytogeneticfinding suggestedthat tumorsuppressor genes, important in the development of AML,
may be located in these chromosomal regions. We analyzed
a total of 60 patients, 43 with primary MDS/AML de novo
and 17 with t-MDS/t-AML. Leukemia cells were evaluated
for restrictionfragment length polymorphisms (RFLPs). Leukemia cell genotypes were compared with lymphoblastoid
cell genotypes from the same patients. Two cases of loss of
heterozygosity were identified from chromosomes lacking
visible deletions: one involving chromosome 5 in a patient
with AML de novo who had a visible deletion of 5q at a later
stage of the disease, and one involving chromosome 7 in a
patient with t-AML. We conclude that allele loss from loci on
chromosomes 5 and 7 in MDS/AML, when it occurs, usually
results from major deletion or simple chromosome loss,
rather than from mitotic recombinationor chromosome loss
with duplication of the remaining homologue.
o 1992by The American Society of Hematology.
T
mosome loss and mitotic recombination, would also lead to
loss of heterozygosity of other markers on the same chromosome?’ These same strategies currently are being applied to
studies of other types of solid tumors, such as small cell
carcinoma of the lung22 and neurobla~toma,2~
which are
associated with deletions of the short arms of chromosomes
3 and 1, respectively.
There are fewer data implicating tumor-suppressor genes
in the development of leukemia. The TP53 gene on 17p has
been found to be rearranged in leukemia cells from a
significant fraction of chronic myeloid leukemia (CML)
patients, predominantly in blast crisis?e26 Recently, rearrangements of the R B I gene have been reported in two
patients with MDSZ7and in four patients with lymphoid
malignancies?8
The observation that deletions of chromosomes 5 and 7
are frequent in t-AML/t-MDS suggests that there may be
tumor-suppressor genes involved in the development of this
leukemia. By cytogenetic analysis of patients with a del(5q)
(105 patients), we previously identified a region of 5q,
HERAPY-RELATED malignant myeloid disorders develop in a proportion of patients who have received
chemotherapy and/or radiotherapy for other primary malignancies.’ The risk has been estimated at 3% to 7% at 10
years after treatment for Hodgkin’s disease, non-Hodgkin’s
lymphoma, and multiple myeloma, depending on the type
(eg, chemotherapy, radiotherapy, or both) and amount of
treatment and the age of the patient? Clonal chromosome
abnormalities are found in the bone marrow cells of the vast
majority of patients with therapy-related acute myeloid
leukemia (t-AML) or myelodysplastic syndrome (t-MDS).
Up to 80% of the patients with these diseases have loss of
material from chromosomes 5 and/or 7, with either loss of
an entire chromosome (-5 or -7), an interstitial deletion
of the long arm [del(5q) or del(7q)], or an unbalanced
translocationgs4(LeBeau MM, Rowley JD, Larson RA, et al,
unpublished data). This karyotypic pattern is quite different from that found in most patients with primary MDS
or AML de novo; other abnormalities, including specific
translocations, are characteristic of different subtypes with
distinctive morphologies. A small proportion of patients
with primary MDS/AML de novo do have loss of material
from chromosomes 5 and/or 7; approximately 39% of the
individuals with these abnormalities have had documented
occupational exposure to possibly mutagenic agents?7
Tumor-suppressor genes have been implicated in the
development of several types of solid tum0rs.8,~Those that
have been identified so far include the retinoblastoma
(RBI) gene,”.” DCC (“deleted in colorectal car~inoma”)’~
and MCC (“mutated in colorectal ~arcinoma”),’~
WTI,
which is involved in the development of Wilms’
and TP53, which is altered in many types of tum01-s.’~
The
first step in the localization of the RBl and Wilms’ tumor
genes was the identification of recurring deletions of
specific chromosome
Based on this information,
investigators discovered loss of heterozygosity at polymorphic loci in these same regions in patients without cytogenetic deletions?’ The rationale for this approach was the
hypothesis that expression of recessive mutations in a gene
critical to the development of cancer could result from
genetic events leading to homozygosity or hemizygosity of a
single mutant allele; some of these events, including chroBlood, Vo179, No6(March 151,1992: pp 1501-1510
From the Departments of Medicine, Pathology, Pediatrics, and
Radiation and Cellular Oncology, University of Chicago, Chicago, IL;
and the Department of Pathology, University of North Carolina at
Chapel Hill.
Submitted March 1,1991; accepted November 8, 1991.
