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6q Deletions in Acute Lymphoblastic Leukemia and Non-Hodgkin’s Lymphomas
By Mats Merup, Teresa Calero Moreno, Mats Heyman, Kristina Rönnberg, Dan Grandér, Rut Detlofsson,
Omid Rasool, Yie Liu, Stefan Söderhäll, Gunnar Juliusson, Gösta Gahrton, and Stefan Einhorn
Deletions on the long arm of chromosome 6 are frequently
found in acute lymphoblastic leukemia (ALL) and nonHodgkin’s lymphomas (NHL). We have used polymerase
chain reaction analysis to study loss of heterozygosity of 16
microsatellite markers on chromosome 6 in 74 ALL and 54
NHL patients. Our results show that deletions of 6q in ALL
are more frequent than what has been reported in previous
studies, occurring in at least 32% of the patients. The
corresponding figure for NHL patients is 7%. Our results
define a region of minimal deletion in ALL of less than 500 kb
between markers D6S1709 and D6S434. The common region
of deletion in NHL is located telomeric of this region. Thus,
two different tumor suppressor genes on chromosome 6q
seem to be relevant for the development of lymphoid
malignancies.
r 1998 by The American Society of Hematology.
N
panning followed by a 2-week culture of normal T cells as previously
described.14 For CLL cases, normal cells were also obtained from
granulocytes or from T cells after E-rosette separation. DNA was
extracted according to standard procedures as previously described.12
PCR analysis. Twenty microsatelite markers from chromosome 6
were used to screen the patients for 6q deletions. The PCR conditions
and sequences for PCR primers are shown in Fig 1. One of the primers
was labeled with g35S-dATP by T4 polynucleotide kinase. After PCR
amplification the products were electrophoresed on 6% denaturing
polyacrylamide gel. The gels were transferred to Whatman 3MM paper
(Maidstone, UK), dried, and autoradiographed on radiograph film.
Sharply descreased intensity or abscence of one allele was interpreted
as LOH. Cases with decreased intensity but not complete loss of one
allele in all microsatellite markers were not evaluated as LOH.
Southern blot analysis and DNA probes. Southern blotting and
hybridization was performed as previously described.13 Scanning
densitometry was used to quantitate the band intensity. The 149b19
probe obtained from UK Human Genome Mapping Project Resource
Centre was assigned immediately centromeric to the D6S468 marker on
chromsome 6. The BCL-1 probe located at 11q13 was used as a control
of DNA quantity.
Chromosome analysis. Cytogenetic analysis was performed in cells
from blood and BM as described previously.14-15
ONRANDOM CHROMOSOMAL deletions reflect loss of
genetic material and suggest the presence of tumor
suppressor genes that are inactivated during the process of
malignant transformation. Chromosomal deletions on the long
arm of chromosome 6 have been found in a variety of human
malignancies including malignant melanoma,1 renal cell carcinoma,2 salivary gland adenocarcinoma,3 ovarian carcinoma,4
and in hematologic malignancies.
In hematologic malignancies, deletions on chromosome 6
occur most frequently in acute lymphoblastic leukemia (ALL)
and non-Hodgkin’s lymphomas (NHL). Cytogenetic studies
have shown deletions in 4% to 13% in ALL5 and in 13% to 15%
in various subtypes of NHL.6-8 Previous studies of loss of
heterozygosity (LOH) have shown LOH in 7% to 13% of
ALL9,10 and in 22% of NHL.11
In this study we have analyzed LOH in polymorphic microsatellite markers on chromosome 6 using polymerase chain
reaction (PCR). We have screened 128 patients with ALL and
NHL. Our results show a higher frequency of chromosome 6
deletions in ALL than has been reported in previous studies and
define a region of minimal deletion localized at D6S283. Also,
our results suggest that a different region located more telomeric is deleted in NHL.
