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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2294-2302
BCL-6 Gene Mutations in Posttransplantation Lymphoproliferative
Disorders Predict Response to Therapy and Clinical Outcome
By
Ethel Cesarman,
Amy Chadburn,
Yi-Fang Liu,
Anna Migliazza,
Riccardo Dalla-Favera, and
Daniel M. Knowles
From the Department of Pathology, The New York Hospital-Cornell
Medical Center; and the Department of Pathology and the Department of
Genetics and Development, College of Physicians and Surgeons, Columbia
University, New York, NY.
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ABSTRACT |
Posttransplantation lymphoproliferative disorders (PT-LPDs)
represent a heterogeneous group of Epstein-Barr virus-associated lymphoid proliferations that arise in immunosuppressed transplant recipients. Some of these lesions regress after a reduction in immunosuppressive therapy, whereas some progress despite aggressive therapy. Morphological, immunophenotypic, and immunogenotypic criteria
have not been useful in predicting clinical outcome. Although
structural alterations in oncogenes and/or tumor suppressor genes identified in some PT-LPDs correlate with a poor clinical outcome, the presence of these alterations has not been a consistently useful predictor of lesion regression after reduction of
immunosuppression. We examined 57 PT-LPD lesions obtained from 36 solid
organ transplant recipients for the presence of mutations in the BCL-6
proto-oncogene using single-strand conformation polymorphism and
sequence analysis, followed by correlation with histopathologic
classification and clinical outcome, which was known in 33 patients.
BCL-6 gene mutations were identified in 44% of the specimens and in
44% of the patients; none were identified in the cases classified as
plasmacytic hyperplasia. However, mutations were present in 43% of the
polymorphic lesions and 90% of the PT-LPDs diagnosed as non-Hodgkin's
lymphoma or multiple myeloma. BCL-6 gene mutations predicted shorter
survival and refractoriness to reduced immunosuppression and/or
surgical excision. Our results suggest that the BCL-6 gene structure is a reliable indicator for the division of PT-LPDs into the biological categories of hyperplasia and malignant lymphoma, of which only the
former can regress on immune reconstitution. The presence of BCL-6 gene
mutations may be a useful clinical marker to determine whether
reduction in immunosuppression should be attempted or more aggressive
therapy should be instituted.
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INTRODUCTION |
IMMUNOSUPPRESSED SOLID organ transplant
recipients are prone to the development of a clinically heterogeneous
group of lymphoid proliferations referred to as posttransplantation
lymphoproliferative disorders (PT-LPDs). PT-LPDs were originally
believed to be non-Hodgkin's lymphomas (NHLs), but their malignant
status has been questioned because they frequently regress after a
reduction of immunosuppressive therapy.1 However, disease
behavior has been difficult to predict. It is now known that PT-LPDs
are most frequently Epstein-Barr virus (EBV)-driven B-cell
proliferations, which can be poly-, oligo-, mono-, or multiclonal based
on phenotype2,3 or genotype.4-8 Furthermore,
PT-LPDs are also morphologically heterogeneous; they do not resemble
any of the recognized entities which fall under the broad category of
lymphoid hyperplasia, and they have been difficult to classify using
the standard lymphoma classification schemes, mainly due to the
frequent polymorphic appearance of the lymphoid cells in these lesions.
In an attempt to distinguish benign and malignant lesions, Frizzera et
al3 divided PT-LPDs into the morphological categories of
polymorphic B-cell hyperplasia (PBCH) and polymorphic B-cell lymphoma
(PBCL). Subsequently, Nalesnik et al2 recognized two
principal categories of PT-LPDs, which they designated polymorphic and
monomorphic. However, neither morphology nor clonality has proven
capable of reliably predicting disease behavior.
