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Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4365-4374
By
From the Hematopathology Section, Laboratory of Anatomic Pathology,
and Department of Hematology, Hospital Clínic, Institut
d'Investigacions Biomèdiques "August Pi i Sunyer"
(IDIBAPS), University of Barcelona, Barcelona, Spain; the Department of
Cellular Biology and Physiology, Autonomous University of Barcelona,
Barcelona, Spain; the Laboratory of Anatomic Pathology, Hospital Virgen
de la Salud, Toledo, Spain; and the Department of Hematology, Hospital
Clínico, Universidad de Salamanca, Salamanca, Spain.
Mantle cell lymphomas (MCLs) are characterized by 11q13 chromosomal
translocations and cyclin D1 overexpression. The secondary genetic and
molecular events involved in the progression of these tumors are not
well known. In this study, we have analyzed 45 MCLs (32 typical and 13 blastoid variants) by comparative genomic hybridization (CGH). To
identify the possible genes included in the abnormal chromosome
regions, selected cases were analyzed for P53,
P16INK4a, RB, C-MYC, N-MYC, BCL2, BCL6,
CDK4, and BMI-1 gene alterations. The most frequent
imbalances detected by CGH were gains of chromosomes 3q (49%), 7p
(27%), 8q (22%), 12q (20%), 18q (18%), and 9q34 (16%) and losses
of chromosomes 13 (44%), 6q (27%), 1p (24%), 11q14-q23 (22%),
10p14-p15 (18%), 17p (16%), and 9p (16%). High-level DNA amplifications were identified in 11 different regions of the genome,
predominantly in 3q27-q29 (13%), 18q23 (9%), and Xq28 (7%). The CGH
analysis allowed the identification of regional consensus areas in most
of the frequently involved chromosomes. Chromosome gains
(P = .02) and losses (P = .01) and DNA
amplifications (P = .015) were significantly higher in
blastoid variants. The significant differences between blastoid and
typical tumors were gains of 3q, 7p, and 12q, and losses of 17p. CGH
losses of 17p correlated with P53 gene deletions and mutations.
Similarly, gains of 12q and high-level DNA amplifications of 10p12-p13
were associated with CDK4 and BMI-1 gene
amplifications, respectively. One of 2 cases with 8q24 amplification
showed C-MYC amplification by Southern blot. Alterations in 2p,
3q, 13, and 18q were not associated with N-MYC, BCL6, RB, or
BCL2 alterations, respectively, suggesting that other genes may
be the targets of these genetic abnormalities in MCLs. Increased number
of gains (0 v 1-4 v >4 gains per case) (P = .002), gains of 3q (P = .02), gains of 12q
(P = .03), and losses of 9p (P = .003) were
significantly associated with a shorter survival of the patients. These
results indicate that an increased number of chromosome imbalances are
associated with blastoid variants of MCLs and may have prognostic significance.
MANTLE CELL LYMPHOMA (MCL) is a malignant
lymphoproliferative disorder derived from naive pregerminal center
CD5+ B cells expressing IgM/D.1
Morphologically, the typical variant of MCL is composed of a monotonous
proliferation of small to medium-sized lymphocytes with irregular
nuclei and relatively low proliferative activity.1 Several
studies have also recognized a blastoid variant in which the cells may
show either rounder nuclei with fine and disperse chromatin resembling
lymphoblasts or larger, deeply indented, and pleomorphic nuclei with
occasional nucleoli.2,3 These blastoid variants have a
higher proliferative activity and a more aggressive biological behavior
than the typical variants of the tumor.2-4
Genetically, MCLs are characterized by chromosomal translocations
involving the 11q13 region and its molecular counterpart bcl-1
rearrangement, which results in the overexpression of cyclin D1
gene.5,6 The identification of this translocation in
virtually all cases of MCL and the constant cyclin D1 overexpression in the tumors indicate that these molecular phenomena are important mechanisms in their pathogenesis.7,8 Experimental studies have shown that cyclin D1 may function as an oncogene in the malignant transformation of different cell types. However, the tumorigenic and
transforming properties of cyclin D1 seem to be less effective than
other oncogenes.9 On the other hand, cyclin D1 transgenic animals do not develop spontaneous lymphomas, and lymphomagenesis in
these animals requires the cooperation with other oncogenes such as
C-MYC.10 All of these findings suggest that other
mechanisms, in addition to cyclin D1 deregulation, may participate in
the development and progression of MCLs.
