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From the Department of Medical Sciences, Section of Hematology, University of Modena and Reggio Emilia, Modena, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hepatitis C virus (HCV) is actually considered as the major etiological factor of type II essential mixed cryoglobulinemia, a disease associated with an underlying B-cell clonal proliferation that, in a minority of cases, may evolve into a frank NHL.5,6 HCV is known to be both a hepatotropic and a lymphotropic virus and it has been suggested that it may play a role in the pathogenesis of clonal proliferation of B-cells.7 Several studies have reported a higher prevalence of chronic HCV infection in patients with B-cell NHL compared with the general population, at least in certain geographical areas.7-9 In particular, in a survey of HCV-positive NHL from the United States NMZL has been recognized as the most common histological type.10
A role of chronic immunological stimulation in the development and maintenance of MZL, and in particular of EMZL of MALT-type, has been suggested by a variety of observations. MZL frequently originates in a setting of chronic inflammation triggered by chronic infection or autoimmune disorders, such as Helicobacter pylori gastritis, Sjögren’s syndrome, and Hashimoto’s thyroiditis.11-13 The etiological link between gastric EMZL and H. pylori infection has also been shown by the regression of some cases by antibiotic therapy.14,15 A role of chronic immunological stimulation is also suggested by sequence analysis of the tumor-related immunoglobulin gene rearrangement that identified a preferential use of the immunoglobulin heavy chain variable region (VH) genes associated with autoimmune disorders suggesting that tumor cells may arise from autoreactive marginal zone B-cells.16,17 Moreover, the analysis of somatic mutations of VH genes revealed a variable but significant level of mutations supporting the hypothesis of a germinal center (GC) of a post-GC origin of MZL.18,19 Nevertheless, these data have been obtained in a significant number of EMZL whereas the tumor-related immunoglobulin chain gene (IgH) status has not yet been determined in the recently identified subset of NMZLs.
In the present study we analyzed the nucleotide sequence of the tumor-related rearranged variable immunoglobulin heavy chain genes in 10 cases of NMZL. The cases examined have been well characterized from a clinical point of view to exclude any cases with extra-nodal involvement. Therefore, we considered this cohort of patients represented of true NMZL in accord to World Health Organization classification. Moreover, we also analyzed the data obtained on the basis of the presence of chronic HCV infection to identify peculiar aspects of the IgH genes distinguishing between HCV-positive and HCV-negative cases.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 10 cases of NMZL evaluated in this study were selected among
18 cases, collected between 1990 and 1998, for which DNA or nitrogen
liquid-frozen lymph node tissues were available for molecular studies.
Diagnosis of monocytoid-marginal zone B-cell lymphoma was documented in
all cases by morphology and immunohistochemistry performed on
formalin-fixed paraffin-embedded tissue according to the proposed
"Revised European-American Classification of Lymphoid
Neoplasms."1
In particular, the presence of
characteristic clear cells with a relatively abundant pale cytoplasm
(positive for CD20 and CD79a and negative for CD5 antigens) recognized
as marginal/monocytoid B cell was evident in all cases (Figure 1)
. Moreover, bone marrow and peripheral
blood double-immunofluorescence labeling with anti-CD5 and anti-CD19
was performed in all cases to exclude B-cell chronic lymphocytic
leukemia. Complete clinical records and follow-up were available for
all patients included in the study; as long as a case had involvement
of a mucosal or other extra-nodal nonhemopoietic site by MZL it was
classified as EMZL of MALT-type and discarded. On the basis of these
criteria 8 out of the 18 cases initially selected were discarded from
the study because of the presence of an extra-nodal localization of the
lymphoma, in particular of a gastric involvement in seven cases and of
the parotid gland in one case. All patients were evaluated on the basis
of physical examination, endoscopy, and neck-thorax-abdominal computed
tomographic scans.
