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Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1145-1151
De Novo CD5+ Diffuse Large B-Cell Lymphomas Express
VH Genes With Somatic Mutation
By
Masanori Taniguchi,
Kouji Oka,
Atsunori Hiasa,
Motoko Yamaguchi,
Toshiyuki Ohno,
Kenkichi Kita, and
Hiroshi Shiku
From The Second Department of Internal Medicine, Mie University
School of Medicine, Tsu, Japan.
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ABSTRACT |
To clarify the cellular origin of de novo CD5+ diffuse
large B-cell lymphoma (CD5+ DLBL), particularly in
comparison with other CD5+ B-cell neoplasms such as
chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), we
analyzed the nucleotide sequence of the Ig heavy chain variable region
(IgVH) genes of de novo CD5+ DLBL cases. All 4 cases
examined had extensive somatic mutations in contrast with CLL or MCL.
The VH gene sequences of de novo CD5+ DLBL displayed
86.9% to 95.2% homology with the corresponding germlines, whereas
those of simultaneously analyzed CLL and MCL displayed 97.6% to 100%
homology. The VH family used was VH3 in 1 case, VH4 in 2 cases, and VH5
in 1 case. In 2 of 4 examined cases, the distribution of replacement
and silent mutations over the complementarity determining region and
framework region in the VH genes was compatible with the pattern
resulting from the antigen selection. Clinically, CD5+
DLBL frequently involved a variety of extranodal sites (12/13) and
lymph node (11/13). Immunophenotypically, CD5+ DLBL
scarcely expressed CD21 and CD23 (3/13 and 2/13, respectively). These
findings indicate that de novo CD5+ DLBL cells are
derived from a B-1 subset distinct from those of CLL or MCL.
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INTRODUCTION |
HUMAN B CELLS CONSIST of two distinct
subsets, B-1 cells and B-2 cells. B-1 cells are originally
characterized by the expression of the CD5 antigen and are distinct
from B-2 cells by anatomical localization, gene usage, function, and
phenotype.1-4 B-1 cells may be developed early in ontogeny
with restricted distribution on anatomical sites and often produce
autoantibodies.1-5 The majority of the antibodies produced
by B-1 cells are coded by relatively restricted repertories of Ig heavy
chain variable region (IgVH) genes with little or no somatic
mutation.2,3,4,6 However, recent analyses of the VH genes
of B-1 cells presented controversial results that B-1 cells are encoded
by hypermutated VH gene.7,8
CD5 is expressed in most cases of chronic lymphocytic leukemias (CLLs)
and mantle cell lymphomas (MCLs) and in some cases of diffuse large
B-cell lymphomas (DLBLs).9-11 Among the DLBLs, the
CD5+ DLBLs have been recognized as Richter's syndrome as a
consequence of a morphologic transformation of CLL.12
However, some patients with CD5+ DLBL have no past history
of lymphoproliferative disease (including CLL), and their DLBL is
considered to arise de novo. We previously reported that patients with
de novo CD5+ DLBL lacked cyclin D1 gene overexpression, a
critical characteristic of MCL.13 Bcl-6 rearrangement,
which is not seen in DLBL associated with Richter's syndrome, was
exhibited in half of the cases of de novo CD5+ DLBL
examined.14 These findings indicate that de novo
CD5+ DLBL is distinct from other CD5+ B-cell
lymphomas such as the CLLs and MCLs.
The VH genes are nonmutated in the majority cases of the CLLs and
MCLs,15-17 although some exceptional cases of CLL with an isotype switch or with VH5-51(VH251) gene were
reported.18,19 For the further clarification of the
cellular origin of B-1 cell neoplasms, it is indispensable to know
whether the VH genes of de novo CD5+ DLBL patients resemble
those expressed in CLL and MCL. We therefore attempted to analyze the
VH genes sequences of de novo CD5+ DLBL. We also evaluated
the clinical and immunohistologic features of 13 patients with de novo
CD5+ DLBL.