Molecular genetic characterization of PT-LPDs, including an assessment
of the presence of structural alterations in some oncogenes and tumor
suppressor genes, has proven to be more informative in predicting
clinical outcome. It was first noted by Locker and Nalesnik6 that those monomorphic PT-LPDs displaying a
strong clonal immunoglobulin gene rearrangement band on Southern
blotting and a c-myc gene rearrangement exhibit disease progression. We subsequently showed that PT-LPDs are divisible into three distinct categories exhibiting unique morphological and molecular
characteristics as follows: (1) plasmacytic hyperplasia (PH): usually
polyclonal by immunoglobulin gene rearrangement analysis, and
polyclonal or faint monoclonal by EBV terminal repeat analysis; (2)
polymorphic PT-LPD, including PBCH and PBCL: monoclonal or oligoclonal
by immunoglobulin gene rearrangement or EBV terminal repeat analysis, without other identifiable genetic alterations; and (3) NHL/multiple myeloma (MM): monoclonal with structural alterations of the N-ras, p53,
and/or the c-myc genes.8 According to this scheme,
the PT-LPDs classified as PH and polymorphic PT-LPD were more likely to
regress with a reduction in immunosuppression or to undergo resolution
with more aggressive therapy than those classified as NHL or
MM.9,10 Although molecular analysis in conjunction with
careful morphological evaluation permitted the classification of
PT-LPDs into categories with clinical relevance, these criteria were
not helpful in predicting response by the polymorphic PT-LPDs to a
reduction in immunosuppression. We have now extended these studies to
include analysis of the BCL-6 oncogene.
The BCL-6 gene codes for a nuclear protein that is normally expressed
at high levels only in lymphoid cells, and predominantly in mature B
cells and CD4+ T cells within germinal
centers.11-14 The BCL-6 protein has six C-terminal zinc
finger motifs and one N-terminal POZ motif, and functions as a
sequence-specific transcriptional repressor.15-17 BCL-6 can
bind Stat6 DNA binding sites and block Stat6-mediated interleukin-4
signaling.18 Experiments using BCL-6 knock-out mice have
shown that this protein controls germinal center formation as well as
T-helper cell type 2-mediated inflammatory responses.18,19 The BCL-6 gene was identified because of its involvement in chromosomal translocations affecting band 3q27, which is a frequent breakpoint site
in diffuse large-cell lymphomas.20-24 Rearrangements of
this gene can be identified by Southern blot analysis in 30% to 45% of diffuse large-cell lymphomas, in 6% to 10% of follicular
lymphomas, and in 20% of acquired immunodeficiency syndrome
(AIDS)-related diffuse large B-cell lymphomas.25-28 The
majority of rearrangement breakpoints cluster within a 4-kb region
spanning the BCL-6 promoter and first noncoding exon, resulting in the
fusion of BCL-6 coding sequences (exons 2 to 10) to heterologous
promoters from other chromosomes, leading to deregulated expression by
the mechanism of promoter substitution.29 Subsequent
analysis of BCL-6 has shown the presence of point mutations
and/or small deletions in 70% of diffuse large-cell lymphomas
and 45% of follicular lymphomas, as well as in 58% of AIDS-associated
NHLs.30,31 These mutations occur within a 4-kb region
spanning the first exon, but tend to cluster in the 5 region of
the first intron, and overlap with the major cluster of chromosomal
breakpoints. Analyses of both BCL-6 gene rearrangements and mutations
have shown that structural alterations in the 5 noncoding region
of BCL-6 are present in virtually all cases of diffuse large-cell
lymphoma and the majority of cases of follicular and AIDS-related
NHLs,30,31 but are absent from all nonhematopoietic tumors
examined thus far. The frequency, clustering, and disease-association
of these alterations suggest that they may contribute to B-cell
lymphomagenesis, presumably by altering BCL-6 gene expression.
Therefore, we analyzed a panel of 57 PT-LPD lesions obtained from 36 patients for the presence of rearrangements and/or mutations in
the BCL-6 proto-oncogene, and correlated the results with the outcome
of the 33 patients for whom complete clinical information was
available.
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MATERIALS AND METHODS |
Pathological specimens.
Fifty-seven PT-LPD specimens were collected from 36 solid organ
transplant recipients (1 lung, 11 kidney, and 24 heart) during the
course of clinical evaluation using standard diagnostic procedures. Cases were processed at or submitted in consultation to the
Columbia-Presbyterian Medical Center and/or The New York
Hospital-Cornell Medical Center. The cases were selected based on the
morphological diagnosis of a lymphoproliferative disorder in a solid
organ transplant recipient and the availability of cryopreserved cells
or tissue blocks. The morphological and molecular analysis of 21 of the
57 lesions, as well as the clinical outcome of 32 patients, have been
previously reported.8,10,32 Involved tissues included
biopsies from lymph nodes, tonsils, adenoids, lungs, colon, liver,
brain, breast, bone marrow, and soft tissues. Representative portions
of the solid specimens were snap-frozen and stored at  70°C.
