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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2424-2432
Mutator Phenotype in Human Hematopoietic Neoplasms and Its
Association With Deletions Disabling DNA Repair Genes and bcl-2
Rearrangements
By
Stefano Indraccolo,
Sonia Minuzzo,
Laura Nicoletti,
Elisabetta Cretella,
Martin Simon,
Georg Papakonstantinou,
Rüdiger Hehlmann,
Marta Mion,
Roberta Bertorelle,
Jelena Roganovic, and
Luigi Chieco-Bianchi
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.
 |
ABSTRACT |
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.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
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.
| |
MATERIALS AND METHODS |
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 [ 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.
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 70°C using x-ray film with double high-speed
intensifying screens.
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.
For single-strand conformation polymorphism (SSCP) analysis, exons 9 and 16 of MLH1 and exons 5 and 13 of MSH2 were amplified by PCR
according to previously reported procedures,29 using exon-specific oligonucleotide primers reported by
others.30,31 Briefly, 25 PCR cycles were run in 50 µL
standard reaction mixture containing 0.2 µg of DNA, 0.4 µmol/L of
each primer, and 2 U Taq polymerase (Perkin Elmer) as follows: 1 minute
denaturation at 94°C, 30 seconds annealing at 55°C, and 30 seconds extension at 72°C in a DNA thermal-cycler (Perkin Elmer). A
total of 10 µL of the amplified products was mixed with 0.1 µCi
 -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 70°C for 24 to 72 hours.
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
| |
RESULTS |
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.
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| Fig 1.
Microsatellite alterations in adult acute leukemias. PCR
amplification of the indicated microsatellites was performed as
described in Materials and Methods on genomic DNA of AML patients at
diagnosis (L) or relapse (Rel), and the resulting banding patterns were
compared with those obtained at remission (R). Tumor-specific
alterations consisted either of gain of new-length alleles (MSI),
indicated by arrows, or loss of bands detected in constitutional DNA
(LOH).
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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).
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| Fig 2.
LOH as clonal marker in ALL. PCR analysis in 1 B-ALL case
(74) detected identical LOH patterns in leukemic cell DNA in 2 consecutive relapses (lanes 06/90 and 05/91, respectively). This was
observed with 2 different microsatellite markers, respectively, on
chromosome 3 (D3S1611) and 15 (D15S161). R, remission; Rel, relapse.
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To determine whether microsatellite alterations might be a marker of
impending relapse, 4 consecutive DNA samples from another ALL patient
(patient 63) were analyzed for MSI involving markers vWA and D2S123
(Fig 3). MSI could be shown in both markers
during impending and full-blown relapse and consisted of faint bands of
increased size, compared with the alleles detected at remission (Fig
3). Conventional bone marrow cytology of this patient showed hypercellularity with 50% blasts at initial relapse and 90% blasts at
full relapse. In view of these findings, we attempted to measure the
sensitivity of the PCR assay used to detect MSI. To this end, genomic
DNAs from 2 healthy individuals showing a different allele for marker
D18S61 were mixed in different proportions, amplified with
D18S61-specific primers, and analyzed on acrylamide gel. As shown in
Fig 4, the B-specific allele of 1 individual could be detected by this method when his DNA represented at
least 3% of the total DNA used as template. According to our estimate, therefore, 3% leukemic cells in the bone marrow sample represent our
current limit of detection in disease affected tissues. Even though
these observations are limited to 2 cases, they suggest that the assay
might potentially be applied to monitor minimal residual disease and
disease progression in those leukemia patients who present with
microsatellite alterations at diagnosis.
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| Fig 3.
MSI as impending relapse marker in ALL. In 1 ALL case
(63), MSI was observed by PCR analysis in 2 different loci (vWA,
D2s123) at both early (lane 12/90) and full (lane 04/91) relapse.
Tumor-specific alterations are indicated by arrows. R, remission; Rel,
relapse.
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| Fig 4.
Sensitivity of the PCR-based assay in detecting
microsatellite alterations. Two DNAs from healthy donors (A and B)
showing a different allele for the D18S61 marker (arrowed) were mixed
at the indicated percent to obtain a constant final amount of 100 ng
DNA as a PCR template. A content of 3% of the donor B DNA showing the
lower allele is required for a faint band to appear in the
autoradiography. This corresponds to a sensitivity of about 1.5% novel
allele DNA in a given sample under these PCR conditions.
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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  2
test) cannot be accounted for by differences in lymphoma histotype between MSI+ and MSI patients (Table 6)
and suggests an association between MSI and bcl-2 rearrangements in
NHL. Finally, in view of the possibility that lymphoma cells might
carry multiple genetic alterations, we also addressed the presence of
mutations involving the p53 gene. As previous reports showed that most
p53 mutations in NHL cluster in exons 5 to 8,34,35 these
exons were analyzed by reverse transcriptase (RT)-PCR in those NHL
patients with MSI; no mutations were found in these patients: genomic
DNA from colon cancer patients, with known mutations for the exons
considered, was also analyzed and served as a positive control for the
SSCP assay. In our patients, therefore, the presence of MSI and bcl-2 rearrangements did not correlate with p53 abnormalities.