The secondary genetic and molecular events involved in the pathogenesis
of these tumors are not well known. Recent studies indicate that
blastoid variants of MCLs harbor more frequent bcl-1 rearrangements at
the major translocation cluster locus2 and have a higher
incidence of P53 gene mutations11,12 and
P16INK4a deletions than typical
variants.13,14 Classic cytogenetic studies have identified
additional chromosome abnormalities besides the 11q13 translocations in
some MCLs.15-19 However, these studies are limited, and no
correlations with the morphologic variants or the biological behavior
of the tumor have been described. Chromosomal banding techniques are
useful for the identification of chromosomal aberrations and
imbalances, but they are less reliable for the recognition of
potentially amplified regions. On the other hand, these techniques
require the analysis of metaphases upon cell culture that may induce a
certain subclone selection and underrepresentation of tumor clones. The
relatively new technique of comparative genomic hybridization (CGH)
allows a rapid analysis of chromosomal imbalances within the tumor
genome including mapping of high-level DNA amplifications without the
requirement of cell culturing and metaphase preparation.20
The aims of this study were to determine the secondary chromosomal
imbalances and high-level DNA amplifications that may play a role in
the development and progression of MCLs, to analyze the potential
involvement of specific genes located in the altered chromosomal
regions in the different variants of MCLs, and to determine the
clinical and pathological relevance of these genetic alterations.
Case selection.
Tumor specimens from 45 MCLs were included in the study. There were 33 males and 12 females. A total of 29 cases were obtained from the
Hospital Clínic Provincial of Barcelona; 10 cases from the
Hospital Clínico Universitario, Salamanca; and 6 from the Hospital Virgen de la Salud, Toledo, Spain. A total of 32 cases were
classified as typical MCLs and 13 as blastoid variants of MCLs,
according to previously described criteria.1-3 All cases were studied at diagnosis and were reviewed and classified by three of
us (E.C., M.A.P., and T.F.). The immunophenotype of the tumors was
analyzed using immunohistochemistry on tissue sections and/or cell
suspensions by flow cytometry. These studies included Ig light and
heavy chains, several B-cell (CD19, CD20, CD22, CD45RA, and CD79a) and
T-cell (CD2, CD3, CD5, CD7, CD4, CD8, CD45RO, and CD43) markers, CD10,
and CD23. Cyclin D1 expression was examined in all cases by Northern
blot analysis and/or immunohistochemistry.7 Bcl-1
rearrangement was also examined in all cases by Southern blot analysis
or polymerase chain reaction (PCR) according to a previously described
method.21 Cytogenetic analysis could be performed in 7 cases. All tumors included in the study had a B-cell phenotype and all,
except one blastoid case, coexpressed CD5. The only CD5 DNA extraction.
High molecular weight DNA was extracted from 42 lymph nodes and 3 involved peripheral blood with the use of the standard Proteinase K/RNAse treatment and phenol-chloroform extraction. Normal DNA was
obtained from 4 male and 1 female healthy blood donors. DNA was diluted
to a concentration of 40 to 60 ng/µL, and 1 µL of each sample was
analyzed in a 0.8% agarose gel and stained with ethidium bromide to
verify its quality and concentration.
CGH.
Normal and tumor DNA were labeled with Spectrum Red-dUTP and Spectrum
Green-dUTP by nick translation using a commercial kit (Vysis, Downers
Grove, IL). Subsequently, equal amounts of normal and tumor labeled
probes (500 ng) and 10 µg of Cot-1 DNA were coprecipitated with the
use of ethanol. The precipitated DNA was dissolved in 12 µL of
hybridization buffer and denatured at 74°C for 8 minutes. Normal
metaphase spreads (Vysis) were denatured for 5 minutes at 74°C and
hybridized with the DNA mixture in a moist chamber for 2 to 3 days.
Slides were washed according to the protocol supplied by the
manufacturer. Chromosomes were counterstained with
4,6-diamino-2-phenylindole (DAPI), resulting in a G banding-like pattern that was used for chromosome identification.
Southern blot analysis.
Genomic DNA (15 µg) was digested with EcoRI, HindIII,
and/or BamHI restriction enzymes (BRL, Gaithersburg, MD),
separated on 0.8% agarose gels, and transferred to Hybond-N membranes
(Amersham, Buckinghamshire, UK). The membranes were prehybridized;
hybridized with the P53, P16INK4a,
BCL6, BCL2, C-MYC, N-MYC, RB, CDK4, BMI-1, and
Probes.
The P53 probe was a 2.0-kb EcoRI-BamHI fragment
of the p1A65 (pArgSP53) cDNA clone containing the entire coding region
of the human P53 gene, which was kindly provided by Dr L.V.