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MATERIALS AND METHODS |
Patients/samples.
Two hundred twenty-five B-cell non-Hodgkin's lymphomas of Japanese
patients were selected from the lymphoma case files from the 10-year
period between 1984 and 1993 in our laboratory. We studied the paraffin
sections stained with hematoxylin and eosin. The snap-frozen specimens,
fixed in 4% paraformaldehyde-ethanol, were immunophenotyped using a
labeled avidin-biotin alkaline phosphatase technique with the following
antibodies: Leu1 (CD5), Leu4 (CD3), and CR2 (CD21) obtained from Becton
Dickinson (San Jose, CA); CALLA (CD10), L26 (CD20), CD23, and anti-IgD
from Dakopatts (Glostrup, Denmark); and biotinylated goat anti-human
IgM,  ,  antibodies from Tago (Burlingame, CA), as previously
described.13 We diagnosed 133 DLBLs according to the REAL
classification.20 Finally, 13 of 133 DLBL cases showed
CD5+ phenotype (Table 1). None
of these 13 patients had a past history of lymphoproliferative
disorder. We obtained tissue specimens from 9 cases of these patients
after their informed consent was obtained. The DNA and total RNA were
extracted from the snap-frozen specimens. The cyclin D1 gene
overexpression of these cases had been already
investigated.13
Polymerase chain reaction (PCR) amplification.
The cDNA was synthesized from 3 µg of lymphoma tissue-derived RNA
using random hexamer nucleotides and Moloney murine leukaemia virus
reversed transcriptase (Bethesda Research Laboratories, Gaithersberg,
MD). One-fifteenth of the cDNA of these samples was amplified using a
Program Temp Control System PC-800 (ASTEC, Fukuoka, Japan) with a 50 pmol specific upstream primer corresponding to one of the six human VH
family leaders (VHL1, 5 -CCATG GACTG GACCT
GGAGG-3; VHL2, 5-ATGGA CATAC TTTGT TCCAG
C-3; VHL3, 5-CCATG GAGTT TGGGC
TGAGC-3; VHL4, 5-ATGAA ACACC TGTGG
TTCTT-3; VHL5, 5-ATGGG GTCAA CCGCC
ATCCT-3; and VHL6, 5-ATGTC TGTCT CCTTC CTCAT-3)21 and a 40 pmol downstream primer (LJH,
5-TGAGG AGACG GTGAC C-3)22 designed to match
the 3 ends of the six JH segments in a 50 µL volume (10 mmol/L
Tris-HCl, pH 8.3, 50 mmol/L KCl, 1.5 mmol/L MgCl2, 0.001%
[wt/vol] gelatin, 200 µmol/L each dNTP, and 2.5 U AmpliTaq GOLD
[Perkin Elmer, Branchburg, NJ]). After 10 minutes at 95°C, a
26-cycle PCR was performed under the following conditions: a
denaturation step at 94°C for 30 seconds, an annealing step at
60°C for 45 seconds, and an extension step at 72°C for 60 seconds (7 minutes in the last cycle). Fifteen microliters of the
reaction product was analyzed by electrophoresis in a 4% NuSieve GTG
Agarose gel (FMC, Rockland, ME) and visualized by staining with
ethidium bromide.
A seminested PCR was also performed with 500 ng genomic DNA. Samples
were first amplified in a final volume of 100 µL reaction buffer (10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 1.5 mmol/L MgCl2,
0.001% [wt/vol] gelatin, and 200 µmol/L each dNTP) containing 2.5 U of AmpliTaq GOLD, 50 pmol of an upstream primer [FR2A,
5-TGG(A/G)T CCG(C/A)C AG(G/C)C(T/C)
(T/C)CNGG-3]22 designed using a sequence of
framework region 2 (FR2), and 40 pmol of a downstream primer (LJH).
