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Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2424-2432
By
From IST-Biotechnology Section, Padova; the Department of Oncology
and Surgical Sciences, University of Padova, Padova, Italy; and III
Medizinische Klinik, Klinikum Mannheim der Universität
Heidelberg, Mannheim, Germany.
As mice carrying mutations of the DNA mismatch repair genes MSH2 and
MSH6 often develop lymphoid neoplasms, we addressed the prevalence of
the replication error (RER+) phenotype, a manifestation
of an underlying defect of DNA mismatch repair genes, in human lymphoid
tumors. We compared microsatellite instability (MSI) at 10 loci in 37 lymphoid tumors, including 16 acute lymphoid leukemias (ALL)
and 21 non-Hodgkin's lymphomas (NHL), and in 29 acute myeloid
leukemias (AML). Significant differences in MSI prevalence between AMLs
and ALLs emerged, and MSI occurrence was more frequent in the NHLs
versus AMLs. Indeed, only 3 of 29 (10%) AMLs exhibited MSI, thus
confirming its paucity in myeloid tumors, while 10 of 37 (27%)
lymphoid tumors, 6 ALLs and 4 NHLs, disclosed an RER+
phenotype. In 1 ALL patient, the same molecular alterations were observed in correspondence with a relapse, but were not detected during
remission over a 14-month follow-up; in another ALL patient, findings
correlated with impending clinical relapse. These results suggest that
the study of MSI in lymphoid tumors might provide a useful molecular
tool to monitor disease progression in a subset of ALLs. To correlate
MSI with other known genetic abnormalities, we investigated the status
of the proto-oncogene, bcl-2, in the lymphoma patients and found that 4 of 4 NHL patients with MSI carried bcl-2 rearrangements, thus linking
genomic instability to enhanced cell survival in NHL; moreover, no p53
mutations were found in these patients. Finally, we addressed the
putative cause of MSI in hematopoietic tumors by searching for both
mutations and deletions affecting DNA repair genes. A limited genetic
analysis did not show any tumor-specific mutation in MLH1 exons 9 and
16 and in MSH2 exons 5 and 13. However, loss of heterozygosity (LOH) of
markers closely linked to mismatch repair genes MLH1, MSH2, and PMS2
was demonstrated in 4 of 6 ALLs and 1 of 3 AMLs with MSI. These
observations indicate that chromosomal deletions might represent a
mechanism of inactivation of DNA repair genes in acute leukemia.
MICROSATELLITES are highly polymorphic
genetic markers dispersed in the human genome and comprise di-, tri-,
and tetranucleotide repeats. Microsatellite instability (MSI) was first
observed in subjects with hereditary nonpolyposis colorectal cancer
(HNPCC) as new alleles generated through errors of DNA
replication.1,2 Later work showed that MSI may be
attributed to an underlying defect of the DNA mismatch repair genes,
including MLH1, MSH2, PMS1, PMS2, and MLH6.3-6 The overall
findings of several studies that addressed MSI occurrence in
hematologic neoplasms indicate that MSI is uncommon in acute myeloid
leukemia (AML), as it is generally detected in less than 10% of
patients.7-12 This is not surprising because the
predominant type of genetic damage in hematopoietic tumors consists of
specific translocations juxtaposing genes that are normally distant in
the genome, thereby activating proto-oncogenes and creating new fusion
proteins.13 Nevertheless, alterations in DNA repair genes
might play a role in defined subsets of hematoproliferative disorders,
and in particular, lymphoid tumors. Indeed, studies in MSH2- and
MSH6-deficient mice clearly establish a link between mismatch repair
gene defects and development of lymphoid tumors.14-16 Consistent with this, MSI has been detected in lymphomas from human
immunodeficiency virus (HIV)+ subjects,17 and
MSH2 mutations have been reported in adult lymphoblastic
lymphoma,18 thus suggesting that a similar connection might
also exist in man. Our study explores this hypothesis by investigating
microsatellite alterations in human lymphoid neoplasms, and addressing
their relationship to rearrangements of the proto-oncogene, bcl-2,
often involved in the pathogenesis of lymphoid neoplasms as an
inhibitor of apoptosis (reviewed by Chao and Korsmeyer19).