A sample of aspirated bone marrow was collected in a heparinized
syringe. A viable mononuclear cell suspension was prepared from this
sample by Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation. The cells were cryopreserved in
dimethylsulfoxide and fetal calf serum at 170°C. Portions of
each of the specimens were routinely fixed for histopathologic
examination.
DNA extraction.
Genomic DNA was extracted from cryopreserved mononuclear cell
suspensions and tissue blocks using a salting-out
procedure.33
Histopathologic classification.
The 57 PT-LPD lesions were classified as described8 by two
of the authors (A.C. and D.M.K.), without knowledge of the molecular features or the patient's clinical outcome, into one of four
morphological categories as follows: (1) PH, lesions exhibited
retention of the underlying architecture and expansion of the
interfollicular area or diffuse infiltration of other tissue by a
predominant population of plasmacytoid lymphocytes, associated with
plasma cells and sparse immunoblasts; (2) PBCH, lesions produced
extensive disturbance of organ architecture and were composed of a
mixture of lymphoid cells with prominent plasmacytoid differentiation and abundant immunoblasts without cytological atypia; in these lesions
necrosis was limited to single cells or small foci; (3) PBCL, lesions
were composed of a mixture of lymphoid cells lacking prominent
plasmacytoid differentiation and displaying significant cytologic
atypia, atypical immunoblasts, and contained large confluent areas of
coagulative necrosis; (4) NHL/MM, lesions were composed of a
monomorphic collection of cytologically malignant large lymphoid cells
or plasma cells, respectively.
Southern blot hybridization analysis.
Five microgram-aliquots of genomic DNA were digested with
BamHI and XbaI, respectively (Boehringer-Manheim,
Indianapolis, IN), electrophoresed in 0.8% agarose gels, denatured
with alkali, neutralized, and transferred to nitrocellulose filters
according to Southern analysis. The filters were hybridized to a
32P-labeled BCL-6 probe (Sac4.0) as previously
described.20
Oligonucleotide primers.
Five sets of primers were used, spanning a 741-bp region which is
altered in almost 70% of diffuse large-cell lymphomas. These sets have
been designated E1.9 through E1.13 as follows: E1.9: 5-GGGTTCTTAGAAGTGGTG-3 and
5-CAAAGCATTTGGCAAGAG-3; E1.10: 5-ctcttgccaaatgctttg-3 and
5-TAATTCCCCTCCTTCCTC-3; E1.11: 5-AGGAAGGAGGGGAATTAG-3 and
5-AAGCAGTTTGCAAGCGAG-3; E1.12: 5-TTCTCGCTTGCAAACTGC-3 and
5-CACGATACTTCATCTCATC-3; E1.13: 5-GATGAGATGAAGTATCGTG-3 and
5-ACACTGAAAGGCATCGCA-3.
Single-strand conformation polymorphism (SSCP) analysis.
Polymerase chain reactions (PCRs) were performed with 100 ng of genomic
DNA, in the presence of 10 pmol of each primer, 25 µmol/L dNTPs, 1 µCi of [ -32P]dCTP (NEN Life Science Products,
Boston, MA; specific activity, 3,000 Ci/mmol), and 1.5 mmol/L MgCl2. Thirty cycles of denaturation (94°C),
annealing (56°C for E1.10, 58°C for E1.11, and 54°C for E1.10), and extension (72°C) were performed. The reaction mixture (2 µL) was diluted 1:25 in 0.1% sodium dodecyl sulfate, 10 mmol/L EDTA, and further mixed 1:1 with a sequencing stop solution (95% formamide, 20 mmol/L EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol). Samples were heat denatured and electrophoresed in a 6%
acrylamide gel containing 10% glycerol.
Sequencing of PCR products.