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| Fig 5.
Southern blot analysis of genomic DNA. Autoradiography
after hybridization with 32P-labeled DNA probes for
detection of bcl-2 gene rearrangements in major (lanes 23, 52, 11) and
minor breakpoint region (lane 298). Lanes 23, 52: follicle center
lymphomas; lane 11: diffuse large B-cell lymphoma; lane 298: mantle
cell lymphoma. Dashes indicate the germline bands and arrowheads the
rearranged fragments. The molecular-weight marker is indicated on the
left.
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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).
As chromosomal rearrangements in hematologic neoplasms are a major
cause of gene disruption,37 we advanced
that deletions affecting chromosomes 2, 3, and 7 carrying the MSH2 and
PMS1, MLH1, and PMS2 genes, respectively, might occur, and lead to
their inactivation in some patients. To test this hypothesis, genomic DNA from patients showing MSI was amplified with primer pairs for
D3S1611, CA21, D2S117, and D7S517, which are microsatellite markers
closely linked to MLH1, MSH2, PMS1, and PMS2,
respectively.27 All mutated NHL and leukemia patients
except patients 213 and VR5 were informative for D3S1611 and CA21; all
patients, except VR5, were informative for D2S117, and all except
patients 619, 52, and 298 for D7S517. 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 microsatellite alterations. In particular, among the ALL patients, patient 32 presented LOH at the D3S1611 site
(MLH1), patient 63 at the CA21 site (MSH2), patient 74 at the D3S1611
and D7S517 sites (MLH1 and PMS2), and patient 509 at the D2S117 (PMS1)
site; AML patient 213 presented LOH at the D7S517 site (PMS2)
(Fig 6). On the other hand, neither 6 RER ALLs, nor 6 RER AMLs and 4 RER+ NHL presented LOH at the same chromosomal loci (data
not shown). To evaluate whether the observed LOH was confined to a
particular marker or was indicative of larger choromosomal deletions,
LOH at mismatch repair gene loci was analyzed in further detail in 3 patients (63, 74, and 213). To this end, microsatellite markers mapping
close to D3S1611, CA21, and D7S517 were chosen for amplification, as
reported in Materials and Methods. In the case of patient 74, this
analysis disclosed that LOH extended to markers located both centromeric (D3S1561 and D7S531) and telomeric (D3S1298 and D7S503) to
D3S1611 and D7S512 (Fig 7); marker D7S481
was not informative in patient 74. On the other hand, in the case of
patients 63 and 213, no additional LOH was demonstrated at these loci.
These findings suggest that in some leukemia patients MSI is linked to
inactivation of DNA repair genes by chromosomal deletions.
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| Fig 6.
LOH involving mismatch repair genes loci; some
representative alterations are shown. Genomic DNA from ALL patients 63, 74, and AML patient 213 was PCR-amplified for microsatellites D3S1611,
CA21, and D7S517, 3 genetic markers respectively associated with the
MLH1, MSH2, and PMS2 genes, as reported in Materials and Methods. R,
remission; Rel, relapse.
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| Fig 7.
LOH involving loci flanking D3S1611(MLH1) and D7S517
(PMS2) in ALL patient 74. Genomic DNA was PCR-amplified for
microsatellites D3S1561, D3S1298, D7S531, and D7S503, as reported in
Materials and Methods. R, remission; Rel, relapse. A schematic
chromosome map indicating the location of the markers used and their
approximate distances in centimorgans is also
shown.28 Marker D7S481 was not informative in patient 74 (data not shown).
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DISCUSSION |
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 compared the prevalence of microsatellite alterations in lymphoid
versus myeloid tumors and found a higher prevalence of MSI in ALL than
in AML. Although this finding was expected on the basis of previous
observations, an explanation for it is presently not forthcoming.
Indeed, it is known that knockout mice lacking the murine equivalent of
mismatch repair genes MSH2 and MSH6 develop lymphoid malignancies
resembling human lymphoblastic lymphoma with high
frequency.14-16,18 Furthermore, a screening of human cell
lines established from leukemia/lymphoma for MSI disclosed genetic
instability exclusively in lymphoid cell lines.36 Finally, it is also known that HNPCC kindreds develop an excess of lymphoid tumors.40,41 Overall, these observations and our findings
suggest a connection between inactivation of mismatch repair genes and subsequent MSI on 1 side and lymphoid tumorigenesis on the other.