MATERIAL AND METHODS
Patients. Seventy-four children with diagnoses of ALL and 54
patients with NHL were investigated. The distribution of immunphenotypes among the ALL samples were 58 pre-B ALL, 14 T-ALL, and 2
clones with B-ALL. In the NHL group, 32 patients had chronic
lymphocytic leukemia (CLL), 10 had centroblastic-centrocytic NHL, 3
had hairy-cell leukemia, and 3 patients had high-grade NHL. Diagnosis
was made on bone marrow (BM) and/or lymphnode biopsies and in
most cases confirmed with flow cytometry.
Cell separations and DNA extraction. ALL samples were collected
from peripheral blood or BM and mononuclear cells were seperated on
a Lymphoprep gradient (Nycomed, Oslo, Norway). In cases where
fluorescence-activated cell sorter (FACS) analysis showed a significant
number of normal cells, malignant cells were purified by positive or
negative panning using monoclonal antibodies for CD7, 10, or 19.12
CLL cells collected from peripheral blood were separated on Lymphoprep. T and B cells were separated through erythorocyte (E)-rosette
sedimentation or by separation on nylon wool.13 The remaining NHL
samples were collected from BM or lymphonode biopsies. After these
procedures the cell populations were greater than 90% pure for ALL and
greater than 95% pure for NHL.
Normal DNA was obtained from peripheral blood in cases where
FACS analysis showed no malignant cells in blood, or from peripheral
blood or BM after remission had been obtained. In some cases
malignant cells were excluded from peripheral blood by negative
Blood, Vol 91, No 9 (May 1), 1998: pp 3397-3400
RESULTS
ALL. LOH of at least one microsatellite marker on chromosome 6 was found in 24 of 74 analyzed patients (32%) and the
distribution was similar in the different immunophenotypic
ALL groups. Six patients had LOH in every evaluable mircrosatellite marker, suggesting loss of one complete chromosome.
The 18 patients with limited deletions on chromosome 6 are
shown in Fig 2. A region of minimal deletion (RMD) was found
involving marker D6S283. The RMD was limited by marker
D6S1709 on the centromeric side (retained in patients 11, 26,
33, 55, and 66) and by marker D6S434 on the telomeric side
From Radiumhemmet and the Childhood Cancer Research Unit,
Department of Pediatrics, Karolinska Hospital, Stockholm; the Department of Medicine, Karolinska Institute at Huddinge University Hospital, Huddinge; and the Department of Hematology, University Hospital
Linköping, Linköping, Sweden.
Submitted July 28, 1997; accepted December 29, 1997.
Address reprint requests to Mats Merup, MD, PhD, Department of
Medicine, Karolinska Institute at Huddinge University Hospital, 141
86, Huddinge, Sweden.
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.
r 1998 by The American Society of Hematology.
0006-4971/98/9109-0003$3.00/0
3397
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3398
MERUP ET AL
Fig 1. Primarsequence and cytogenetic location of 16 microsatellite markers used in the study.
The distance in centimorgans between the markers is indicated.
The PCR conditions included denaturation in 95° for 5 minutes.
Denaturation temperature was
always 94°C for 30 to 60 seconds. Annealing time was 30°C
and the temperature is shown
for every microsatelite marker.
Elongation temperature was
72°C for 30 to 40 seconds. The
number of cycles is shown for
every marker. Amplification was
always concluded with 10minute final extension at 72°C.
Marker D6S15433 and D6S1709,
located between D6S468 and
D6S283, and marker D6S434 and
D6S1028, located between
D6S283 and D6S301, are not
shown in the figure.
(retained in patients 11, 20, and 66). The distance between the
markers has been estimated to be less than 1 cM.16
In one patient (no. 13) with LOH from marker D6S275 to
D6S301, there was also an LOH in marker D6S305 with retention of
two alleles in at least five intervening microsatellite markers.
To confirm LOH data, Southern blot hybridizations using
DNA from two ALL patients with 6q LOH and one case without
detectable LOH were performed. The 149b19, located immediately centromeric of the RMD, showed hemizygous deletions,
detected as a reduction of signal intensity by approximately
50%, in the two ALL patients with LOH, whereas no deletion
was detected in the patient without LOH (Fig 3).