Eight representative PCR products were cloned in the pCR2.1 vector
using the TA Cloning Kit (Invitrogen Corp, Carlsbad, CA) following the
manufacturer's instructions. Sequence of the inserts was performed
with a Taq DyeDeoxy Terminator Cycle Sequencing method with an ABI 373A
automated DNA sequencer (Applied Biosystems, Foster City, CA). The two
strands and at least four independent clones were sequenced in each
case to exclude mismatches due to polymerase mistakes, and to analyze
the status of both alleles.
Classification of clinical outcome.
Clinical follow-up was available in 33 patients. The clinical outcomes
with respect to their PT-LPDs were categorized using a modification of
the guidelines suggested by Nalesnik et al.2 A more
extensive review of the clinical features of 32 of these patients has
been reported separately.10 "Regression"
was defined as disappearance of PT-LPD lesions after reduction in
immunosuppression, with or without acyclovir, as evaluated by physical
examination (28 patients), endoscopy (3 patients), and/or
radiographic studies (21 patients). In these instances, surgical
intervention was limited to biopsy or partial excision of the PT-LPD
only. "Surgical resolution" refers to cases in which the entire
tumor was surgically excised. Two cases in which adjuvant radiation was
administered were included in this category. "Medical resolution"
refers to cases in which the tumor disappeared after chemotherapy.
Progression of PT-LPD in spite of therapeutic intervention was
considered a "no response." Four cases in which there was no
clinical documentation of either regression or progression of the
PT-LPD were classified as "not evaluable." With one exception
(Case 19), all patients who received chemotherapy initially had a
reduction of immunosuppression, and thus a "medical resolution"
implies a lack of response to reduction in immunosuppression in the
majority of cases. In three patients there was a recurrence in a
different site, of different clonal origin and of different
histological category. In these instances, only the outcome after the
most recent lesion was considered for statistical analysis.
Statistical methods.
The Fisher's exact test was used to determine if a particular
morphological category and clinical outcome of patients with PT-LPD was
associated with the presence of mutations in the BCL-6 gene. If a
significant association was found, then pairwise comparisons with a
Bonferroni correction were performed to determine where the difference
actually existed.34 Formal analysis of survival (Kaplan-Meier estimate) was performed with regard to the presence or
absence of BCL-6 gene mutations using the LIFETEST
Procedure.
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RESULTS |
Histopathology.
Histological sections from the 57 PT-LPD specimens included in this
study were classified as follows: (1) PH, 11 specimens from 10 patients; (2) PBCH, 8 specimens from 7 patients; (3) PBCL, 29 specimens
from 13 patients; (4) NHL/MM, 9 specimens from 8 patients. Nine
patients had multiple (2 to 12) PT-LPD lesions that were evaluated in
this study. In 6 of these patients (Cases 6, 23 through 26, and 31) all
lesions corresponded to the same histopathologic category, and in 3 patients (Cases 34 through 36) the PT-LPD lesions were of different
histopathologic subtype (Table 1).
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Table 1.
BCL-6 Gene Structural Alterations and Relationship to
Other Molecular Features in Posttransplantation Lymphoproliferative
Disorders
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Analysis of BCL-6 gene rearrangements.
Southern blot analysis was performed to assess the presence of
rearrangement in the 5 region of the BCL-6 gene. Representative examples of each category were evaluated, and included a total of 27 lesions from 21 patients. All of the PT-LPD cases examined showed a
distinct band in the germline configuration, and no rearrangements were
identified using this method (Table 1).
Analysis of BCL-6 gene mutations.
Mutations and/or small deletions in the 5 noncoding
region of BCL-6 were detected by SSCP analysis in a total of 25 PT-LPD specimens (44%) in 16 (44%) patients (Table 1).
Representative experiments are shown in Fig
1. Cases were scored as positive for mutations only when the migration
in the SSCP gel was different from that observed in a negative control
as well as from that of known polymorphic controls as
described30 (Fig 1). Some specimens showed abnormal
migration of one of the five fragments amplified (E1.9 through E1.13)
to cover the BCL-6 mutation cluster region, whereas other cases
(underlined in Table 1) showed abnormal migration in several of these
fragments, reflecting the presence of multiple mutations. There was no
evidence for involvement of both alleles, because all cases with
abnormal bands also retained the wild-type pattern bands, although
these may be derived from infiltrating, reactive cells.
