Blood, 15 January 2003, Vol. 101, No. 2, pp. 699-702
NEOPLASIA
Brief report
Genetic analysis of de novo CD5+ diffuse large B-cell
lymphomas suggests an origin from a somatically mutated
CD5+ progenitor B cell
Tiemo Katzenberger,
Andreas Lohr,
Stephan Schwarz,
Martin Dreyling,
Julia Schoof,
Christina Nickenig,
Stephan Stilgenbauer,
Jörg Kalla,
M. Michaela Ott,
Hans Konrad Müller-Hermelink, and
German Ott
From the Institute of Pathology, University of
Würzburg, Department of Medicine III, University Hospital
Großhadern/LMU München, Würzburg, Germany;
Clinical Cooperative Group "Leukemia," GSF National Research Center
for Environment and Health, and Department of Internal Medicine III,
University of Ulm, Ulm, Germany.
 |
Abstract |
CD5+ diffuse large B-cell lymphomas (DLBLs) have
recently been described as a particular subgroup of DLBLs. Classical
banding and interphase cytogenetic analyses targeting ATM,
TP53, and P16INK4a genes and the
D13S25 locus from 13 CD5+ DLBLs were compared
with 55 CD5
DLBLs. Additionally, analysis of somatic
mutations of the immunoglobulin heavy chain variable region
(IgVH) genes were performed in CD5+ DLBLs. CD5+
DLBLs were somatically mutated (7 of 8 cases) and were negative for
t(11;14)(q13;q32) and t(14;18)(q32;q21), whereas t(3;14)(q27;q32) was found in only one tumor. Trisomy 3 and gains on chromosomes 16/16p
and 18/18q were significantly overrepresented in CD5+
DLBLs. No ATM deletions were detected. The
prevalence of deletions at the D13S25 locus was
significantly higher in CD5+ DLBLs (4 of 12 [33%])
compared with CD5 DLBLs (4 of 42 [10%]), as were
p16INK4a deletions (33% versus 8%). On the
basis of these findings, CD5+ DLBLs are likely to arise
from the same progenitor cell as the mutated variant of
CD5+ lymphocytic lymphoma/B-cell chronic lymphocytic
leukemia (B-CLL).
(Blood. 2003;101:699-702)
© 2003 by The American Society of Hematology.
| |
Introduction |
Diffuse large B-cell lymphomas (DLBLs) are the most
common type of non-Hodgkin lymphoma (NHL).1 They represent
a heterogeneous category with respect to morphology, immunophenotype,
cytogenetics, and gene expression profiles.2-4 Studies
identified de novo DLBL with CD5 expression (CD5+ DLBL) as
a particular subgroup, suggesting that these tumors may be different
from both CD5+ DLBL developing in the setting of small
lymphocytic lymphoma/chronic lymphocytic leukemia (Richter syndrome)
and CD5 DLBL.5-9 Their clinical
characteristics are elderly onset, female predominance, frequent
involvement of extranodal sites, and a poor clinical
course.9 No BCL1 rearrangements or CyclinD1
expression were demonstrated in those tumors, excluding a possible
association with the blastoid variant of mantle-cell lymphoma
(MCL).5,7,10 Moreover, most CD5+ DLBLs have
been shown to carry somatic mutations in their immunoglobulin heavy
chain variable region (IgVH) genes, further separating them from
MCL.11 Interestingly, in one study, the extent of somatic mutations in de novo CD5+ DLBL was determined to be similar
to that of CD5+ (mutated) B-cell chronic lymphocytic
leukemia (B-CLL), thus allowing for speculations on the
derivation of CD5+ DLBL and CD5+ B-CLL from the
same cell of origin, the B-1 lymphocyte.6
Until now, only rare genetic data,5 and especially
no cytogenetic data on CD5+ DLBL, have been reported. In
this work, we describe classical cytogenetic results in 13 CD5+ DLBLs. Because deletions at the D13S25
locus and of the ATM gene are frequent findings in
CD5+ B-CLL,12 we analyzed these chromosomal
regions by interphase cytogenetics (fluorescence in situ
hybridization [FISH]) and also performed FISH for
TP53 and P16INK4a genes, the
inactivation of which has been strongly associated with lymphoma
progression.13 These data were compared with respective findings in 55 nodal CD5 DLBLs.