In hereditary and sporadic solid tumors, MSI is generally linked to
point mutations of DNA mismatch repair genes, and gene inactivation by
deletions is uncommon.3-6 This relationship, however, has
been rarely if at all addressed in hematopoietic tumors. As chromosomal
aberrations are commonly found in leukemia and lymphoma, we
investigated their contribution to the inactivation of MLH1, MSH2,
PMS1, and PMS2 genes; we report that loss of heterozygosity (LOH) involving microsatellite markers closely
associated with mismatch repair genes occurs in some hematopoietic
tumors with MSI.
Patients and DNA isolation.
We analyzed 66 samples of tumor DNA obtained from the Third Medical
Clinic of the University of Heidelberg (Heidelberg, Germany) and from
the Divisione di Ematologia of the University of Verona (Verona,
Italy). These samples corresponded to 45 cases of leukemia (29 AML, 16 acute lymphoid leukemia [ALL]), and 21 of non-Hodgkin's lymphoma
(NHL), as detailed in Table 1. Tumor DNA
was extracted from bone marrow biopsies or peripheral blood in the case
of leukemias and from bone marrow or lymph nodes in the case of NHL.
Control constitutional DNAs were extracted either from the leukocytes or bone marrow of the same patients during remission or from
lymphoma-unaffected bone marrow samples of some NHL patients. The AMLs
were classified morphologically according to the
French-American-British classification,20 and the NHLs
according to the Revised European-American Lymphoma (REAL)
classification.21 ALLs were classified according to their immunophenotype.22 High-molecular-weight DNA was isolated
by standard procedures.23 Cytogenetic analysis was
performed on short-term cultures of bone marrow or peripheral blood
cells. Chromosomes were analyzed using a modified GAG-banding
technique, as described elsewhere.24
Microsatellite analysis.
DNA was amplified by polymerase chain reaction (PCR), as previously
described,8 in a volume of 25 µL containing 0.1 µg of
genomic DNA as template, 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.3),
1.5 mmol/L MgCl2, 0.2 mmol/L of each deoxynucleoside triphosphate, 0.4 µmol/L of each sense and antisense primer, and 1 U
of Taq Polymerase (Perkin Elmer, Norwalk, CT). The primer pairs listed
in Table 2 were used. Forward primers were
end-labeled with [
Molecular analysis of bcl-2 rearrangements.
Ten micrograms of high-molecular-weight genomic DNA was digested
independently with 2 restriction endonucleases (EcoRI and HindIII) under conditions recommended by the supplier
(Boehringer Mannheim, Mannheim, Germany). After electrophoresis on 1%
agarose gels, DNA was transferred to nylon membranes (Bio Rad, Munich, Germany) according to the manufacturer's instructions. For the hybridization of Southern blots, denatured 32P-labeled DNA
probes were independently used for the major (MBR) and the minor (mcr)
breakpoint cluster regions, PFL1 and PFL2, respectively,25,26 both kindly supplied by Dr M.L. Cleary
(Stanford University School of Medicine, Stanford, CA).
The labeling of the DNA probes by random priming was performed under
conditions recommended by the supplier (Amersham). Autoradiography was
performed at Mismatch repair genes analysis.
We performed LOH studies on the MLH1, MSH2, PMS1, and PMS2 DNA repair
genes.3-5 Briefly, LOH was investigated by the same PCR
assay described above, using the D3S1611, CA21, D2S117, and D7S517
microsatellite markers to study the MLH1, MSH2, PMS1, and PMS2 genes,
respectively. The sequence of these primers has been reported
elsewhere.27,28 The intensity of the bands corresponding to
the 2 alleles of a given marker was quantified by analysis with
Instantimager (Packard, Grove Hills, IL). In a set of experiments, the
microsatellite markers D2S119, D2S136, D2S288, D2S378, D3S1561, D3S1298, D7S531, D7S481, and D7S503 were used28 to
characterize the LOH involving the MLH1, MSH2, and PMS2 genes.