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| Fig 1.
SSCP analysis of representative first intron segments of
the BCL-6 gene in PT-LPDs. Samples corresponding to radiolabeled,
PCR-amplified fragments from the E1.10 (top) and E1.11 (bottom) regions
of BCL-6 are shown. Numbers above the lanes indicate the corresponding
cases. Among the patients with more than one PT-LPD lesion are Cases 23 and 36. The histological classification of each case is indicated above
the corresponding lanes. Several identical bands were seen in all of
the samples in the cases containing the wild-type sequence. Three
different wild-type patterns are seen in the E1.11 region (bottom) due
to a known polymorphism; polymorphic cases are indicated with a P
beneath the corresponding lanes (Cases 11, 15, 17, and 18 are
heterozygous and Case 13 is homozygous for this polymorphism). Cases
15, 27, and 36 showed a different pattern (arrows) in region E1.10
(top), and Cases 12, 15, 16, and 36 showed a different pattern (arrows)
in region E1.11 (bottom), as a result of the presence of mutations in
the corresponding region, indicated with an M beneath the corresponding
lanes. ND, amplified DNA that was not denatured before electrophoresis;
 , the same PCR reaction in the absence of DNA; C, positive control
known to contain BCL-6 mutations in the region examined.
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In patients containing multiple PT-LPD lesions, several different
patterns were identified. Case 35 had two lesions, 4 months apart and
in different anatomic sites, one of which was classified as PBCH, and
the second one as PBCL. However, both of the lesions seemed to have the
same clonal origin, as evaluated by Ig gene rearrangement and EBV
terminal repeat analysis, and the same BCL-6 mutation. Case 24 had
three separate lesions at the same time, all classified as PBCL, and
all derived from the same original clone. However, two of the lesions
contained distinct BCL-6 mutations. Case 25 had multiple lesions in the
bowel, all classified as PBCL, but each derived from distinct original
clones.35 In this patient, different BCL-6 gene mutations
were identified in 8 of 12 separate tumors examined. Cases 34 and 36 presented with a polyclonal PH, and 57 and 13 months later developed
lesions classified as PBCL and MM, respectively. In both cases, only
the second lesion contained mutations in the BCL-6 gene.
To confirm the results obtained by SSCP analysis, 10 representative PCR
products, of which 6 had altered mobility patterns on SSCP analysis and
4 did not, were cloned and subjected to sequence analysis. Lesions from
which these PCR fragments were obtained are identified in Table 1 by an
asterisk. Mutations identified were as follows, where base numbering
corresponds to the first intron of the BCL-6 gene: Case 15, region
E1.9: C insertion (117), C T (134), TA (141); Case
15, region E1.10: AT (225), AG (244), AC
(262), CG (323), AC (333); Case 16, region E1.11:
GC (485), CG (502); Case 28, region E1.11:
TG (526), GC (552); Case 30, region E1.11:
CT (596); and case 36B, region E1.11: TG (503), TG (504), CG (586). Mutations were frequently
multiple, as seen by the abnormal migration patterns in more than one
PCR fragment (Table 1), or by the presence of multiple mutations within
one sequenced PCR fragment. Wild-type clones were obtained from all six
cases with abnormal SSCP migration, which may derive from a normal
allele or from normal cells infiltrating the PT-LPD lesion. No cases
were detected in which heterozygous mutations were present in both
alleles. In the three cases for which PCR fragments with normal SSCP
migration patterns were sequenced, either no mutations were identified
in any of the four clones examined (one case) or single-base pair
mutations in only one clone were observed (two cases), which could not
be confirmed by sequencing of additional clones. These changes may
represent Taq polymerase mistakes rather than true mutations. However,
the possibility of the presence of rare tumor cells carrying mutations
that cannot be identified by SSCP analysis cannot be excluded.
Correlation of BCL-6 gene mutations and histopathologic category.
None of 11 PH specimens (0%), 3 of 8 PBCH specimens (37%), 14 of 29 PBCL specimens (48%), and 8 of 9 (87%) NHL/MM specimens contained
alterations in this region. These differences were statistically significant (P = .0048; Table 2).