Supported by National Institutes of Health Grants No. CA49039
(R.A.F.), CA425.57 (J.D.R.),and CA40046 (Program Project), a grant
from the North Carolina Biotechnology Center (R.A.F.),and Contract
No. DE-FG02-86ER-60408fiom the Depamnent of Energy (J.D.R.).
WLN was supported by US Public Health Service Postdoctoral
Training Grant No. CA09273. M.M.L. is a Scholar of the Leukemia
Society of America. C.M.R. is a Pew Scholar in the Biomedical
Sciences.
Address reprint requests to Rosann A . Farber, PhD, Department of
Pathology, University of North Carolina at Chapel Hill, CB#7525
Brinkhous-BullittBuilding, Chapel Hill, NC 27599-7525.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1992 by TheAmerican Society of Hematology.
0006-497119217906-0OO4$3.00/0
1501
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1502
consisting of 5q31, that was deleted in all patients exam(Le Beau MM, unpublished data). Fewer patients
ined4229
with a del(7q) have been examined (44 patients), since
monosomy for the entire chromosome is more common in
AML; however, there appear t o b e two distinct critical
regions on chromosome 7, one a t band 7q22 and one a t
7q32-q34 (Le Beau MM, Thangavelu M, unpublished data).
We have used restriction fragment length polymorphism
(RFLP) markers on 5q and 7q t o determine whether loss of
heterozygosity has occurred in the leukemia cells of t-MDS/
t-AML patients who lack abnormalities of these chromosomes, using markers that span the critical regions. We
have also studied individuals with leukemia that is not
therapy-related, since abnormalities of chromosomes 5 and
7 a r e found in a fraction of patients with AML d e novo of
nearly every subtype, or with primary MDS.
NEUMAN ET AL
Southem 6lotting. Leukemia cell samples used in this study
consisted of mononuclear fractions from Ficoll-Hypaque gradients
of bone marrow or peripheral blood samples that contained at least
70% malignant cells. High-molecular weight DNA was isolated
from leukemia cells and lymphoblastoidlines by phenol extraction
and ethanol precipitation. DNA was digested with restriction
enzymes according to instructions from the manufacturer (New
England Biolabs, Bedford, MA), using a 7.5-fold excess of enzyme.
Blotting and hybridization were performed as previously described? except that in some cases yeast RNA (500 kg/mL) was
substituted for salmon sperm DNA and DNA was bound to filters
by treatment with UV irradiation?’ rather than by baking.
Whenever possible, the polymorphism analysis was performed
using DNA from the same leukemia cell sample that was analyzed
cytogenetically. Of 12 exceptions, four were cases in which the
sample available for DNA isolation was obtained during progression of the leukemia, between the dates of two other samples that
were karyotyped and had nearly identical cytogenetic profiles. In
eight cases, the sample available for DNA isolation was obtained
MATERIALS AND METHODS
after the last sample that was analyzed cytogenetically;in each of
these individuals, the disease had continued to progress up to the
Clinical and morphological analysis. The patients included in
time that the sample used for DNA extraction was obtained.
this study were evaluated at the University of Chicago Medical
Probes. The probes used on Southern blots were: chromosome
Center and were each assigned a unique patient number? The
5, ~105-798Rb(D5S78), ~105-153A(D5S39), C l l p l l (D5S71),
diagnosis of t-MDS, t-AML, primary MDS, or AML de novo was
pEW5.5 (D5S86), pTP5E (D5S70),“2,
CRI-L1265 (D5S52),
made by morphologic study of bone marrow specimens and
pJ0205E-C and pJ0205H-C (D5S22), and hMS8 (D5S43); chromoperipheral blood. Each patient with t-MDS or t-AML had received
some 7, pJ5.11 (D7S10), TM102L (D7S135), pRMU7.4 (D7S370),
cytotoxic therapy (chemotherapy, radiotherapy, or both) for an
NJ-1 and NJ-3 (COLIAZ), pJ3.11 (D7S8), pmetH and pmetD
antecedent disease. The diagnosis of t-MDS was made when the
(MET), pB79a (D7S13), 7C22 (D7S18), pXV-2c (D7S23), CRIpatient’s peripheral blood and bone marrow showed features of
S194 (D7S104), JURP-2 (TCRB), TCRG cDNA, pTHH28
dyspoiesis as defined by the French-American-British (FAB)
(D7S371), pYNB3.1R (D7S372) and hg3 (D7S22); and, chromocriteria for MDS and described by us and others as characteristicof
some 2, pYNH24 (D2S44).38
the changes in t-MDS.”’” Patients classified as having t-MDS had
Polymerase chain reactions. KM-19 and CS.7 (D7S23) and
less than 30% blasts in the marrow, whereas the diagnosis of overt
APOB 3’VNTR (variable number of tandem repeat) polymort-AML was made when the percentage of blasts was greater than
phisms were assayed by polymerase chain reaction (PCR). (Note
30%, as determined from marrow aspirates or as judged from
that three different RFLP sites within the D7S23 locus were
marrow biopsy sections when increased reticulin prevented aspirastudied, one of which, XV-~C,
was analyzed by Southern blotting.)
