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RAPID COMMUNICATION
Mutator Phenotype in a Subset of Chronic Lymphocytic Leukemia
By Ronald Gartenhaus, Michael M. Johns 111, Ping Wang, Kanti Rai, and David Sidransky
The replication error phenotype (RER'), characterized by widespread microsatellie instability, is an important feature of tumors from patients with herediry nonpolyposis colorectal
carcinoma (HNPCC).This widespread instability affects repeat
tracts of all lengths and is usually attributed to mutations of
critical mismatch repair genes. Recently, several reports described occasional microsatellite alterations in tumors not associated with HNPCC. However, a true mutator phenotype
C
HRONIC LYMPHOCYTIC leukemia (CLL) is the
most common leukemia in the Western world.' Ten
thousand new cases are diagnosed annually in the United
In contrast to chronic myelogenous leukemia
(CML) and acute leukemias, CLL is not associated with
exposure to radiation, chemicals or alkylating agents4 Although cytogenetic analysis of CLL has identified two major
chromosomal abnormalities, trisomy 12 and deletions of
13q14 occurring in a minority of cases, the molecular mechanisms underlying development of this leukemia are still
poorly understood.'-' Recently, several groups have identified widespread microsatellite instability manifest as expansions or deletions of simple repeated sequences in primary
cancers.'-" In contrast to neoplasms with rare alterations,
primary tumors with the mutator phenotype (RER') display
microsatellite alterations at the majority of mononucleotide
and dinucleotide tracts. These primary tumors arise in
HNPCC kindreds and many harbor mutations of critical mismatch repair genes including hMSH2, hMLH-1, PMS-I,
PMS-2,''.l5 and the newly described GTBP gene.16
A true mutator phenotype is rare outside of HNPCC-associated tumors and is observed predominantly in nonhematologic ma1ignan~ies.I~
However, recently a subset of human
immunodeficiency virus (H1V)-associated lymphomas were
found to harbor diffuse microsatellite instability." In this
study, we examined 29 cases of CLL for evidence of microsatellite instability and found a true mutator phenotype in a
subset of these cases.
From the Division of Hematology-Oncology, Long Island Jewish
Medical Center, Campus of the Albert Einstein College of Medicine,
New ffyde Park, NY; and the Division of Head and Neck Cancer
Research, the Department of Otolaryngology, Johns Hopkins University, Baltimore, MD.
Submitted September 12, 1995; accepted October 9, 1995.
Supported by a research grant from United Leukemia Fund, Inc.
R.G. is a Scholar of The Helena Rubinstein Leukemia Foundation.
Address reprint requests to Ronald Gartenhaus, MD, Department
of Medicine, Long Island Jewish Medical Center, 269-1 1-76th Ave,
New Hyde Park, NY 11042.
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 1996 by The American Society of Hematology.
0OO6-4971/96/8701-0041$3.00/0
38
(RER') is very rare outside of HNPCC-associatedmalignancies.
We examined 29 cases of chronic lymphocytic leukemia (CUI,
the most common leukemia in the Western world for evidence
of microsatelliteinstability. We identifieda mutator phenotype
in 12/29] 7% of the cases studied. These data suggest that the
mismatch repair pathway may be altered in at least a subset
of patients with CLL.
0 1996 by The American S ~ h t of
y Hematology.
MATERIALS AND METHODS
Purijcation of CLL and T cells. Peripheral blood mononuclear
cells (PBMC) were isolated from patients with CLL and viably
frozen as previously described." The clinical data of these 29 patients with CLL is shown in Table l . A sequential magnetic bead
separation scheme was devised to purify two populations of cells;
a doubly positive CD5, CD19 cell (B-cell CLL phenotype) or a
doubly positive CD3, CD4 (T-cell phenotype) referred to as tumor
or normal cell, respectively. Thawed cells were washed then resuspended in 2 mL of "complete" RPMI 1640 media containing 10%
fetal bovine serum, 50 mg/mL penicillin, 50 pg/mL streptomycin,
100 mg/mL neomycin, and 2 pmol/L L-glutamine (GIBCO, Grand
Island, NY). One-milliliter aliquots of cells were incubated with
magnetic beads coated with either anti-CD5 or anti-CD3 antibodies
(Perceptive Diagnostics, Inc, Cambridge, MA) at a bead/target cell
ratio of approx. 1:1. After a 30-minute incubation on ice the magnetic
bead coated cells were separated using a magnet and resuspended
in 5 mL of fresh media. To permit the magnetic particles to fall
away from the cells because of cell-surface turnover, an additional
30 to 40 hours of incubation at 37°C was performed. The cells were
next pelleted and resuspended in 1 mL of media, with the detached
beads separated by magnet. After transferring the cells to a new test
tube, anti-CD19 or anti-CD4 coated beads (Perceptive Diagnosis,
Inc, Cambridge, MA) were added to either CD5' or CD3+ cells,
respectively. An additional 30-minute incubation on ice was performed and magnetic bead coated cells were isolated by magnet and
used for subsequent DNA preparation. Flow cytometric analysis
confirmed the successful positive selection of the two distinct cell
populations.