In contrast to the results of Pabst et
al,11 we detected MSI much more frequently than LOH; this
discrepancy might probably be explained, at least partially, by
differences in the microsatellite markers used, as well as the patient
population. Interestingly, 2 of 6 ALLs with MSI belong to the rare
immunophenotypic group of mature B-cell ALL, which constitute about 4%
of ALLs: this might indicate a nonrandom association between the 2. Clearly, only investigation of MSI prevalence in this rare subset of
ALLs and in the closely related B-lymphoblastic lymphoma will
definitively resolve this issue. In our patients, MSI seemed to be an
adverse prognostic factor. Two of the 6 leukemic patients with MSI
detected at diagnosis (patients 702 and 619) relapsed within 15 months, and the relapse was refractory to chemotherapy. In patient 702, the
leukemic cells harbored a translocation t(8;21) that normally associates with a favorable outcome.42 Furthermore, the 4 NHL patients with MSI also had an overall poor response to
chemotherapy. This contrasts with findings in colorectal cancer, where
MSI seems to be a good prognostic indicator2; 1 possible
explanation for this discrepancy might be that MSI in leukemia is
correlated with resistance to chemotherapy, which usually is the
first-line therapy for hematologic neoplasms. Intriguingly, human cell
lines with mutator phenotype carrying MLH1 mutations are tolerant to some DNA-alkylating agents, thus suggesting that a functioning mismatch
repair system might favor the cytotoxic effects of these drugs.43 A future area of investigation will try to address whether MSI detected at diagnosis might represent a negative prognostic indicator for the outcome of chemotherapy.
Of particular relevance is the finding that in NHL patients MSI is
associated with rearrangements of bcl-2, a proto-oncogene whose product
is involved in inhibition of apoptosis.44 Chromosomal translocations resulting in high-level bcl-2 expression have been detected in the majority of follicular neoplasms, in about one third of
diffuse large B-cell lymphomas and less commonly in other NHL.33 In agreement with this, we did not detect MSI in
those lymphoma types, which are not commonly associated with bcl-2
rearrangements (Table 6). In view of the pathogenic relevance of bcl-2,
it is tempting to speculate that an increased cell survival conferred by bcl-2 overexpression and genomic instability due to DNA repair gene-defects might cooperate in facilitating the inclusion of new
mutations in the genome, possibly increasing the malignancy of the
tumor. We also looked for other associated genetic abnormalities and
could not detect p53 mutations in the RER+ NHL patients.
This finding, however, was not completely unexpected, as a negative
association between MSI and p53 gene mutation in colorectal cancer was
previously reported.45
Given the relative frequency of MSI in ALL, we believe that
microsatellite analysis might be useful in monitoring the disease course in some leukemia patients. Leukemias constitute
a unique opportunity to study tumor progression because the neoplastic cells are easily accessible, and the bone marrow is routinely sampled
for conventional cytologic evaluation. Although observed in only a few
patients, our study shows that the same microsatellite alterations can
be consistently detected during follow-up and might be predictive of
impending relapse. The advantage of an early detection of leukemia
relapse by microsatellite analysis would be to begin chemotherapy at an
early stage, when the tumor burden is still low. However, the limited
sensitivity of this technology is a critical factor; some investigators
suggest that it might be feasible to increase it to detect 1 neoplastic
cell among 500 normal cells.46 Compared with some other
tumor-specific molecular markers, such as K-ras mutations (detected in
1 of 10,000 cells), this is still low, but might be sufficient in many
situations. While specific genetic changes are very sensitive tumor
markers, including the BCR-ABL, MLL-AF4, E2A-PBX1, and TEL-AML1 fusion genes in ALL, and AML1-ETO, CBF -MYH11, and PML-RAR in
AML,13 unfortunately they are not always observed.
Microsatellite analysis is easy to perform and also reproducible by
nonradioactive methods47; these features should facilitate
its application in future studies aimed at assessing the relevance of
microsatellite alterations as a tumor marker in lymphoid malignancies.
Although many studies describe MSI in hematologic neoplasms, its
relationship to genetic alterations of DNA repair genes has only been
marginally explored. To address this issue, we looked for point
mutations in a limited number of exons of MLH1 and MSH2, but found
none. Admittedly, this was not surprising given the complexity of these
genes and the reported absence of clusters of mutations in
RER+ tumors.30,31 To fully characterize
mutations of DNA repair genes in human hematologic malignancies, it
would be necessary to analyze all of the exons of MLH1 and MSH2 and
perhaps extend these investigations to the PMS1 and PMS2 genes. On the
other hand, our analysis of the chromosomal loci of these genes using closely linked microsatellite markers showed LOH in 4 of 6 RER+ ALLs, and 1 of 3 RER+ AMLs, thus showing
that chromosomal deletions might contribute to inactivate mismatch
repair genes, at least in some leukemia cases. As defects in mismatch
repair genes are apparent only in completely knocked-out
cells,27,48 it could be speculated that the remaining
allele in leukemias positive for LOH carries disabling mutations. We
advance that chromosomal deletions might also
contribute to leukemogenesis by targeting and inactivating genes
involved in DNA repair.
| |
ACKNOWLEDGMENT |
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.
| |
FOOTNOTES |
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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INTRODUCTION