tion. Clinical data were collected, and details concerning the
Amplifications were performed in a 1OO-kL reaction mixture
primary treatment were obtained from a review of each patient’s
containing 1 kg genomic DNA, 25 to 75 pmol of each oligonuclemedical history. Radiation therapy ports, the dose of each treatotide primer, and each of the four dNTPs at 200 kmol/L, in 10
ment course in cGy, and the doses and duration of each chemothermmol/L Tris, pH 8.3, 50 mmol/L KCI, 2.5 mmol/L MgCI,, 0.01%
apy course were determined whenever possible for each patient.
gelatin. This mixture was heated for 7 minutes at 95”C, after which
The diagnosis of primary MDS or AML de novo was made
2.5 U Taq polymerase (Perkin-Elmer Cetus, Emeryville, CA ) was
according to the FAB riter ria.""^ None of these patients had
added. Primer sequences and times, temperatures, and numbers of
received cytotoxic treatment for a previous disorder, and none had
cycles for each of the reactions were as given by Feldman et a139
developed leukemia as a terminal phase of a chronic myeloprolifer(KM-19), Williams et ala (CS.7), and Boerwinkle et a14’ ( M O B
ative disorder.
3’VNTR), with the following exceptions: (1) for KM-19, denaturAll samples were obtained with informed consent, according to
ation time was 2 minutes and extension time was 3 minutes (10
the guidelines approved by the Institutional Review Board of the
minutes for the final extension), and (2) the primers used for CS.7
University of Chicago.
and 5’-GGTITTAwere 5’-GGGAGAGAAGCGAAGCAATG-3’
Cytogenetics. Cytogenetic analyses of bone marrow cells or
GACACGGGTGCATGA-3’. KM-19 and CS.7 products were
peripheral blood cells were performed as previously described.‘
treated with restriction enzymes (PstI or HhaI, respectively)before
Epstein-Barr virus transformation of B lymphocytes. Mononuelectrophoresis. Amplification products were visualized on ethidclear cells were separated from peripheral blood by density
centrifugation over Ficoll-Hypaque by the method of B~yum.’~ ium-bromide-stained 2% agarose gels.
Epstein-Barr virus (EBV) was harvested from the marmoset
RESULTS
lymphoblastoid cell line B95-8. Supernatant from cultures at a cell
density of approximately 106/mL was filtered twice through a
Electrophoretic patterns of polymorphic loci o n the long
0.45-km HA Millipore (Bedford, MA) filter. Mononuclear cells
arms of chromosomes 5 and 7 were assayed in leukemia
(2 x lo6) were suspended in 2 mL of filtrate in a 15-mL conical
cells and lymphoblastoid lines derived from each patient;
tube (without cyclosporine) and incubated at 37°C. After 2 days,
the lymphoblastoid cell DNA was used t o determine the
the EBV-containing culture medium was removed, and cells were
normal constitutional genotypes. The chromosomal posiresuspended in 5 mL complete medium (RPMI 1640 suppletions of the genetic markers used in this study, relative to
mented with 10% fetal calf serum, antibiotics, and 4 mmol/L
the critical regions of deletions observed in MDSIAML
L-glutamine) in T-25 flasks. Characteristic clusters of transformed
patients, are shown on the maps in Fig 1.
cells were visible 10 to 60 days later.
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1503
LOSS OF HETEROZYGOSITY IN AML/MDS
+COLI
Fig 1. ldiograms showing cytogenetic map positions of polymorphic markers relative to critical regions of deletions observed in MDS/AML patients:
(A) chromosome 5, (6) chromosome7.