NHL. Four out of 54 patients (7%) had LOH shown by
microsatelite analysis (Fig 5). Two patients, L3 with a highgrade follicular B-cell lymphoma and L4 with a high-grade
T-cell lymphoma, had limited deletions extending from marker
D6S301 and D6S261, respectively. Both patients retained
marker D6S301, which was found to be the border for the RMD
in ALL. In patients L1 and L2, both with CLL, the microsatelite
analysis showed extensive deletions on chromosome 6. Patient
L2 retained the most distal marker D6S281. These patients were
evaluated cytogenetically showing del(6)(q16-21) in patient L1
and del(6)(q21) in patient L2. An additional CLL patient
Fig 2. Eighteen ALL patients
with LOH on chromosome 6. Six
ALL patients with LOH in very
evaluable marker on chromosome 6 are not shown in the
figure. Filled box (j) indicates
retention of two alleles. White
box (h) indicates LOH. Diagonally striped box (•) indicates
that the marker was not informative and reversed stripes (§) that
the marker was not evaluable or
not done. MSI indicates microsatellite instability.
showed an aberrant chromosome 6 with a translocation
t(6;6)(q23;q27), but the microsatellite analysis did not reveal
any LOH.
DISCUSSION
Our study shows that deletions on chromosome 6 are a more
frequent phenomenon in ALL than has been reported in
previous studies. Using PCR of microsatelite markers we found
deletions in 32% of analyzed cases. The deletions clustered
around marker D6S283 and our results indicate a minimal
region of deletion limited by marker D6S1709 and D6S434.
The cytogenetic localization of D6S283 is 6q16-q21. In the
published radiation hybrid map for chromosome 6, marker
D6S283 and D6S434 are localized on the same fragment,
indicating that they are closely related. Also, markers D6S1709
and D6S283 are localized on the same PI-derived artificial
chromosome (PAC) clone, suggesting the distance between
these two markers to be less than 100 kb. These data suggest
that the minimal region of deletion defined by this study does
not exceed 500 kb. This region is likely to harbour a tumor
suppressor gene relevant for ALL.
Most published studies are based on cytogenetic analysis
which is often complicated in ALL because of poor yield of
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6q DELETIONS IN LYMPHOID MALIGNANCIES
3399
one additional tumor suppressor gene located on chromosome 6
that is inactivated during the development of NHL entities.
In previous reports based on cytogenetics, the incidence of
abnormalities on chromosome 6 has been 13% to 15% in
various subtypes of NHL.6-8 In CLL, deletions on chromosome
6 seem to be less frequent than in other NHL entities, occurring
in 4% of CLL cases.18 Molecular studies have shown 6q
deletions in 19% of acquired immunodeficiency syndrome–
related NHL19 and in 22% of other NHL entities.11
The low frequency of 6q deletions in this series can partly be
explained by the large proportion of CLL patients (60%).
However, another possibility could be that chromosome 6
deletions are limited to a subclone of the malignant cells and
that the number of cells with two alleles of the microsatellite
markers thus will be amplified using a PCR method.
In previous cytogenetic and molecular studies it has been
possible to associate specific histological subtypes with specific
deleted regions on 6q. Deletions in 6q21 are associated with
high-grade lymphomas. Deletions of 6q25-27 are associated
with intermediate-grade lymphomas. Additionally, deletions in
6q23 are commonly found in low-grade NHL without t(14;
18).11,20 It has been suggested that the cluster of deletions in
6q21 frequently found in high-grade lymphomas overlaps to the
deleted region found in ALL.11,21 However, in our material the
Fig 3. Autoradiographs of Msp I—digested DNA, hybridized to the
149b19 probe on chromosome 6 and a BCL-1 probe. ALL patient 56
(lane 1) and patient 64 (lane 3) had LOH on chromosome 6 in the PCR
analysis. Lane 2 contains normal DNA and lane 4 contains DNA from
an ALL patient without LOH. There is a reduced signal in DNA from
patients 56 and 64, indicating a hemizygous deletion of the 149b19
marker.