Using pairwise comparisons, these differences were not significant
between PBCH and PBCL. Grouping these two categories into one of
polymorphic PT-LPDs, the correlation of the presence of BCL-6 gene
mutations with histopathologic category increased in statistical
significance (P = .0022).
Correlation of BCL-6 gene mutations and clinical outcome.
To determine the clinical significance of BCL-6 gene mutations, we
compared clinical outcome of 33 patients with the presence of mutations
in the BCL-6 gene in their PT-LPD lesions. The presence of these
mutations had a significant correlation with time to death (P = .02); patients with lesions lacking BCL-6 gene mutations had a longer
survival as seen in the Kaplan-Meier analysis shown in
Fig 2. There was also a high correlation
between the presence of BCL-6 gene mutations and the different clinical
outcome categories. None of the cases with lesions that regressed after
reduction of immunosuppression had BCL-6 gene mutations. Moreover, the
absence of such mutations had a predictive value for regression when
compared with resolution (P = .026) and no response (P = .007), with a significant overall predictive value of BCL-6 mutations
when regression versus resolution versus no response were compared
(P = .008) (Table 3). Cases that
showed surgical resolution also lacked BCL-6 mutations, in contrast to
those with medical resolution (P = .0001).
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| Fig 2.
Survival function estimates of BCL-6 gene mutations in
PT-LPDs. Formal analysis of survival (Kaplan-Meier estimate) with
regard to the presence or absence of BCL-6 gene mutations is shown.
There was a statistically significant (P = .02) decrease in
time to death in the patients with PT-LPDs containing BCL-6 gene
mutations. The continuous line indicates a wild-type BCL-6 as assessed
by SSCP, and the discontinuous line indicates a mutant BCL-6.
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Because the cases belonging to the PH category have been shown to have
a benign clinical course with regression after reduction of
immunosuppression, and the monomorphic cases classified as NHL/MM to
behave as aggressive malignant neoplasms with a poor clinical
outcome,10 we performed the same statistical analysis including only the polymorphic categories (PBCH and PBCL) to determine whether the presence of BCL-6 mutations predicted clinical behavior among these cases. Survival analysis could not be performed because of
the small sample size of 17 patients. However, a statistically significant predictive value for the presence of BCL-6 gene mutations was observed for regression versus resolution versus no response (P = .02) (Table 3), which was because of the predictive value for regression when compared with resolution (P = .044) and no response (P = .048).
When cases were grouped by either of two clinical outcomes, one
including regression and surgical resolution, and the second including
medical resolution and no response, the statistical significance of the
presence of BCL-6 gene mutations was P = .00001. When the same
analysis was performed for only the polymorphic categories, the
predictive value for the presence of BCL-6 gene mutations was still
statistically significant (P = .0004)
(Table 4). None of the lesions in the first
outcome category (regression or surgical resolution) contained BCL-6
mutations. In contrast, PT-LPD lesions that showed medical resolution
or no response had BCL-6 gene mutations in all but two patients (Cases
19 and 33). Case 19 was the only case in which chemotherapy was
administered without previous reduction of immunosuppression, and thus
it is unknown whether this lesion would have regressed. The second
exception, Case 33, was unusual in that it was an EBV-negative tumor
classified morphologically as a diffuse large-cell lymphoma which
presented 10 years after renal transplantation. Thus, the lack of
mutations in the BCL-6 gene is a very good predictor of regression or
resolution with local therapy, whereas the presence of mutations in
this gene predicts the need for more aggressive and systemic
therapeutic intervention.
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Table 4.
Predictive Value of Absence of BCL-6 Gene
Mutations in PT-LPDs: Response to Reduction of
Immunosuppression or Surgical Excision Versus Medical Resolution or
No Response
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Relationship of BCL-6 gene mutations with other molecular features.