Before the 30-cycle PCR, samples were heated at 95°C for 10 minutes. The cycling protocol was a denaturation step at 96°C for
15 seconds, annealing at 60°C for 45 seconds, and extension step at
72°C for 45 seconds. Reamplification of a 1-µL aliquot (1%) of
the first PCR product with 40 pmol FR2A and 60 pmol VLJH
(5-GGTGA CCAGG GTCCC TTGGC CCCAG-3)21 was performed for 24 cycles under the following conditions: after 10 minutes at 95°C, a denaturation step at 96°C for 15 seconds, an
annealing step at 63°C for 45 seconds, and an extension step at
72°C for 45 seconds (7 minutes in the last cycle) in a reaction volume of 100 µL. The final concentrations of reagents in the solution were the same as described above except for 2.0 mmol/L MgCl2. The PCR products were electrophoresed as described
above.
Cloning and sequencing of PCR products.
The PCR products were purified by electrophoresis and recovered from
the gel with QIAquick Gel Extraction Kits (QIAGEN, Chatsworth, CA). The
recovered DNA was ligated into the pCR2.1 vector (TA cloning kit;
Invitrogen, San Diego, CA), which was used to transfect INV F
competent cells. Using the method of blue-white selection with X-Gal
(TaKaRa Biochem Co, Kyoto, Japan), 5 to 10 white colonies were picked
at random and grown overnight in 2.5 mL of LB (Luria-Bertani) medium.
Minipreps of plasmid were prepared from culture by alkaline lysis and
purification on DNA affinity columns (QIAprep Spin Plasmid kit; QIAGEN)
following the manufacturer's protocol. The double-stranded plasmid was
sequenced in an automatic DNA sequencer (Applied Biosystems 373;
Applied Biosystems, Foster City, CA) by using the dye terminator cycle
sequencing method for usage solely with this system. For each patient,
the same procedures of PCR and sequencing were performed with genomic
DNA or total RNA at least two times independently.
Analysis of mutations.
The sequences obtained for each patient were compared with germline
sequences using the BLAST (Basic Local Alignment Search Tool; National
Center for Biotechnology Information, Bethesda, MD)
homology search program to determine the usage of VH segments and to
estimate the somatic mutations compared with the most proximal germline
sequences. We then evaluated the numbers of mutations with amino acid
replacement (R) or silent mutation (S) observed in the FR or
complementarity determining region (CDR) of the VH gene. A
sensitive binomial probability model was used to evaluate whether the
observed replacement mutation in the CDR were selective.23
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RESULTS |
Clinical and histologic features.
Table 1 summarizes the clinical and histologic features of the 13 de
novo CD5+ DLBL patients studied. Six of the 13 patients
were men, and the age range was 54 to 81 years (median, 63 years). None
of them showed a high titer for human T-cell lymphotropic virus type 1 (HTLV-1) or clinical findings of human immunodeficiency virus infections. All patients except 1 (case no. 11) showed extranodal involvement at various sites (the stomach, testis, bone marrow, spleen,
liver, central nervous system, Waldeyer's ring, ileum, orbital mucosa,
skin, lung, breast, kidney, and adrenal gland). Two patients (cases no.
1 and 9) presented localized extranodal disease without lymph node
involvement.
Histologically, in all cases, malignant lymphoid cells were large-sized
and diffusely infiltrated. None of the samples had a proliferation
center as seen in CLL or a residual germinal center as observed in MCL
(Fig 1).
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| Fig 1.
De novo CD5+ DLBL (case no. 1, stomach).
(A) Diffuse infiltration of large neoplastic cells is observed
(hematoxylin and eosin). (B) Frozen section stained for CD5 shows
strong staining of atypical large cells and concomitant normal T
cells.
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Phenotypic findings.