| |
Study design |
Thirteen CD5+ DLBLs were compared with 55 CD5 DLBLs according to their morphologic features,
classical and interphase cytogenetic data, and clinical
characteristics. In addition, the molecular configuration of the IgVH
genes in CD5+ DLBL was determined. Classification of the
tumors was performed on high-quality Giemsa-stained slides according to
the World Health Organization (WHO) classification.2
Cytogenetic investigations were done following established
protocols,14 and metaphases were evaluated according to
the International System for Cytogenetic Nomenclature (ISCN)
guidelines.15
Bicolor interphase FISH for TP53, D13S25,
and BCL1 (cases 9 and 10) was carried out according to the
manufacturer's advice (VYSIS, Stuttgart, Germany). The
P16INK4a deletion status was analyzed by a
cosmid contig of approximately 200 kb that had previously been shown to
reliably detect genomic deletions of the INK4a cluster
region.16 For deletions affecting ATM,
biotin-dUTP (deoxyuracil triphosphate)-labeled P1-derived artificial chromosome (PAC) probes specific for the ATM gene
locus17 were applied as previously
described.18,19 Signal visualization was accomplished by
using a Zeiss Axioskop2 fluorescence microscope (ZEISS, Jena, Germany),
and illustrations were made by using the ISIS imaging system
(MetaSystems, Altlussheim, Germany).
Genomic DNA of 8 CD5+ DLBLs was extracted from
cryopreserved tissue blocks. VH-DH-JH gene rearrangements were
amplified by polymerase chain reaction (PCR) by using
family-specific VH forward primers and consensus JH reverse primers
according to the modified protocol of Küppers et
al20 and Campbell et al.21 PCR products were
ligated into pPCR-Script Amp SK (+) cloning vector and transformed into
Epicurian Coli XL-Gold Kan ultracompetent cells (Stratagene, La Jolla,
CA). Usually, 9 clones per case were sequenced bidirectionally and
compared with the nucleotide sequence database Entrez Blast (www.ncbi.nlm.nih.gov/blast/). VH-DH-JH rearrangements were analyzed by
using DNAPLOT (www.dnaplot.de/).
| |
Results and discussion |
On morphology, the series presented here displayed a
remarkable heterogeneity. The CD5+ DLBLs consisted of 8 centroblastic and 3 immunoblastic DLBLs. Two tumors (cases 9 and 10)
displayed particular features: their neoplastic cells were medium- to
large-sized with scant cytoplasm and slightly irregular nuclei, giving
the morphologic impression of blastoid MCL (Figure
1). All cases were CD5+ and
negative for CD23 and CyclinD1. IgD expression was found in 5 of 13 (39%) CD5+ tumors. In the CD5 DLBLs, 8 of 39 cases tested were IgD+ (difference not statistically
significant). Cases 6 and 12, in addition, stained weakly for CD10.
Nuclear expression of BCL6 was demonstrated in more than 10% of cells
in 12 of 12 CD5+ DLBLs and in 46 of 54 (85%)
CD5 DLBLs. There was also no statistically significant
difference between the groups, if only high BCL6 expression (> 60%
of cells) was taken into account (4 of 12 [33%] CD5+
DLBL versus 29 of 54 [54%] CD5 DLBL).
View larger version (102K):
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| Figure 1.
The morphologic spectrum of CD5+
DLBLs.
(A) DLBL centroblastic. (B) DLBL immunoblastic. (C) DLBL
unclassified. Giemsa stain, original magnifications
× 400.
|
|
All 68 patients enrolled in this study were treated by a conventional
anthracycline-containing regimen (cyclophosphamide, hydroxydaunomycin,
vincristine, and prednisone [CHOP]) with or without
radiotherapy. In contrast to recently published clinical data,9 there was no statistically significant difference
in survival between the CD5+ and CD5 DLBLs.