Crawford (Imperial Cancer Research Foundation, Cambridge,
UK).22 The P16INK4a probe was a
fragment of exon 2 obtained by PCR with the use of primers previously
described.13 The BCL6 probe was a 1.4-kb EcoRI-Bgl II fragment of the partial cDNA clone of
BCL6 gene, which was kindly provided by Dr B.W. Baron
(University of Chicago, Chicago, IL).23 The BCL2
probe was a 1.5-kb HindIII fragment of the partial cDNA clone
of BCL2 gene, which was kindly provided by Dr J. Boix
(University of Lleida, Lleida, Spain).24 The
C-MYC probe was a 1.4-kb Cla I-EcoRI fragment
containing the third coding exon, which was kindly provided by Dr R. Dalla Favera (Columbia University, New York, NY). The N-MYC
probe was a 1.0-kb EcoRI-BamHI coding fragment of exon
2 (Oncor, Gaithersburg, MD). The RB probes were Rb0.9 and Rb3.8
representing the 5' and 3' portions of RB cDNA, which was
kindly provided by Dr R.A. Weinberg (Whitehead Institute, Cambridge,
MA).25 The CDK4 probe was a 1.2-kb
BamHI/Sma I full cDNA, which was kindly provided by Dr
M. Serrano (Centro Nacional Biotecnologia, Madrid,
Spain).26 The BMI-1 probe was a 1.5-kb Pst
I fragment of the partial cDNA, which was kindly provided by Dr M. van
Lohuizen (The Netherlands Cancer Institute, Amsterdam, The
Netherlands).27 Probes were radiolabeled using a random
primer DNA labeling kit (Amersham) with [ Statistical analysis.
Differences among the histologic variants and other initial and
evolutive characteristics of the patients in terms of the CGH
imbalances were compared by the Fisher's exact test (two-tailed). The
differences observed between means of gains, losses or amplifications, and the histologic subtype were compared using the Student's
t-test when the data fulfill the criteria for parametric
statistics. Nonparametric tests were used when necessary
(U-Mann-Whitney). The actuarial survival analysis was performed
according to the method described by Kaplan and Meier,28
and the curves were compared by the log rank test.29
CGH.
Forty of the 45 patients (89%) showed gains (total, 124) or losses
(total, 109) of genetic material (Figs 1 and
2). All of the
altered cases, except for 5, showed more than one chromosome imbalance.
The single alterations identified in these cases were trisomy X (case
35), trisomy 3 (case 17), monosomy 14 (case 41), gain of 18q23 (case
31), and trisomy 20 (case 8). Irrespective of the morphology of the
lymphoma, the most frequent imbalances were gains of chromosomes 3q
(49%), 7p (27%), 8q (22%), 12q (20%), 18q (18%), and 9q34 (16%)
(Table 1). High-level DNA amplifications were identified in 11 different regions of the genome, predominantly in
3q27-q29 (13%), 18q23 (9%), and Xq28 (7%) (Table
2). Eight of the 11 (73%) amplifications
were localized in chromosomal regions in which known fragile sites have
been identified (3q27-q29 and FRA3C, Xq28 and FRAXF, 8q24 and FRA8C,
2p25 and FRA2C, 7p22 and FRA7B, 13q31-q32 and FRA13D, 3q24-q25 and
FRA3D, and 17q23-q25 and FRA17B). The most frequent losses were on
chromosome 13 (44%), 6q (27%), 1p (24%), 11q14-q23 (22%), 10p14-p15
(18%), 17p (16%), and 9p (16%) (Table
3).
Comparison of CGH results with Southern blot analysis.
To identify the possible genes included in abnormal chromosome regions,
selected cases were analyzed for P53,
P16INK4a, C-MYC, N-MYC, BCL6,
BCL2, RB, CDK4, and BMI-1 gene alterations. The results are
summarized in Tables 4 and
5. The status
of the P53 gene was studied by Southern blot and
single-stranded conformational polymorphism (SSCP)
analysis in the 6 blastoid MCLs with 17p losses. Tumors with an
anomalous SSCP pattern were sequenced. Two of these cases showed
homozygous deletions of the gene (cases 5 and 15; Table 4 and Fig
3A), and the other 4 tumors (cases 1, 2, 3, and 38) had point mutations associated with loss of the remaining
allele. Sixteen additional cases with normal chromosome 17 profile were
also examined molecularly, and no P53 alterations were observed
in any of them. Southern blot analysis confirmed a homozygous deletion
of P16INK4a gene in a case with loss of 9p
by CGH (case 12). Two cases (cases 2 and 5) with a 25% reduction in
the CGH profile of chromosome 9p showed around 20% reduction of the
P16INK4a signal by Southern blot. However,
CGH did not detect loss of 9p in 2 tumors (cases 4 and 6) in which
homozygous deletions of P16INK4a gene were
detected by Southern blot. Fifteen additional cases with normal
chromosome 9 profile were also examined and no
P16INK4a gene alterations were observed
(Table 5).