As shown in Table 2, in all cases, the
neoplastic cells showed B-cell phenotypes bearing the pan-B-cell
antigen CD20 and lacking CD10. The cells also stained for the
pan-T-cell antigen CD5, but failed to stain for CD3. Ten cases were
negative for CD21 and 11 cases were negative for CD23. Eleven cases
expressed the IgM+/IgD or
IgM+/IgD+ phenotype, but 2 (cases no. 5 and 12)
showed IgM/IgD.
Nucleotide sequence analysis of VH genes of tumor cells.
VH genes were amplified in both genomic DNA and cDNA by the use of the
corresponding primer sets as described in the Materials and Methods. In
cDNA, a monoclonal pattern of PCR products were obtained in 4 (cases
no. 1, 2, 6, and 11) of 9 cases (cases no. 1, 2, 3, 4, 6, 8, 11, 12, and 13), whose examples are shown in Fig 2.
Sequencing of those PCR products presented clonal sequences of VH
segments in all of those 4 cases, as shown in
Fig 3. PCR amplifications and sequencings
were independently repeated two to three times, by which essentially
identical clonal sequences were obtained.
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| Fig 2.
Examples of PCR amplification of de novo
CD5+ DLBLs with genomic DNA of cases no. 1, 2, and 11 (A)
and with cDNA of cases no. 1, 2, 6, and 11 (B). PCR products were
electrophoresed on a 4% agarose gel, stained with ethidium bromide,
and visualized under UV light. Lane M, molecular weight standard
 X174/Hae III. Lane N, negative control. (A) Cases no. 1, 2, and 11 all presented monoclonal patterns whose VH genes were amplified.
(B) VH1 through VH6 were amplified with corresponding VH1 to VH6 gene
family-specific leader primer and a JH primer, respectively. In cases
no. 1, 2, and 11, monoclonal patterns were obtained only in one of VH
families, whereas in case no. 6, they were obtained in two VH families
(VH3 and VH5). However, PCR products of VH3 in case no. 6 were
polyclonal, as confirmed by sequencing.
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We were also able to examine genomic DNA of 7 cases (cases no. 1, 2, 3, 4, 11, 12, and 13). Clonal PCR products were obtained in 3 cases (cases
no. 1, 2, and 11), as also shown in Fig 2. Independently repeated PCR
and subsequent sequencings of PCR products of cases no. 1, 2, and 11 showed clonal VH sequences exactly compatible with those obtained in
the analysis of cDNA samples. Genomic DNA of case no. 6 was not
available for this analysis.
Table 3 shows the comparison of the
rearranged VH segments found in our samples with the most proximal
germline genes reported. Three different VH families (VH 3, VH 4, and
VH 5) were used, and the VH genes of two patients (cases no. 2 and 11)
were closest to the VH4-34 gene.
The VH genes of de novo CD5+ DLBLs were extensively
mutated, showing 86.9% to 95.2% identities to their corresponding
germlines. This finding is in contrast with VH gene sequences of
simultaneously examined CLL and MCL cases, in which either no or much
less somatic mutations were observed as previously reported (Table 3).
In all de novo CD5+ DLBL patients, the sequences of clones
derived from independent bacterial plaques were essentially identical,
with 1 base difference if there was any, which suggests that
intraclonal variability is improbable. These reproducible observations
of extensive mutations in CD5+ DLBL, in contrast with
mutations in CLL and MCL samples, argue against PCR errors in the
sequencing procedures.
Distribution of mutations in VH sequences.
The distribution of the somatic mutations in VH segments is shown in
Table 4. In cases no. 1 and 2, the R/S
ratios in the FRs were smaller than 1.5. The R/S ratios in the CDRs of
cases no. 1, 2, 6, and 11 were greater than 2.9, and the probabilities of the clustering of R mutations in the CDRs were significant in cases
no. 1, 2, and 11 according to the binomial model.