Analysis of somatic hypermutations of IgVH genes revealed a germ line
pattern in one case and somatically mutated or hypermutated IgVH genes
in 3 (37%) and 4 (50%) of 8 cases, respectively (Table 1). VH4 gene family usage was
demonstrated in 7 of 8 (88%) tumors, and VH4-34 gene usage in 4 of 7 cases. Only case 6 showed ongoing somatic mutations. These findings are
in accordance with published data, suggesting that the cell of origin
of CD5+ DLBL predominantly is of postgerminal
origin.6,8,11
On banding analysis, the majority (11 of 13 [85%]) of
CD5+ DLBLs exhibited complex karyotypic alterations with 12 structural or numerical aberrations on average. All cases were negative
for the t(11;14)(q13;q32) and t(14;18)(q32;q21) chromosome
translocations, and t(3;14)(q27;q32) targeting the BCL6 gene
was found in only 1 tumor (case 2). In contrast, CD5
DLBLs harbored BCL2 and BCL6 rearrangements in 9 of 55 (16%) and 4 of 55 cases (7%), respectively. The sole recurrent
structural aberration was deletion del(10)(q22q24) in cases 5 and 8. Trisomies for chromosomes 3 (7 of 13 [54%] versus 7 of 55 [13%]),
16/16p (5 of 13 [38%] versus 8 of 55 [15%]), and 18/18q (6 of 13 [46%] versus 7 of 55 [13%]) were significantly more frequent in
CD5+ DLBLs than in CD5 DLBLs
(P < .05).
FISH analysis failed to reveal ATM deletions, being strongly
associated with B-CLL12 and MCL,17 in both
CD5+ and CD5 DLBLs. In contrast, the
prevalence of deletions at the D13S25 locus was
significantly higher in CD5+ DLBLs (4 of 12 [33%])
compared with CD5 DLBLs (4 of 42 [10%],
P < .05). Deletions of TP53 were equally distributed in both groups, whereas p16INK4a
hemizygous deletions were more frequent in CD5+ tumors (4 of 12 [33%]) than in the CD5 group (3 of 38 [8%],
P < .05).
Deletion of D13S25 is a frequent finding in B-CLL and
MCL, but it is rarely found in other malignant lymphomas of B-cell
type.12,22,23 The strong association of D13S25
deletions with CD5+ DLBL may, therefore, indicate that this
aberration hits a CD5+ progenitor B cell at the same
differentiation level as in B-CLL. Interestingly, deletions of
ATM commonly found in B-CLL12 were not
detectable in CD5+ DLBLs. It recently turned out, however,
that ATM deletions in B-CLL occur almost exclusively in the
unmutated variant of the disease,24 whereas
D13S25 deletions cluster in mutated B-CLL.25 Because most of our CD5+ cases displayed mutated IgVH
genes, their cell of origin is likely to be different from the
pregerminal progenitor cell of the unmutated B-CLL cases that are
frequently characterized by ATM inactivations. CD5+ DLBLs
may, therefore, be viewed as an early transformed aggressive DLBL
variant related to CD5+ lymphocytic lymphoma with
additional transforming events having occurred before the clonal
expansion of the low-grade neoplasm. According to the results of this
first study comparing cytogenetic alterations in CD5+ and
CD5 DLBLs, one likely candidate gene for this early
transforming event is the loss of one p16INK4a
allele. The frequent association of p16INK4a
inactivations with transformed low-grade lymphomas13 would be in excellent agreement with this hypothesis.
| |
Acknowledgments |
We thank Mrs H. Brückner, Mrs A. Trumpfheller, and Mrs
N. Hemmrich for technical assistance and Mr E. Schmitt for artful photographic work. The Resource Center/Reference Library of the German
Human Genome Project (Berlin, Germany) generously provided the
P1-derived artificial chromosome (PAC) probes specific for ATM (PAC ATM-1, LLNLP704G18220Q19 and PAC ATM-2,
LLNLP704O01298Q19). These clones were part of the libraries RPCI 1, RPCI 3-5, originating from Roswell Park Cancer Institute, created by
Pieter de Jong and Panayiotis A. Ioannou.
| |
Footnotes |
Submitted June 11, 2002; accepted August 8, 2002.
Prepublished online
as Blood First Edition Paper, August 29, 2002; DOI
10.1182/blood-2002-06-1726.
Supported by the European Commission "Growth program, Research
project Molecular and biological risk factors in mantle cell lymphoma" (Contract no. QLG1-CT-2000-00687).
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Tiemo Katzenberger, Institute of Pathology,
University of Würzburg, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany; e-mail:
path054{at}mail.uni-wuerzburg.de.
| |
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Abstract
Introduction