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Polymerase Chain Reaction (PCR) Amplification of the Rearranged IgH and IgK
DNA was extracted from lymph node specimens using a commercial kit (Easy-DNA; Invitrogen, Carlsbad, CA). One µg of DNA was added to PCR buffer (10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 1.5 mmol/L MgCl2), containing 200 µmol/L dNTPs, 100 nmol/L of each primer and 1.5 U of AmpliTaq Gold polymerase (Perkin Elmer, Norwalk, CT) in a total volume of 50 µl. Forty cycles of amplifications were performed at the following condition: 1 minute at 95°C, 1 minute at 60°C, and 1 minute at 72°C with 10 minutes of an initial denaturation step at 95°C and 7 minutes of a final extension step at 72°C. Initially, PCRs were performed in separate reactions with seven family-specific FR1 sense primers together with a 3' primer complementary to a germline JH consensus sequence. If the region from FR1 to JH could not be amplified, the amplification experiments were performed at the same conditions using 5' oligonucleotides specific for each leader sequence of the VH1 and VH7 families together with the same JH antisense primer. For each DNA sample a DNA-free control was used to check contamination. Each amplification experiment was performed in duplicate. After agarose gel electrophoresis, a predominant band was obtained in samples 1, 4, 7, 8, 9, and 10 using the VH FR1-specific family primers and in samples 2, 3, 5, 6, and 9 using VH leader-specific family primers. The segment initially obtained with FR1 primers in sample 9, belonging to VH3 family, was an out-of-frame rearrangement.
5' oligonucleotides complementary to FR2 and FR3 consensus sequences were also used together with the 3' JH primer at the same PCR conditions with the exception of an annealing temperature of 50°C in each cycle.
The light chain gene region was amplified using VK
family-specific primers and a mixture of JK primers at the
same PCR condition used for VH family-specific
amplifications. The sequence of the oligonucleotides used for the PCR
experiments is reported in Table 1
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PCR products were excised from 1% low-melting agarose gel, further purified with Wizard PCR prep purification kit (Promega, Madison, WI) and directly sequenced using the BigDye terminator cycle sequencing kit and a DNA sequencer (ABI prism 310; PE Applied Byosystem, Foster City, CA). Sequencing reactions were performed with the same oligonucleotides used in the PCR amplifications by both sense and antisense primers. The nucleotide sequences of the amplification products representative of the IgH rearrangements were compared with the VBASE directory (VBASE Sequence Directory, I.M.; Tomlinson, MRC Center for Protein Engineering, Cambridge, UK; URL: www.mrc-cpe.cam.ac.uk/imt-doc)20 using the DNAPLOT analysis software. A diversity (D) germline segment was assigned to the longest stretches with the highest nucleotide homology with a minimum of six successive matches or seven matches interrupted by one mismatch.
Somatic Mutation Analysis
Mutations in the variable region were identified by comparing the nucleotide sequence of each tumor with the closest germline VH sequence. Two nucleotide exchanges in one codon were considered as one replacement (R) mutation. Mutations at the joining site of the VH segment were not regarded as mutations. The number of expected replacement (R) mutations in the complementary determining region (CDR) or framework region (FR) was calculated using the formula: Exp R (CDR or FR) = n x (CDR Rf or Fr Rf) x (CDRrel or FRrel); where n is the total number of observed mutations, CDRrel or FRrel is the relative size of CDRs or FRs and Rf is the inherited replacement frequency to CDRs or FRs sequences. Rf value was calculated for each individual VH gene sequence using the InhsusCalc1.0 software, kindly provided by Dr Paolo Casali (Department of Pathology, Weill Medical College, Cornell University, NY). According to the binomial distribution model, the probability that excess or scarcity of R mutations in CDRs or FRs resulted on the basis of chance alone was calculated using the formula: p = {n!/[k!(n - k)!]} x qk x (1 - q)n-k; where k is the number of observed mutations in the CDRs or FRs and q is the probability that a R mutation will localize to CDRs or FRs (q = CDRrel x CDR Rf or FR rel x FR Rf).21
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IgH rearranged genes were amplified from DNAs extracted
from lymph node biopsies using family-specific VH FR1 or,
alternatively, family-specific VH leader primers coupled
with a 3' heavy-chain joining (JH) primer. After agarose gel
electrophoresis and staining with ethidium bromide a predominant band
was observed in all cases. All samples were analyzed in duplicate.
These amplification products were directly sequenced on both strands
with the same sense and antisense primers used in the PCR experiments.