In addition, all samples were assayed for genotypes at the
highly polymorphic D2S44 and/or APOB loci, both of which
are located on chromosome 2, in order to verify that pairs of
DNA samples from leukemia cells and lymphoblastoid lines
were actually derived from the same patient. This precaution was taken since the EBV-transformation process is
several weeks in duration, allowing opportunities for sample mix-ups or cell-line cross-contamination.
t-MDS/t-AML patients. Chromosome 5 and 7 RFLP
data on these patients are shown in Table 1. Data on
patients without visible abnormalities of chromosomes 5
and 7 were included in Table 1 only if at least one marker
on each chromosome was informative (ie, lymphoblastoid
cells were heterozygous); data on patients with a deletion of
chromosome 5 were included if at least one marker on
chromosome 7 was. informative, and vice versa. When
possible, markers within cytogenetically detectable deletions were assayed as well. (In therapy-related cases, it was
often possible to obtain only a limited amount of DNA,
since the bone marrow in these patients is frequently
hypocellular or fibrotic and, therefore, difficult to aspirate.)
Samples were obtained either at diagnosis or from patients
with residual disease.
Patients classified as having an abnormality of chromosome 5 or 7 all had the abnormality present in the majority
of the leukemia cells at the stage of the disease when DNA
was extracted, with the exception of the one patient (2087)
indicated in Table 1, who had the abnormality in only 35%
of cells karyotyped. In those cases where translocations are
specified, they were unbalanced with loss of material distal
to the breakpoint on chromosome 5 or 7. Many of the
patients had abnormalities of other chromosomes, which
are not delineated here.
A summary of the data on patients with t-MDS/t-AML is
given in Table 2. The two patients designated “atypical”
had been treated for prior malignancies, but in both cases
the leukemia clinically resembled AML de novo.
All of the losses of heterozygosity observed reflected
abnormalities that were detected cytogenetically, with one
exception (patient 2092). This patient was studied at three
different times, as indicated in Table 1. She presented with
t-AML at the time of the first sample, having been previously treated successfully with chemotherapy for breast
cancer. Throughout the remainder of her life there was no
further evidence of breast cancer. She was subsequently
treated for the leukemia and experienced a brief remission;
she then relapsed with an MDS, which after 8 months
progressed again to overt t-AML. The second sample was
obtained during this transition period from t-MDS to
A2
~%%~D7S13.D7518,
D7523)
075104
t-AML and the third after progression to t-AML. Cytogenetic analysis was performed on both the first and third
samples; although clonal abnormalities were detected, they
did not involve chromosomes 5 or 7.
Figure 2 shows the Southern blots of the markers for
which loss of heterozygosity occurred in patient 2092, along
with some examples from patients in whom there was no
loss of heterozygosity observed for the same markers. Allele
loss was observed in all three leukemia samples at D7S23
(in the CF region), TCRB, D7S372, and D7S22; whether
this resulted from mitotic recombination or chromosome
loss and duplication could not be determined, since the
lymphoblastoid cell genotype was not informative for additional markers on chromosome 7, including D7S371 and
COLIA2, located proximal to the CF region on 7q, and
D7S10, D7S135, D7S370, and TCRG on 7p.
We previously reported a patieht (2095) with t-AML
whose cells showed loss of heterozygosity for several 7q
markers, including loci in the CF region, D7S372, and
D7S22, with an increase in the proportion of cells with
allele loss as the leukemia pr~gressed.~’
Although the
patient’s bone marrow showed no aberrations of chromosome 7 at the time of diagnosis (March 1987, 93 cells
examined), cytogenetic analysis of serial samples showed
the evolution of an unbalanced 7;13 translocation, which
resulted in loss of 7q distal to q11.2 [-7,+der(7)t(7;
13)(qll.%q14)].Thirty percent of metaphase cells analyzed
9/87 were -7,+der(7), whereas 91% of cells analyzed 6/88
had the der(7). Loss of heterozygosity was detectable only
at CF loci (D7S8 [data not shown] and D7S23) in the
earliest sample from which DNA was extracted (October
1987), but was clearly evident at all informative chromosome 7 loci in a sample obtained in January 1988, and
appeared to be complete in a sample obtained in May 1988.
Thus, the proportion of cells showing loss of heterozygosity
by RFLP analysis and the translocation by cytogenetic
analysis was similar at various stages and increased with
time. Autoradiographs of Southern blots on markers for
which allele loss was observed in this patient are shown in
Fig 3. These results demonstrate that it is important to
perform parallel cytogenetic and molecular analyses to
establish mechanisms of loss of heterozygosity.