P53 gene mutation analysis.
P53 gene mutations were detected by PCR-SSCP analysis of exons 5 to 8 with specific primers as described above.32
Microsatellite alterations in leukemias and NHL.
We looked for microsatellite alterations in 10 microsatellite markers
scattered on 7 different chromosomes, as detailed in Table 2 and in
Materials and Methods. MSI was defined as a gain of new alleles at a
given locus; LOH was a significant attenuation (>50%), or loss of 1 normal allele in the tumor, compared with constitutional DNA; some
representative alterations found in leukemias are shown in
Fig 1. The bands indicating MSI, in some
cases, were not equimolar to the germ line bands, likely due to
contaminating normal cells, as also suggested by cytological analysis
(data not shown), or heterogeneity within the leukemic poulation. PCR analysis disclosed molecular changes in only 3 of the 29 AML patients analyzed (10%), thus confirming the scarcity of MSI in adult myeloid leukemia.8-10 Interestingly, however, these alterations
were detected in 10 of 37 lymphoid tumors (27%), including 6 of 16 ALL
patients (37%) and 4 of 21 NHL patients (19%). Thus, in our study
population, MSI was more frequently observed in ALL than AML patients
(P < .05, Fisher's exact test). Molecular alterations,
listed in Table 3, were found on 1 to 3 loci in 9 of 13 cases; on the other hand, patient 213 (AML), and
patients 32, 509, and VR5 (all with ALL) presented widespread genetic
instability involving most of the loci analyzed and consisting of both
MSI and LOH. Overall, MSI was more frequently detected, accounting for
33 of 44 molecular alterations found, while the remaining 11 were LOH
at defined loci (Table 3). MSI was found in tumor DNA obtained at
diagnosis in 6 leukemia patients (702, 739, 619, 32, 509, and VR5), and 2 NHL patients (11, 23), thus indirectly suggesting that alterations in
mismatch repair genes might already occur in the early steps of the
malignancy (Table 4). In the remaining 5 patients, alterations were detected in tumor DNA obtained at relapse,
after chemotherapy. Overall, MSI in leukemia was detected in 3 of 9 patients studied at relapse (33%) and in 6 of 36 patients studied at
diagnosis (16%) (Tables 4 and 5). However,
because DNA samples at diagnosis were not available, it could not be
established whether the same alterations were already present at
disease onset or had developed during tumor progression.
Microsatellite alterations as a disease marker in leukemia.
Given the relatively frequent occurrence of microsatellite
alterations in ALL, it would be important to establish whether they
might be useful as molecular markers of disease. To address this point,
serial DNAs collected from a B-ALL patient (patient 74) over a 14-month
follow-up were analyzed by PCR. Alterations in marker D15S161,
consisting of LOH, were first shown in DNA collected during a relapse
that occurred in June 1990 (Fig 2). As
expected, LOH disappeared when this patient achieved remission (August
1990), and was no longer detected at different time points when the
patient was still in remission (Fig 2). However, this same molecular
alteration was again detected during a second relapse, which occurred
about 1 year later and then disappeared after effective therapy and
achievement of remission (Fig 2). Marker D3S1611, which is closely
associated with mismatch repair gene MLH1, showed the same behavior
(Fig 2).
Association between MSI, bcl-2 rearrangements, and p53 mutations in
NHL patients.