To determine whether the presence of BCL-6 mutations had any
relationship to other molecular features previously
reported,8,10,36,37 we compared our results with these
previous molecular analyses as shown in Table 1. None of the polyclonal
cases contained BCL-6 gene mutations, but only a subset of cases in
which clonality was shown by the presence of Ig heavy chain
rearrangements and/or EBV terminal repeat analysis contained
BCL-6 gene mutations. The type of EBV and the presence of EBV-LMP 1 gene deletions did not show any correlation with the presence of
mutations in the BCL-6 gene. A subset of cases with BCL-6 gene
mutations, specifically, all but one of those classified as NHL or MM,
contained structural alteration in one of the oncogenes/tumor
suppressor genes examined (c-myc, p53, and N-ras), and all cases
containing structural alterations in one of these genes had mutations
in the BCL-6 gene.
| |
DISCUSSION |
Lymphoproliferative lesions occurring after solid organ transplantation
have been difficult to classify into categories that reflect their
pathobiology and behavior. Here we report the frequent presence of
somatic mutations in the BCL-6 gene in PT-LPDs. The presence of these
mutations is revealing in terms of understanding the biology of these
lymphoid proliferations as well as mechanisms of tumor progression. Our
findings show that BCL-6 gene mutations are a consistent step in the
progression from a PT-LPD lesion that can be controlled by a
reconstituted immune system to one that will require more aggressive
therapeutic intervention. This is the first report of an objective
parameter that apparently can distinguish between "hyperplastic"
and "neoplastic" categories of PT-LPDs. This distinction is even
true among the polymorphic lesions which, despite considerable
histopathologic heterogeneity, possess indistinguishable
immunophenotypic and molecular features. Therefore, we show that
assessment of the presence of structural alterations in the BCL-6 gene
is useful in predicting clinical responses and in guiding informed
therapeutic strategies.
Our previous studies suggested that PT-LPDs are divisible into three
clinicopathologic categories: (1) PH at the benign end of the spectrum;
(2) NHL/MM at the malignant end; and (3) the polymorphic lesions, which
include the histopathologic categories of polymorphic hyperplasia and
polymorphic lymphoma, in the middle.8 However, we have not
been able to identify molecular or clinical differences that
consistently correlate with the morphological subdivision of the
polymorphic lesions into hyperplasia and lymphoma categories. Age,
stage of disease, time from transplantation to development of PT-LPD,
and clinical outcome were not statistically significantly different
between the two histopathologic subcategories of polymorphic
PT-LPD.9 Furthermore, these polymorphic lesions are all
clonal as determined by Ig gene rearrangement and EBV terminal repeat
analysis, and they lack structural alterations in most of the genes
involved in other lymphoid neoplasms, including c-myc, p53, ras, BCL-1,
and BCL-2.8 We now show that BCL-6 gene mutations are
common in the polymorphic PT-LPDs, but the presence of these mutations
is not preferentially associated with the morphological subcategories
of hyperplasia or lymphoma. However, the results of BCL-6 gene mutation
analysis correlated well with clinical outcome, suggesting that these
molecular studies may be more accurate than morphology in dividing
PT-LPDs into the biological categories of hyperplasia and malignant
lymphoma.
In this study, rearrangements of the BCL-6 gene were not identified by
Southern blot analysis, distinguishing PT-LPDs from diffuse large
B-cell lymphomas. However, because only a small number of PT-LPD
lesions classified as NHL were examined, we cannot exclude the
possibility that BCL-6 gene rearrangements occur more frequently among
PT-LPDs than is reflected in this report. Our results confirm previous
studies demonstrating that mutations can occur independently of
rearrangements in the BCL-6 locus, and show that mutations are much
more common than rearrangements in PT-LPDs.
A subset of PT-LPDs which remain poorly understood are those lacking
EBV, which account for only 10% or less of all lesions. It seems that
these frequently occur late after organ transplantation, and it has
been suggested that these have poor clinical outcomes.38 Among the cases included in this study, three cases lacked EBV, classified as PH (Case 8), PBCH (Case 12), and NHL (Case 33), respectively. Although there was no obvious difference between the
first two EBV-negative cases and their EBV-positive counterparts, Case
33, classified as NHL, was different from other PT-LPDs in several
respects. This case occurred 10 years after a kidney transplant, which
is the longest interval we have seen between transplantation and
appearance of the first PT-LPD, with all other cases having occurred
between 6 weeks and 8 years with a median of 18.5 months. Furthermore,
this is the only case of NHL in which we could not detect the presence
of BCL-6 gene mutations, and the only case without these mutations
which showed no response to therapy. It is possible that this case does
not represent a true PT-LPD but rather a sporadic lymphoma presenting
coincidentally in a patient with a history of renal
transplantation. Although this case was nevertheless included for
statistical purposes, this observation suggests that the presence or
absence of BCL-6 gene mutations in EBV-negative cases may not be
clinically significant. Studies of larger numbers of PT-LPD lesions,
especially those negative for EBV, should help to clarify this issue.