mitoses and fuzzy-appearing chromosomes. The frequency of
6q deletions in cytogenetic studies has been 4% to 14%.5,6,17
Our study, using PCR to overcome the problems associated with
cytogenetic analysis of ALL, shows that deletions of chromosome 6 are more common. The PCR method is based on an
evaluation of signal intensity of heterozygous alleles and one
possible explanation for alteration in signal intensity could be
extra copies of chromosome 6. In the patients with deletions in
this study this possibility could be excluded, as a result of the
fact that the LOH did not include all of chromosome 6, or of the
complete or almost complete disappearance of one of the bands.
In previous LOH studies of childhood ALL, deletions of 6q
have been found in 7% to 13% of the patients.9,10
Patient 13 had LOH in two separate regions with retention of
two alleles in the intervening microsatellite markers. The larger
deletion included the common region of deletion around marker
D6S283. The second deleted region could be a result of genetic
instability rather than an indication of a second cluster for
deletions on chromosome 6 in ALL.
In the NHL group, deletions were found in 4 out of 54
patients (7%). In two high-grade lymphomas, the deleted
markers were located telomeric on 6q, and two alleles were
retained of the microsatellite markers in the RMD found in
ALL. However, in two CLL patients there were extensive
deletions that were not distinctly separated from the RMD
discovered in ALL. These results suggest that there is at least
Fig 4. Microsatellite analysis of patient 11 with ALL and patient L4
with high-grade T-cell NHL. For each microsatellite marker the PCR
products of the malignant sample is shown to the left and of the
normal sample to the right. For ALL patient 11, there is LOH in marker
D6S283. Patient L4 has a LOH in marker D6S311 and D6S437.
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3400
MERUP ET AL
Fig 5. Four NHL patients with LOH on chromosome 6. Filled box (j) indicates retention of two alleles. White box (h) indicates LOH.
Diagonally striped box (•) indicates that the marker was not informative or reverse stripes (§) that the marker was not evaluable or not done.
two high-grade lymphomas were found to have deletions in a
region distinctly separated from the RMD found in ALL.
In a recent study using micro—fluorescence in situ hybridization it was shown that what seem to be terminal 6q deletions by
cytogenetic analysis can be interstitial deletions or nonreciprocal translocations.22 A new RMD at 6q11 was suggested for
follicular lymphomas. Our study supports the finding that
deletions on 6q can be misinterpreted on cytogenetic analysis.
In patients L1 and L2 the cytogenetic analysis indicated
terminal deletions with breakpoints at 6q16-21 and 6q21,
respectively. However, the PCR analysis indicated that patient
L2 had an interstitial deletion from marker D6S283 (6q16.3-21)
to D6S270 (6q22.3-23.3) and retention of two alleles in D6S281
(6q27). In patient L1 there was LOH in all evaluable markers on
6q on PCR, indicating a more extensive loss than what was
revealed by cytogenetics.
This study defines a region of less than 500 kb on chromosome 6 that is likely to harbor a tumor suppressor gene relevant
for the development of ALL. We also confirm that a second
region of chromosome 6 seems to be deleted in NHL. However,
in NHL the picture is still complex and more molecular studies
are needed to clarify what the recurrent breaks on 6q represent.
ACKNOWLEDGMENT
The skillful technical assistance of Elisabeth Anderbring, Birgitta
Stellan, and Kristina Friberg is gratefully acknowledged.
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1998 91: 3397-3400
6q Deletions in Acute Lymphoblastic Leukemia and Non-Hodgkin's
Lymphomas
Mats Merup, Teresa Calero Moreno, Mats Heyman, Kristina Rönnberg, Dan Grandér, Rut Detlofsson,
Omid Rasool, Yie Liu, Stefan Söderhäll, Gunnar Juliusson, Gösta Gahrton and Stefan Einhorn
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