In an attempt to correlate MSI with genetic alterations of known
oncogenes, the DNA from all 21 NHL patients was analyzed by Southern
blotting, as detailed in Materials and Methods, for rearrangements of
the bcl-2 gene, whose involvement in lymphomagenesis is well
established.25,26,33 Strikingly, bcl-2 rearrangements were
detected in 4 of 4 patients with MSI, but only in 3 of 17 patients
without MSI (Fig 5 and
Table 6). This difference, which is
significant (P < .05, Yate's corrected
Detection of LOH involving mismatch repair genes in some leukemia
patients showing MSI.
Because MSI in solid tumors is linked to the functional inactivation of
mismatch repair genes, including MLH1, MSH2, PMS1, PMS2, and, recently,
MSH6,3-6 we investigated whether this might also be true
for MSI in leukemias and lymphomas. We looked for mutations affecting
the MLH1 and MSH2 genes; given the complexity of these genes, we
focused on exons 9 and 16 of MLH1, and exons 5 and 13 of MSH2, all of
which have been reported to be mutated in some solid and also lymphoid
tumors.18,36 We examined samples from all the patients who
were positive for MSI and were unable to demonstrate tumor-specific
mutations by SSCP analysis. Genomic DNA from a healthy donor,
containing a known mutation in the second domain of the CD4
gene,29 was also simultaneously analyzed by SSCP and served
as a control for our SSCP experimental conditions (data not shown).
The last few years have witnessed major advances in the decoding of the
molecular basis of acute leukemias; indeed, in many cases (up to 50%)
a specific chromosomal translocation, often involving genes that encode
transcription factors, is involved in the pathogenesis of the
disease.13,37 In the other cases, however, translocations
are random or do not occur at all, thus leaving the origin of the
malignancy unexplained. Therefore, other types of genetic damage, for
example those affecting tumor suppressor genes and DNA repair genes,
might occur and participate in the leukemic transformation. In view of
this likelihood, a number of recent studies addressed the occurrence of
microsatellite alterations in hematologic malignancies as indirect
evidence of disruption of DNA repair genes.7-12,38 The
overall conclusion reached by most of these studies was that, unlike
findings in some hereditary and sporadic solid tumors, MSI is uncommon
in human leukemia and is detected only in a minority of cases, with the
possible exception of therapy-related leukemia.39
Remarkably, AML was the predominant type of leukemia investigated in
these studies; less is known about MSI occurrence in lymphoid tumors.
We are grateful to Dr R. Zamarchi for statistical analysis, P. Gallo
for artwork, and P. Segato for precious help in the preparation of the
manuscript. We also thank Dr Fabrizio Vinante and the Divisione di
Ematologia of the University of Verona, Italy, for bone marrow samples
from ALL patients.
Submitted June 10, 1998; accepted May 23, 1999.
Supported by grants from Associazione Italiana Ricerca sul Cancro
(AIRC) and Fondazione Italiana Ricerca sul Cancro (FIRC).
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.
Address reprint requests to Stefano Indraccolo, MD, Department of
Oncology and Surgical Sciences, via Gattamelata, 64, 35128-Padova,
Italy; e-mail: indra{at}ux1.unipd.it.
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TOP
ABSTRACT
INTRODUCTION
33P] adenosine triphosphate (ATP)
for 1 hour at 37°C using T4 polynucleotide kinase (Amersham, Little
Chalfont, UK). Reaction conditions consisted of 1 minute at 94°C,
30 seconds at 55°C, and 30 seconds at 72°C for 30 cycles. PCR
products were electrophoresed on denaturing 6% polyacrylamide
formamide-containing gels. After electrophoresis, the gel was dried and
exposed to x-ray film at 
70°C for 24 to 72 hours.
-33P-dATP and 0.1 U Taq polymerase, and 5 additional
cycles were run. To detect DNA mutations, PCR product samples were
heated at 95°C for 5 minutes and then electrophoresed through a 6%
acrylamide gel containing 5% glycerol; the gel was then dried, and
exposed to x-ray film at 
2
test) cannot be accounted for by differences in lymphoma histotype between MSI+ and MSI
patients (Table 
-MYH11, and PML-RAR