Primaly MDSIAML de novo patients. RFLP data on
these patients are given in Table 3 (patients without visible
abnormalities of chromosomes 5 or 7, arranged according
to FAB subtype) and Table 4 (patients with visible abnormalities of chromosome 5 or 7); these data are summarized
in Table 2. In addition to the data shown in Table 2, there
were two patients informative only for markers on chromo-
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Table 1. RFLP Data on t-MDS/t-AML Patients
Locus
Chromosome 5
D5S78
Patient No
D5S39
Abnormality of chromosome 5
2083
[-5,+der(5)t(5;?1 l)(q13;q23)]
2089
IdeI(5)(q13q34)1
D5S70
ADRB2
Chromosome 7
D5522
D5S43
CF*
TCRB
078372
-
12
1.2
182
1.2
1.2
-
2100 [-51
Abnormality of chromosome 7
2085t
D7S22
1.2
1 >2
2087*
[deIV)(q21q34)1
20955 10/87
1/88
5/88
[-7,+der(7)t(7;13)(q11.2;q14)]
1.2
-
1.2
1>2
18 2
1
12
18 2
1
1.2
182
1
2097 [-71
182
2102 [-71
Abnormalities of chromosomes 5 and 7
2090
1.2
[-5,del(5)(ql3q31),
+der($)t(5; 17)(q35;ql1-12),
142
-
112
1.2
del(7)(qllq34)1
2096
[del(5)(qllq35).
-7,+der(7)t(7;?)(qll
-
-
182
-
142
142
1.2
1.2
-
18 2
18 2
18 2
1
1
;?)I
No abnormality of chromosome 5 or 7
2086
2092 1/89
2/90
3/90
2093
-
2099
1.2
2101
1.2
142
14 2
2
142
1,2
-
1.2
1.2
-
1.2
1.2
1.2
1.2
No abnormality of chromosome 5 or 7 (atypical)Y
2074
1.2
[APL-M3 with t(15;17)]
1.2
1.2
1,2
1.2
Numerals indicate alleles present in leukemia cells in informative cases (1, the larger of two allelic bands; 2, the smaller band). The "1.2" genotype
is heterozygous with normal band ratios; a single numeral represents loss of heterozygosity; aberrant band ratios (1>2, etc) also indicate loss of
heterozygosity, but with the presence of both alleles in a fraction of cells (presumably either leukemic clones that have remained heterozygous or
nonleukemic cells). A dash indicates that the marker was not informative. A blank indicatesthat the assay was not done.
*Markers in the region of the cystic fibrosis gene (MET, D7S8, D7S13, D7S18 and/or D7S23).
tTetraploid karyotype, 72% of cells have loss of one copy of chromosome 7 and/or deletion of 7q32-qter or 7q22-qter resulting from unbalanced
translocations.
*Deletion present in only 35% of cells karyotyped.
§Cytogeneticanalysis of samples in September 1987 and June 1988 showed that 30% and 91%. respectively, of metaphase cells had the der(7)
chromosome.
PThese two patients had clinical and pathological features more characteristic of AML de novo, perhaps unrelated to the prior cytotoxic treatment.
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1505
LOSS OF HETEROZYGOSITY IN AMLlMDS
Table 2. Summary of RFLP Data
reduced intensity of one of the allelic bands at the D5S70
locus, as shown in Fig 4. The patient had a complete
remission after treatment and relapsed in 1986; at this time
a de1(5)(q32q35) was detected in 41% of the cells examined. It may be that this deletion was present in a significant
fraction of slowly cycling cells even at the time of diagnosis
and was detected by the RFLP probe. This finding indicates
that the use of appropriate DNA probes may be of
diagnostic value in some cases of AML.
Sensitivity of assaysfor loss of heterozygosity. As indicated
in Tables 1 and 4, there were 15 patients (six patients with
primary MDS/AML de novo and nine with t-MDS/t-AML)
with deletion or loss of chromosomes 5 and/or 7 for whom
markers in the deleted regions were informative. Allele loss
was detected by RFLP analysis in 12 of these 15 individuals
( 75% of these cases), and one of those in whom allele loss
was missed (2087) had a deletion in only 35% of cells
karyotyped. The sensitivity with which we are able to detect
allele losses in samples from patients without cytogen :tic
abnormalities should be comparable to that which we
observed in the patients with such abnormalities.