As anticipated by the PCR analysis, the VH segments involved
in a productive VH-D-JH rearrangement in NMZLs belonged to
the VH4 family in four cases, to the VH1 family
in three cases, to the VH3 family in two cases, and to the
VH2 family in one case. No rearranged segments of the
VH5, VH6, and VH7 family were found to
be involved in this series of NMZL cases. VH nucleotide
sequences have been deposited in the GenBank database (accession
numbers AF355605 to AF355614). All three of the VH1-family
genes identified (cases 1, 4, and 5) were found to be closely related
to the VH1-69 germline gene with a nucleotide homology
ranging from 92.40 to 94.79%. Three of the four lymphoma
VH4-family genes (cases 2, 3, and 6) seem to be related to
the VH4-34 germline gene with a nucleotide identity ranging
from 83.33 to 98.28%, whereas the fourth VH4-family case
(case 9) was assigned to the VH4-30.4 germline segment. The
two VH3-family lymphoma segments seemed to be related to the
VH3-30.3 (case 7) and VH3-48 (case 10) whereas a
rearranged VH2-05 germline segment was observed in case 8
(Table 2)
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. Nevertheless, the sequence analysis
of the VH1-69 segments revealed that the somatic mutations
of the three VH1-69 cases were different, excluding a gross
contamination between the different DNA samples (see Figure 3
).
Moreover, to confirm these data, amplification fragments of the
VH-D-JH rearrangements were obtained with primers specific
to FR3 and FR2 consensus regions of the VH segments. PCR
experiments performed with a degenerate oligonucleotide specific for
FR3 region revealed the presence of a clear band in cases 1 and 4 and
of a smear with a faint band in case 5; of note all PCR fragments
obtained were of the same length. In two out of the three cases (cases
4 and 5) a band of the same length was evident also with a degenerate
primer specific for the FR2 region. In the third case (case 1) a smear
was present probably because of the fact that the nucleotide sequence
of the FR2 region of this case was not susceptible to amplification
with this set of primers (Figure 2)
.
Sequence analysis of the DNA fragments obtained with FR2 and FR3 PCR
experiments revealed a nucleotide composition identical to the sequence
already determined with the family-specific amplification fragments in
all cases. The three cases rearranging a VH1-69 segment were
all positive for HCV infection. The other two HCV-positive cases
rearranged a VH4-30.4 (case 9) or a VH3-30.5
segment (case 7). The three cases using a VH4-34 segment
were all HCV-negative (Table 2)
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Somatic Hypermutations and Statistical Analysis
The rearranged VH sequences were found to carry somatic
mutations in all cases of NMZL studied, with a sequence identity rate
compared with the closest germline gene ranging from 83.33 to 98.28%.
To establish if the pattern of somatic mutations were indicative of
antigen selection, we determined the distribution of replacement (R)
and silence (S) mutations in the FRs and CDRs regions using the method
described by Chang and Casali.21
A preferential cluster of
R mutations in the CDRs region (P < 0.05) was
found only in case 7. In FRs regions the number of R mutations was
significantly lower than would be expected to arise solely by chance in
five cases, probably as a result of selection against R mutations
within the FRs to preserve structure of immunoglobulins. Among this
group of patients cases 1, 5, and 7 were HCV-positive; cases 2 and 6
were HCV-negative (Table 4)
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the cases examined we found that NMZLs harbor numerous point mutations with respect to the closest related germline gene sequences (Table 5) with a rate of homology ranging from 83.33 to 98.28% corresponding to a number of substitutions ranging from 5 to 41 nucleotides (average, 17.8). Because most of the human germline VH genes have been identified, it is unlikely that the majority of nucleotide substitutions found represent VH segments not yet identified or because of sequence polymorphism.20 These data show that the tumor cells are likely to derive from mature B cells that had participated in a GC reaction. We cannot identify if tumor cells have to be considered of GC or of post-GC derivation because the molecular approach used (direct sequencing of PCR products) does not allow the assessment of nucleotide intraclonal variations because of an ongoing mutational process.
The distribution of R and silence S mutations of mutated VH
sequence was analyzed by the method of Chang and Casali.21
Somatic mutations of the our NMZLs were not distributed in a manner
indicative of positive selection or of antigen-affinity maturation in
the majority of cases. In fact, only one of the cases examined (case 7)
showed a statistically significant evidence of accumulation of R
mutations in the CDRs. Nevertheless, five cases showed evidence of
negative selection of R mutations in the FRs (Table 4)
. This aspect is
consistent with a GC selection to preserve the immunoglobulin structure
providing the scaffolding for the antigen-contacting cells.