| |
DISCUSSION |
In the current study, all 4 de novo CD5+ DLBL cases we
analyzed contained somatic mutations in the VH genes. The rearranged IgVH was detectable in 4 of 9 de novo CD5+ DLBL samples
using the optimized PCR-based method. One possible explanation for this
low frequency of detecting the IgVH rearrangement by PCR is the
presence of high somatic diversification at sites complementary to the
primers.22,24 A specific member of the VH4 family, the
VH4-34 gene, was detected in 2 patients. Autoantibodies directed
against the i and I carbohydrate antigen on red blood cells, a
so-called cold agglutinin, are encoded by VH4-34 gene.25 Idiotypes of heavy chains encoded by the VH4-34 gene are present on B
cells in the mantle zone of lymph nodes and CD5+ B cells in
the cord blood.26,27 A relatively high incidence of the
VH4-34 gene has been reported in a minor subset of CLL with an isotype
switch.18,26
The distribution of mutations in the VH regions may reflect the antigen
selection of B cells. It has been suggested that an R/S ratio greater
than 2.9 in the CDR accompanied by a lower ratio in the FR indicates an
antigen selection of the somatically generated substitution in B
cells.28 The results of the present sequence analysis of 2 de novo CD5+ DLBLs (cases no. 1 and 2) were consistent with
the frequently observed mutation patterns resulting from antigen
selection in B-2 cells, and cases no. 6 and 11 did not follow this
pattern. In recent studies, the mutation with unusual R/S ratios was
sometimes observed in human peripheral B-1 cells and in the malignant
B-1 cells of patients with CLL and B-cell lymphoma with acquired
immunodeficiency syndrome.7,18,29 Repeated or chronic
exposure to the same antigen is suggested to result in the accumulation
of neutral mutations that do not affect the affinity of the Ig
molecule, as is the case with allergens and
autoantigens.30,31 Whether somatic mutations of B-1 cells
are developed in mechanisms similar to selected mutations of B-2 cells
requires further analyses.
Most cases of CLLs and MCLs might arise from B-1 cells containing
nonmutated IgVH genes, with some exceptional cases of CLL. In contrast,
de novo CD5+ DLBL might be derived from B-1 cells
containing mutated VH genes. Phenotypically, the neoplastic cells from
CLL and MCL express CD21 and/or CD23,32 whereas
neoplastic cells in our de novo CD5+ DLBL cases seldom
expressed either CD21 or CD23. These differences in cellular
characteristics clearly indicate that de novo CD5+ DLBL are
raised in B-1 cells distinct from those from which MCL and CLL arise.
The clinical characteristics of de novo CD5+ DLBL were also
different from those of CLL and MCL. Patients with CLL and MCL often
have an involvement of peripheral blood, bone marrow, or lymphoid
organs, including lymph nodes, spleen, and tonsils. In contrast, most
of the present de novo CD5+ DLBL patients showed an
involvement of various extranodal sites with lymphadenopathy. These
observations suggest that the precursor of the malignant cells of de
novo CD5+ DLBL represents a subset of normal B-1 cells
localizing at particular anatomical sites, possibly at a more
differentiated stage. Alternatively, de novo CD5+ DLBL may
have originated from B-1 cells of unique lineages. Although more
investigation is necessary, CD5+ DLBLs might constitute a
discrete subgroup that is distinguished from other DLBL derived from
germinal center cells or other B-2 cells.
| |
FOOTNOTES |
Submitted May 20, 1997;
accepted November 13, 1997.
Supported in part by Grants-in-Aid for Cancer Research (9-10) from the
Ministry of Health and Welfare of Japan and Grants-in-Aid for
Scientific Research (08307003) from the Ministry of Education, Science,
Sports and Culture.
Address reprint requests to Hiroshi Shiku, MD, The Second Department of
Internal Medicine, Mie University School of Medicine, 2-174 Edobashi,
Tsu 514, Japan.
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.
| |
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Diffuse low-grade B-cell lymphomas: Four clinically distinct subtypes defined by a combination of morphologic and immunophenotypic features.
Am J Clin Pathol
100:373,
1993[Medline]
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Abstract
Introduction
Abstract