There were two patients with cytogenetic deletions in at
least 50% of karyotyped cells, but loss of heterozygositywas
not observed at an informative locus within the apparently
deleted region. Patient 291 had a de1(5)(q13q35) in 90% of
the cells karyotyped, but the cells remained heterozygous at
the D5S70 locus (5q23-31). Leukemia cells from patient 299
had a de1(5)(q13q34), but retained heterozygosity at the
D5S71 locus (5q15-21);52% of the cells karyotyped had this
deletion. The karyotypes in these patients were complex
with several interchromosomal rearrangements.
No. of Patients
t-MDSI
t-AMLO
RFLP Results
Patients with visible abnormality of
chromosome 5, no allele loss from 7q
Patients with visible abnormality of
chromosome 7, no allele loss from 5q
Patients with no visible abnormality of
chromosome 5 or 7
Allele loss from 5q
Allele loss from 7q
No allele loss from 5q or 7q
Primary
MDSlAML
De Novo
3
6
5
3
It
1*
69
33
15
Totals
43
-
"Two patients with abnormalities of both chromosomes 5 and 7 are
excluded.
tPatient 196, who at a later date had a visible deletion of 5q.
*Patient 2092.
§Two of these are "atypical."
some 5 and 14 patients informative only for markers on
chromosome 7 who did not have karyotypic abnormalities
of these chromosomes; loss of heterozygosity was not
observed in any of those individuals. Among the patients
for whom data are shown in Table 2, the only one lacking
cytogenetic deletions in whom loss of heterozygosity was
observed was patient 196. The leukemia cells from this
patient that were available for DNA isolation were from a
sample that had been stored frozen at the time of diagnosis
in 1983. Abnormalities of chromosomes 5 and 7 were not
detected cytogenetically at that time, but we observed a
B
A
C
D
a
a
a
b
c
d
e
b
c
d
e
f
b
K
f
-TIT=--
a
b
c
d
e
f
-1
D7S23
TCRB
D7S372
D7S22
Fig 2. Autoradiographs of Southern blots of DNA demonstrating allele loss at four loci in patient 2092. Loci are designated under each panel.
Restriction enqmes used for each marker are as follows: 07823 (pXV-2c). Teql; TCRB,Sg/Il; D7S372 and D7S22, Rsal. Alleles are designated t o
the right of each autoradiograph as "1" and "2." (The unmarked band in A is constant in all individuals.) (A, 6, & C) Lanes (a) t o (d) contain the
following DNA samples from patient 2092: (a) leukemia sample obtained at diagnosis in January 1989; (b) leukemia sample obtained during early
relapse (t-MDS) in February 1990; (c) leukemia sample obtained during late relapse (t-AML) in March 1990; (d) lymphoblastoid cells. ID) Patient
2092: lane (a) January 1989 leukemia cells; (b) lymphoblastoid cells. Lanes e (leukemia cells) and f (lymphoblastoid cells) in A, 6, and C show
examples of results on patients in whom loss of heterozygosity at the corresponding loci was not observed: (A) patient 2098; ( 6 )patient 2087; (C)
patient 2086. (See Fig 3 for an example of a patient in whom there was no allele loss at D7S22.)
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NEUMAN ET AL
a
a
b
C
B
A
-c
b
c
d
-1
d
a
-1
- 0 .
. L -
-
o -
o
b
c
d
e
f
- . - m
1
1-
2-
-2
- w 07823
D7S372
2
D7Sn
Fig 3. Autoradiographs of Southern blots of DNA demonstratingallele loss at three loci in patient 2095.Loci am designated under each panel.
Restriction enzymes used for each marker are as follows: D7S23 (pXV-2c). Tsq I; 078372 and D7S22, Rsal. Alleles are designated to the right of
each autoradiograph as "1" and "2." (The unmarked band in A is constant in all individuals.) Lanes (a) to (d) in all three panels contain the
following DNA samples from patient 2095: [a) leukemia sample obtained in October 1987; (b) leukemia sample obtained in January 1988; (c)
leukemia sample obtained in May 1988; Id) lymphoblastoidcells. An example of a patient 1255) in whom loss of heterozygosity was not observed
at D7S22 is also shown in C: (e) leukemia sample; (1) lymphoblastoidcells. (Genotypes at D7S22for the individuals shown differ from each other,
since there are many alleles in the populationat this locus.)
In one patient (2090), apparently monosomic for chromosome 5, loss of heterozygosity was detected at D5S22,
whereas the leukemia cells were heterozygous at D5S39
and D5S78. The leukemia cells from this patient carried a
complex rcarrangement involving the remaining chromosome 5. This derivative chromosome had an interstitial
deletion of bands q13-31. In addition, this chromosome was
involved in an unbalanced translocation with chromosome
17, resulting in loss of 5q35-qter. There were several other
rearrangements present, including a del(7)(ql lq34), within
which loss of heterozygosity was detected.