Whereas functional characterization of BCL-6 is still in its early
stages, BCL-6 is thought to be involved in germinal center formation.18,19 Mutations are always found in a 3.5-kb
region spanning the first noncoding exon, and clustering in the
5 region of the first intron,30 which was the only
fragment analyzed in the present study. It is thought that this region
contains an important regulatory element. Interestingly, although this is the same region in which chromosomal breakpoints in large-cell lymphomas tend to cluster, we have not identified molecular
rearrangements in this region of BCL-6 in PT-LPDs.39
Similar to previous studies,30,31 we have found that
mutations are often multiple. This finding is consistent with the
hypothesis that these mutations are caused by a mechanism similar to
that responsible for Ig variable region gene hypermutation, although
they can occur independently of translocation to Ig loci and in the
absence of any recognizable rearrangement of BCL-6.30 BCL-6
protein is normally expressed in germinal centers. In neoplastic
situations it is expressed in B-cell lymphomas thought to originate in
germinal center or post-germinal center B cells, specifically,
follicular lymphomas, diffuse large B-cell lymphomas, Burkitt's
lymphoma, and lymphocyte-predominance Hodgkin's disease.40
Furthermore, the BCL-6 gene has only been found to be altered in
follicular, diffuse large-cell lymphomas, and AIDS-related NHLs.20,25,30,31,41 PT-LPDs are most likely derived from germinal center B cells. The germinal center is where the Ig variable region gene undergoes somatic hypermutation,42 and
hypermutation in this region suggesting antigen selection has been
reported in a small number of PT-LPDs.43 It remains to be
determined whether mutations in the noncoding region of the BCL-6 gene
are directly responsible for transformation of PT-LPDs, or are simply a
reflection of an altered genetic stability or abnormal hypermutation mechanism, which in turn may result in alterations of other genes that
are responsible for tumor progression. Regardless of the direct
mechanism of transformation, alterations in the BCL-6 gene are the
earliest genetic lesions identified thus far in the progression of
PT-LPDs, occurring after clonal expansion and before mutations and rearrangements in other oncogenes (c-myc and N-ras) and tumor suppressor genes (p53). Thus, the presence of BCL-6 gene mutations seems to be a good early marker of progression from a reactive lesion
that can still be controlled by the immune system to a malignant
lymphoma.
In conclusion, this study suggests that the presence of BCL-6 gene
mutations distinguish posttransplantation-associated lymphomas from
hyperplasias. Furthermore, assessment of the presence of mutations in
the BCL-6 gene provides an evaluable and objective parameter for tumor
progression that does not depend on subjective histomorphological
evaluation. Although our results should be confirmed in larger
prospective studies, they indicate that the presence of mutations in
the BCL-6 gene may be a useful clinical marker to determine whether a
reduction in immunosuppression should be attempted or more aggressive
therapy should be instituted.
| |
FOOTNOTES |
Submitted August 12, 1997;
accepted May 28, 1998.
Supported in part by National Institutes of Health Grants No. CA73531
and CA68939 to E.C.
Address reprint requests to Ethel Cesarman, MD, PhD, The New York
Hospital-Cornell Medical Center, Department of Pathology, 525 E 68th
St, New York, NY 10021; e-mail: ecesarm{at}mail.med.cornell.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We are grateful to Dr Cristina Sison for statistical analysis; to Drs
Jonathan M. Chen, Daphne T. Hsu, Thomas J. Garrett, J. Gregory Mears,
Steven Zangwill, Linda J. Addonizio, Robert E. Michler, and Gabriela
Cesarman for submitting the PT-LPD tissues to our immunopathology
laboratory and providing the relevant clinical information; to Dr
Glauco Frizzera for assistance in the morphological classification of
many of the cases presented in this study; and to Dr Elaine Schattner
for critical review of this manuscript.
| |
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Abstract
Introduction
Abstract