Microsatellite marker analysis. DNA was extracted from purified tumor and normal cells using the Micro-Turbo Gen DNA isolation kit according to the manufacturer's instructions (Invitrogen, San
Diego, CA). One microliter (-50 ng) of DNA was used as polymerase chain reaction (PCR) template in a 50-pL reaction mixture as
described below. Primers used to amplify each locus were obtained
from Research Genetics (Huntsville, AL) or synthesized from sequences obtained in the genome data base (GDB). Loci tested included BAT25, BAT40 (mononucleotides), D13S270, D13S284,
D13S272, and D12S87 (dinucleotides), SAT and MJD (trinucleotides), UT703 and D9S747 (tetranucleotides), and transforming
growth fdCtOrp-RII (TGFP-NI) (poly-A) in selected cases. One
primer was end-labeled with
adenosine triphosphate (New
England Nuclear) using the 5' DNA Terminus Labeling System
according the supplier's instructions (GIBCO BRL, Grand Island,
NY). Both tumor and normal DNA were subjected to 40 cycles of
PCR with automated temperature cycling program performed as
below: step-cycle file for 40 cycles: 94°C X 1 minute, 60°C X 2
minutes, and 72°C x 3 minutes; time-delay file: 72°C X 7 minutes
and soak cycle, 4°C. PCR products were analyzed on denaturing 8
mol/L urea/polyacrylamide gels run at 70 W for 90 minutes with
autoradiography performed for 2 to 12 hours.20
Blood, Vol 87, No 1 (January I), 1996: pp 38-41
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39
CLL MUTATOR PHENOTYPE
D9S747
T N
Table 1. Clinical Characteristics
Patient
CLL 7
CLL 8
CLL 11
CLL 16
CLL 28
CLL 30
CLL 32
CLL 36
CLL 37
CLL 39
CLL 48
CLL 52
CLL 64
CLL 65
CLL 71
CLL 73
CLL 77
CLL 88
CLL 89
CLL 117
CLL 136
CLL 138
CLL 140
CLL 141
CLL 142
CLL 145
CLL 146
CLL 147
Rai Stage
IV
111
I
I
I
111
Richter's
II
IV
0
I
I
111
II
IV
IV
Richter's
II
II
111
I
IV
I
I
I
II
I
I
WBC (X10')
59.0
40.0
31.7
46.8
41.8
30.0
49.4
39.1
125.0
34.8
27.2
53.0
74.0
47.6
5.5
55.0
5.0
59.0
56.0
206.1
100.4
37.8
14.4
17.9
16.2
14.0
24.4
153.0
Treatment
Alkylator
Alkylatorlfludarabine
Alkylator
Alkylatorlfludarabine
Alkylator
Alkylator
ND
ND
Alkylator
ND
Alkylator
ND
Alkylatorlfludarabine
ND
AlkylatorIlFN-a
Alkylator
Fludarabine
Alkylator
Alkylatorlfludarabine
Alkylator
ND
Alkylatorlfludarabine
ND
ND
ND
ND
ND
ND
Abbreviations: ND, no drug; IFN-a, interferon-a.
DSS261
T N
BAT25
T
N
TAlO
T
N
CLL 140
Fig 1. Autoradiographs from microsatellite analysis. CLL 140 (TI
and corresponding normal (NI are shown for microsatellite markers
D9S747 (tetranucleotide repeat), D8S261 (dinucleotide repeat), BAT
25 (mononucleotide repeat), and TAlO (TGFD-RII, mononucleotide
repeat) as indicated. D9S474, D8S261, and BAT 25 show a new lower
allele when compared with normal. TAlO shows an unaltered tumor
poly-A repeat with respect t o normal.
were stage I disease according to the Rai classification. The
dinucleotide marker D13S72 was altered in two additional
cases, one a patient in Richters transformation (CLL 65) and
another with stage I1 disease (CLL 77).
Allelic loss in at least one marker was observed in 9/29
(31 %) cases. There was loss of heterozygosity (LOH) at the
microsatellite marker D9S747 in 6/29 (20%) cases. with five
of these having either stage I or I1 disease. The microsatellite
markers D12S87 and D13S72 had loss of one allele in a
Richter's transformation and stage I, respectively. Finally,
the marker D13S284 displayed LOH in two cases, one with
stage I1 and another with stage I disease. The band patterns
demonstrated clear LOH (reduction of band intensity by
>50%) and were reproducible in replicate assays.