Kuppers and colleagues23 showed in four cases defined as monocytoid B-cell lymphoma a distribution of somatic mutations indicative of a clonal antigen-positive selection driven by antigens. Our data, obtained in a larger number of cases, contrast with these findings. Nevertheless, in their cases the primary localization of the lymphoma was not reported and at least in one case the tumor was localized in the stomach and therefore likely to be considered as an EMZL of MALT-type with monocytoid cells. Thus, the differences between our data and that of Kuppers may be because of different criteria in selecting lymphoma cases.
Zuckerman and colleagues10
have recently reported that
NMZL is the most common histological type found in a series
HCV-infected NHL cases from the United States. They also found that
50% of the NMZL cases examined were HCV-infected. Therefore, the
frequency of HCV chronic infection found in our NMZL cases, selected
only on the basis of the availability of biological material adequate
for molecular studies, confirms Zuckerman’s data. Interestingly, we
also found that the VH gene usage among NMZL is not random.
Different VH segments are preferentially used in
HCV-positive and HCV-negative patients (Table 2)
. Three out of five
HCV-negative NMZLs (cases 2, 3, and 6) use the VH4-34 gene
segment joined with different D and JH segments.
In two cases, the VH4-34 segments appear to be heavily
mutated, with nucleotide sequence homology with the VH4-34
germ-line gene of 83.33% in case 2 and 83.68% in case 6. In both
these cases, despite the high frequency of somatic mutations, aspects
for selection against R mutations in the FRs were present (Table 4)
.
The conservation of the amino acid sequence of the FRs is actually
considered a feature that distinguishes functional from nonfunctional
sequences suggesting that the immunoglobulins encoded by these
VH4-34 MZL cells are functional.24,25
Therefore, neoplastic cells have aspects indicating the origin from B
lymphocytes that have undergone a selection process within the GC
reaction. In case 3, the VH4-34 segment was slightly mutated
(98.28% of homology) without a statistical significance of the pattern
of distribution of R and S mutations. Nevertheless, the low rate of
mutations found in this case determines that the statistical model used
may be not informative. The VH4-34 gene is used in 5% of
healthy adult B lymphocytes26
and is frequently found in
diffuse large-cell lymphoma, primary central nervous system lymphoma,
B-chronic lymphocytic leukemia, and autoimmune disorders, but never in
multiple myeloma.27-32
Interestingly, the
VH4-34 gene is found in virtually all cases of cold
agglutinin disease33-35
and the red blood cell I/i
antigens bind to the FR1 domain of Ig. A restricted usage of
VH genes, as well as binding of an immunoglobulin outside
the CDRs, which are the sites that bind conventional antigens, are
characteristic aspects of B-cell superantigens that are supposed to
directly activate B cells.36
In particular, certain
portions of the FRs seems to be important for superantigen binding and
these would be preserved in a superantigen selection
pressure.37
Interestingly, staphylococcal enterotoxins A
and D, that function as a human B superantigen, are able to rescue B
cell-expressing VH3 and VH4 (including
VH4-34) genes inducing cell survival in in vitro
experiments.38,39
Therefore, the high frequency of the
VH4-34 gene usage joined with different
D and JH segments as well as the
preservation of the FRs sequences, suggests a possible role of yet
unknown B-cell superantigen(s) in driving HCV-negative NMZL development
and proliferation.
Three out five HCV-positive NMZLs revealed the usage of the
VH1-69 gene indicating a highly biased and nonrandom use of
the VH segments in this subtype of tumors. Sequence analysis
of the rearranged VH segments of these three cases revealed
a substantial deviation from the germline VH1-69 sequence
with a degree of mutations very similar among the different cases
indicating that these cells have traversed the GC activating the
somatic hypermutation mechanism. Statistical analysis of the pattern
and distribution of R and S mutations between CDRs and FRs revealed a
statistical significance in two cases (cases 1 and 5) in which the
presence of R mutations in the FRs are lower than expected by chance
only (Table 4)
. This pattern is indicative of a selective pressure to
conserve the functional structure of the immunoglobulin protein.
Moreover, the VH1-69 segments were joined with a
D3-22 and a JH4 segment in all of the three
cases. A D3-22 segment joined with JH4 was also
found in a HCV-negative case (case 8) assembled with a
VH2-05 gene. As a consequence, the CDR3 region encoded for
an amino acid sequence with very strong similarities between the three
VH1-69 different cases. We exclude that this data may be
because of cross contamination because the sequence analysis of the
VH1-69 segments revealed different somatic mutations between
the three samples (Figure 3)
and the same
CDR3 sequences were obtained with different sets of primers specific
for FR3 and FR2 VH consensus sequences (Figure 2)
.