Clinical significance of CGH imbalances.
The clinical characteristics of the current series of MCLs were similar
to those previously reported4: median age of 62 years
(range, 32 to 81 years), male predominance (male/female ratio, 2.7:1),
frequent palpable spleen (53%), advanced stage (stage IV, 90%), and
extranodal involvement (94%), including bone marrow infiltration in
the majority of cases (87%) and high serum LDH levels in 47%.
Patients with losses of 9p showed high serum LDH more frequently than
the reminders (86% v 36%, respectively; P = .03).
No other correlation was found between the clinical and analytical
parameters at diagnosis and the above-mentioned genetic alterations.
After different treatment approaches (polychemotherapy, 34 cases;
monotherapy with alkylating agents, 5 cases; other, 3 cases), the
complete response (CR) rate was 17%. No significant differences were
found in the CR rate according to the presence of the genetic lesions.
Clinical follow-up was available in 42 patients. The overall survival
(OS) was 3.5 years. Median OS for patients with typical histology was
4.8 years, whereas it was 1.6 years for those with blastoid variants
(P = .005). A number of CGH alterations were associated with
a poorer prognosis. The presence of chromosome gains (0 v 1-4 v >4 gains per case) was related to a poor overall survival
(4-year OS: 68%, 58%, and 0%, respectively; P = 0.02; Fig
4A). When the analysis was restricted to
the 29 patients with typical variants, the presence of chromosome gains
(0 gains v 1-4 gains per case v >4 gains
per case) still retained prognostic significance (P = .03).
Moreover, gains of 3q (patients with normal 3q v patients with
gains of 3q; 4-year OS: 63% v 34%; P = .02), gains
of 12q (normal 12q v gains of 12q; 4-year OS: 55% v
18%; P = .03), and losses of 9p (normal 9p v loss of
9p; 4-year OS: 61% v 0%; P = .003) (Fig 4B) were
associated with a significant shorter survival.
In the present study, we have identified a high number of chromosomal
imbalances and DNA amplifications in MCLs. The most recurrent
alterations observed in our series were gains of 3q, 7p, 8q, 12q, 18q,
and 9q34 and losses of chromosome 13, 6q, 1p, 11q14-q23, 10p14-p15,
17p, and 9p, as well as amplification in 11 different sites. Previous
cytogenetic studies had identified different secondary alterations in
MCLs, including gains and trisomies of chromosomes 3, 7, 12, and 18;
losses of 6q, 1p, 11q, and 13q12-q14; and monosomies 9, 13, and
17.15-19 Our study confirms these chromosomes as recurrent
targets in MCLs, but the frequency of the alterations is higher in the
CGH analysis than in conventional cytogenetic studies. In addition, we
have observed certain imbalances, such as gains of 3q and 8q and losses
of 9p, not previously recognized by classic cytogenetics. A recent CGH
study on MCLs showed similar chromosomal alterations in these tumors,
although the number of high-level DNA amplifications was
lower.32
The authors thank Iracema Nayach and Nerea Peiró for their
excellent technical assistance and Eva Cid for her linguistic advice.
Submitted June 29, 1998; accepted February 9, 1999.
Supported by Grant No. SAF 99/20 from CICYT, Grant No. SAF 96/177,
Maratón-TV3 Cáncer, Asociación Española contra
el Cáncer, and Generalitat de Catalunya SGR52/96. S.B., M.P., and
L.H. were fellows supported by Spanish Ministerio de Educación y
Cultura (S.B.), Maratón-TV3 Cáncer (M.P.), and
Fundació Rius i Virgili (L.H.).
S.B. and M.R. contributed equally to this study.
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 Elías Campo, MD,
Laboratory of Pathology, Hospital Clínic, Villarroel 170, 08036-Barcelona, Spain; e-mail: campo{at}medicina.ub.es.
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TOP
ABSTRACT
INTRODUCTION
tumor had a bcl-1 rearrangement at the MTC locus detected by Southern
blot and PCR, and cyclin D1 overexpression by Northern blot and
immunohistochemistry. Cyclin D1 overexpression was observed in all
tumors. Bcl-1 rearrangement was detected in 20 (44%) tumors. In
addition, 4 of the 7 cases examined by cytogenetic analysis had the
t(11;14)(q13;q32) translocation.
-ACTIN probes; and washed as previously
described.
-32P]-dCTP.
To normalize the DNA loading, the blots were hybridized with a