DISCUSSION
RES ULTS
Twenty-nine patients with the diagnosis of CLL were
tested for repeat instability by microsatellite analysis (Table
2). Peripheral lymphocytes were obtained and divided into
T and B cell populations by magnetic bead separation followed by DNA isolation of each fraction. A PCR-based
assay was used to analyze 15 unrelated microsatellite loci
including mononucleotide, dinucleotide, trinucleotide, and
tetranucleotide markers. Normal and neoplastic (B-cell)
DNA was amplified and microsatellite alleles were compared
for evidence of new mobility shifts caused by expansion or
deletion of repeat tracts.
Differences at microsatellite loci of neoplastic DNA when
compared with normal DNA were detected in 4 of 29 (14%)
samples tested (Fig 1). Somatic instability at 7 of the I O
microsatellite loci was detected in case 142 and at 10/10 loci
in sample 140. Although mononucleotide alterations are rare,
both cases displayed instability in at least one poly-A tract.
Moreover, both samples with multiple somatic alterations
Table 2. Mutator Phenotype in CLL
Tumor
Tetra
Tri
Di
Mono
CLL 140
CLL 142
212
212
212
112
414
314
212
112
Indicated is the total number of alterations over the total number
of microsatellite markers tested for each nucleotide repeat catagory.
Our data show that diffuse microsatellite instability occurs
in a subset of patients with CLL. In this series of patients,
both cases that exhibited a mutator phenotype were stage I
disease and neither had a peripheral white blood cell count
greater than 20,000. The widespread microsatellite instability
observed in the two cases of CLL presented here are consistent with the replication error phenotype (RER') tumors
found in hereditary nonpolyposis colorectal cancer
(HNPCC). The incidence of microsatellite instability shown
here (7%) is somewhat less than that reported in the literature
for sporadic colorectal carcinoma ( 1 1.6% to 1 S.O%).x'"
Although tumors outside of HNPCC kindreds harbor microsatellite alterations at large (trinucleotide and tetranucleotide) repeat loci,*"~" poly-A alterations and widespread
dinucleotide instability are rare"." (and unpublished observations, February 1995). Recently, TGFP-I1 receptor polyA mutations were described in 80% of sporadic colorectal
tumors with instability,'' potentially leading to abrogation
of the effects of TGFP. Interestingly, both patients with a
true mutator phenotype in this study did not harbor mutations
in the poly-A tract of this receptor (Fig I).
An interesting observation in our group of patients was the
high rate of allelic loss at the microsatellite marker D9S747.
Although losses on chromosome 9p21 are a common occurrence in human tumors,24the significance of loss at D9S747
will require more extensive analysis of this chromosome
before ascribing it a role in the initiation or progression of
CLL. Deletions of 9p21 with loss tumor suppressor genes
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GARTENHAUS ET AL
40
in these region were recently implicated in T-cell but not Bcell A L L . ~ * . ~ ~
The contribution of this RER+ phenotype to leukemogenesis is unclear at present as is any prognostic significance. One
of the challenges in treating patients with CLL is the difficulty
in predicting the clinical course within a certain stage. It would
be of interest to longitudinally follow patients with early stage
CLL and an RER' phenotype to compare the potential prognostic value with that of the commonly used blood lymphocyte doubling time and pattern of bone marrow infiltration as
well as chromosome karyotype. In HNPCC, patients whose
tumors have an RER+ phenotype appear to have a better
prognosis than those without instability.
Several recent reports have extended the number of tumors
believed to manifest the mutator phen~type.~~.~'-*~
However,
few have included analysis of different sized microsatellites
including the mononucleotide repeats such as the poly-A tracts
(BAT 25 and BAT 40)used in this and other studiesz3More
widespread testing of a variety of microsatellite repeats may
more accurately identify a true mutator phenotype, and consequently tumors likely to harbor mismatch repair gene mutations. However, not all RER+ tumors have been found to
contain deficits in mismatch repair."' Therefore, correlation
of microsatellite instability in CLL with mutational analysis
of these mismatch repair genes may provide a direct link for
inactivation of this critical pathway in the development of
CLL.
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From www.bloodjournal.org by guest on December 29, 2014. For personal use only.
1996 87: 38-41
Mutator phenotype in a subset of chronic lymphocytic leukemia
R Gartenhaus, MM 3rd Johns, P Wang, K Rai and D Sidransky
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