It is most likely in these cases with complex karyotypes
that, when heterozygosity was observed within an apparently deleted region, it reflected the translocation to another chromosome of a small fragment including an allele
of the heterozygous locus.
There are rcports of involvement of B lymphocytes in
myeloid leukemias, as evidenced by the presence of immunoglobulin gene rearrangements in leukemic cell^.^' To
validate the use of lymphoblastoid cells as controls in this
study, we asked whether immunoglobulin rearrangements
were present in leukemia cells from a sample of our
patients, using a heavy-chain J-region probe (JH) on Southem blots of DNA digested with Hind111 and with Hind111
and BumHI. A rearrangement was detected in one of 20
patients (seven with t-MDS/t-AML and 13 with primary
MDS/AML de novo of various subtypes) (Eaton SB, Rios
RB, Farber RA, unpublished data); this proportion is
similar to those reported in other studies of AML patients.-The one patient with a rearrangement (3050) had
AML-MI. As indicated in Table 4, the leukemia cells from
this patient were monosomic for chromosome 7, but the
lymphoblastoid cells were heterozygous for RFLP markers
on this chromosome. Therefore, although the leukemia
may have arisen from a multipotential progenitor cell in this
case, the lymphoblastoid line appears to have been derived
from B-cell clones that were not involved in the leukemia.
These findings indicate that possible lineage infidelity is
unlikely to have obscured our ability to detect loss of
heterozygosityin more than a small fraction of patients.
DISCUSSION
These results show that loss of heterozygosity at loci on
the long arms of chromosomes 5 and 7 in MDS/AML
patients who lack cytogeneticallydetectable deletions is not
common. This conclusion is supported by the work of Kere
et a1,47 who found no loss of heterozygosity for loci on
chromosome 7 (between 7cen and 7q22) in six informative
primary MDS/AML de novo patients without abnormalities of this chromosome.
Chromosome loss and reduplication can be ruled out as a
mechanism for expression of mutations in tumor-suppressor genes in all cases where heterozygositywas retained; in
those cases where markers distal to the critical region were
informative, single reciprocal somatic recombination events
are also ruled out. Other possible mechanisms for expression of a recessive mutation, including small deletions, gene
conversion, or inactivation of an allele by methylation, are
not likely to have been detected with the available markers.
A question of particular interest is whether patients with
cytogenetically detectable abnormalities of either chromosome 5 or 7, but not both, have evidence of loss of
heterozygosity from the other chromosome by mechanisms
besides major deletion or monosomy. The number of
patients with visible abnormalities of both chromosomes is
considerably higher than would be predicted by the numbers of patients with deletions of either chromosome alone,
if the two events are independent. This observation suggests the possibility that lesions of both chromosomes might
be necessary for development of leukemia. The fact that we
never found allele loss from chromosome 5 in individuals
with abnormalities of 7 or vice versa implies either that this
is not the case or that the mutations involving a tumorsuppressor gene on these chromosomes are more subtle.
Alteration of genes on each of these chromosomes may
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1507
LOSS OF HETEROZYGOSITY IN AML/MDS
Table 3. RFLP Data on Primary MDS/AML De Novo PatientsWithout Abnormalities of Chromosomes 5 or 7
Locus
Chromosome 5
PatientNo.
AML-MI
196
242
262
280
323
AML-M2
237
259
260
279
296
297
310
8030
8042
APL-M3
218
267
AMMOL-M4
203
255
258
298
8013
AMOL-M5
272
8043
AMOL-M5B
271
283
292
308
MDS
5051
5052
5053
5054
5055
5056
5057
D5S78
D5S39
D5S71
D5S86
D5S52
Chromosome 7
D5S70
ADRB2
1 <2
-
D5S43
COLlA2
1.2
12
CF'
1,2
1,2
12
1.2
1.2
-
-
D5S22
1.2
1.2
TCRB
D7S372
D7S22
D7S104
12
1,2
1.2
1.2
1,2
1.2
-
-
1.2
-
1.2
1.2
1.2
1.2
-
12
1.2
1.2
1.2
1.2
1.2
12
-
12
1.2
1.2
-
-
1.2
1.2
1.2
1.2
-
1.2
1,2
12
-
12
12
Alleles indicated as in Table 1.