Therefore, these data indicate the role of a common antigenic epitope
involved in the selection and in the expansion of the B-cell clone at
the origin of neoplastic cells. The VH1-69 immunoglobulin
segment is expressed in the restricted repertoire of fetal liver B
lymphocytes and is thought to be involved in natural
immunity.40,41
A productive VH1-69
rearrangement is present in 1.6% of normal B lymphocytes in
adults.42
VH1-69 is rearranged in 10 to 20%
of B-cell chronic lymphocytic leukemia30,43
and a
VH1-69 monoclonal rearrangement is present in the majority
of patients with type II mixed cryoglobulinemia, a typical HCV-related
disorder.44,45
A preferential usage of VH1-69
was also found by Ivanovski and colleagues46
in the
majority of the HCV-positive lymphoplasmocytoid lymphoma/immunocytoma
NHL subtypes secreting monoclonal IgM cryoglobulins. Very recently, a
frequent usage of VH1-69 was also reported by Bahler and
colleagues19,47
in the subset of the salivary gland EMZL
of MALT-type that arise in the course of lymphoepithelial
scialoadenitis associated or not with Sjögren’s syndrome. In one
case they also reported the use of a D3-22 joined with
VH1-69 and JH4 segments. They also report that
VH1-69 salivary gland lymphoma often has CDR3 regions with
similar characteristics, ie, a preferential use of the JH4
segment, a length of 12 to 14 amino acids and the presence of some
amino acids motifs at the VH-D and D-JH
junctions, concluding that the salivary EMZL may be selected by similar
antigen epitopes. Unfortunately, they did not report the status of HCV
infection in the cases examined.
Therefore, in the subset of NMZLs with a chronic HCV infection we find the presence of the VH1-69 segment in the majority of cases examined, that is frequently expressed in other subsets of lymphoproliferative disorders, mainly associated with HCV infection. The almost equal sequences of the CDR3 region of the VH1-69 cases associated to the presence of a chronic HCV infection suggests, in first hypothesis, that a same HCV antigen epitope could be involved in the B-cell selection by a direct antigenic stimulation. This hypothesis is also sustained by the observation that B cells derived from a HCV-infected individual, immortalized as hybridomas, and selected for binding the HCV E2 envelope glycoprotein use the VH1-69 segment in the majority of cases.48
Nevertheless, the possibility that the virus infects and induces the proliferation of a particular fraction of B lymphocytes cannot be excluded. Gastric EMZLs are associated with H. pylori infection in virtually all cases suggesting a possible H. pylori direct antigenic stimulation.13 Nevertheless, in this subset of EMZL, tumor immunoglobulins seem to be autoantigen-related rather than specific to H. pylori and the neoplastic growth seem to be sustained by stimulation through a H. pylori-specific T-cell reaction.49,50 We cannot exclude that HCV could play a role similar to that of H. pylori in gastric EMZL in the subset of HCV-positive NMZL.
Tierens and colleagues18 recently reported an analysis of somatic mutations in a series of MZL occurring at different sites including four cases with primary lymph node localization in which the pattern of somatic mutations was not indicative of positive or negative antigen selection. However, they report in two out of the four cases of NMZL studied the presence of a case rearranging the VH1-69 gene joined with a D3-22 segment and a case with the rearrangement of a VH4-34 segment. Unfortunately, they did not report data about HCV infection in these cases.
In conclusion, these results indicate that NMZLs are originated from GC-experienced B lymphocytes with evidence of antigen selection in half of the cases. The presence of a VH1-69 segment in the subset of the HCV-positive NMZLs with similar CDR3 sequences strongly supports the hypothesis of a common antigen, probably a HCV antigen epitope, involved in the clonal B-cell selection. The involvement of a VH4-34 segment with different characteristics of the CDR3 regions suggests a role of unknown B-cell superantigen(s) in the selection of HCV-negative NMZL.
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Footnotes |
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Supported by a grant from the Associazione Italiana per la Ricerca sul Cancro, Milan, Italy (to M. L.) and by the Associazione Italiana contro le Lucemie, Modena, Italy.
Accepted for publication March 21, 2001.
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X174 HaeIII digested; NC, DNA-free negative
control.