'Markers in the region of the cysticfibrosis gene (Mn, D7S8, D7S13, D7S18, or D7S23).
represent steps in alternative pathways in the progression
of the leukemia; alterations in both pathways might then
enhance the process.
The observation that a high proportion of t-MDS/t-AML
patients have loss or deletions of chromosomes 5 and/or 7
indicates that genes on the long arms of these chromosomes
are probably involved in the development of the leukemia.
Loss of at least one normal copy of one of these genes may
be a critical step. When homozygosity, as well as hemizygosity, of a specific chromosomal region is found for a
particular type of cancer, the existence of a recessive
mutation in a tumor-suppressor gene is suggested. In AML,
loss of one allele without a mutation in the remaining gene
(ie, a change in gene dosage) may be sufficient to complete
this step in the malignant process, which could explain why
deletions are so common.
Alternatively, mitotic recombination may simply occur
with a much lower frequency in myeloid cells than in some
other cell types, and monosomy may be relatively stable in
myeloid cells, such that duplication of the remaining
homologue would be observed rarely, if at all. If these
conditions exist, recessive mutations may be important in
these disorders, but hemizygosity may be observed in the
critical chromosomal regions much more often than homozygosity, as a reflection of the mechanisms giving rise to
expression of mutations.
Much additional work will be needed before we understand the genetic basis of these disorders. More loci within
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1508
NEUMAN ET AL
Table 4. RFLP Data on Primary MDSlAML De Novo Patients With Abnormalities of Chromosome 5 or 7
Probe
Chromosome 5
Chromosome 7
Patient No.
D5S78
D5S39
D5S71
Abnormality of chromosome 5
264 (M2) [-51
-
-
-
142
291 (M4) [del(51(q13q35)1
1.2
-
-
1,2
1,2
299 (M6) [de1(5)(q13q34)]
-
1.2
1,2
-
-
D5S52
D5S70
D5S22
319 (M2) [de1(5)(q13q33)
COLlA2
CF'
TCRB
D7S372
-
1.2
1.2
-
1.2
1,2
-
-
-
5058 (MDS) [de1(5)(q22q33)]
1.2
Abnormality of chromosome 7
269 (M2)
1.2
12
1.2
-
-
12
1.2
-
-
1.2
D7S104
1.2
12
1.2
5003 (MDS) [dei(5)(q15q33)
D7S22
-
-
1,2
1.2
12
142
[ -7,+der(7)t(4;7)(q13;q31)]
-
3050 ( M l ) [-71
5059 (MDS) [-71
-
1.2
1.2
12
-
1,2
2
2
-
1.2
192
Alleles indicated as in Table 1.
"Markers in the region of the cystic fibrosis gene ( M n , D7S8, D7S13, D7S18, or D7S23).
the critical regions of chromosomes 5 and 7 will be
examined to determine whether small deletions have been
missed in any of the patients who did not have visible
abnormalities; the discovery of such deletions would obviously be a very useful step toward the identification of genes
important in AML. There is a striking cluster of cloned
growth factor genes in the critical region of 5q4'; these loci
will be important markers for further studies, although very
few polymorphic sites have been identified in them by
screening with restriction enzymes. Work is in progress to
saturate the cytogenetically defined critical region of chromosome 5 with polymorphic markers and to construct a
high-resolution genetic and physical map of this region.
ACKNOWLEDGMENT
The authors would like to thank D n Y.Nakamura, K-U. Lentes,
B. Weiffenbach, F. Ramirez, J. Schmidtke, M. Dean, J. Wasmuth,
R. Williamson, and T. Mak for DNA probes. Additional probes
were obtained from the American Type Culture Collection (Rockville, MD). We thank the members of the Hematology/Oncology
Cytogenetics Laboratory at The University of Chicago for cytogenetic analysis of patient samples; Karen Daly, RN, and Marjorie
Isaacson for management of clinical and cytogenetic data; Marsha
Guthrie, Mekhala Banejee, and Peter Rubinelli for technical
assistance; and Drs Bernard Strauss and Thomas Petes for helpful
discussions.
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1992 79: 1501-1510
Chromosomal loss and deletion are the most common mechanisms
for loss of heterozygosity from chromosomes 5 and 7 in malignant
myeloid disorders
WL Neuman, CM Rubin, RB Rios, RA Larson, MM Le Beau, JD Rowley, JW Vardiman, JL
Schwartz and RA Farber
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