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Regular Articles |
From the Molecular Immunology Group of the Wessex Immunology
Service*
and the Department of
Pathology,
Southampton University Hospitals
National Health Service Trust, Southampton; and the Department of
Pathology,
Cheltenham General Hospital,
Cheltenham, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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clonal products were identified in
16/22, TCRß clonal products in 16/22 and IgH clonal products
in 6/22 cases. Oligoclonal PCR products were seen for TCR in 3/22 and
for IgH in 3/22 cases. In one biopsy PCR products from all reactions
were polyclonal. Sequence analysis revealed functional TCR,
TCRß, and IgH sequences in 6/12, 9/11, and
8/8 cases, respectively. Functional TCR and/or IgH oligoclones
were detected in 6/20 (30%) cases. In addition, nonfunctional
TCR and IgH sequences were found in 11 cases. EBERs were identified in
18/20 cases varying from occasional to 25 to 30% nuclei staining and
were associated with both T and B cells, although the majority
were of indeterminate phenotype. The presence of EBERs was not
associated with all clonal IgH gene rearrangements but was associated
with B cell oligoclones. Patterns of gene recombinations indicated that
the majority of TCR recombinations used GV1 and GJ1S3/2S3 genes. Six
out of eleven cases used TCR BV4S1 or BV2S1 genes associated with
various BJ and BD1/2 genes. No common IgH gene usage was
identified, but 8 clones had varying degrees of replacement and
silent mutations (0.6–10.1%), consistent with B cell clones
having undergone somatic mutation in the germinal center, and 3
clones harbored unmutated V genes, consistent with naive B
cells. Our data do not support the concept of AILD as a clearly defined
peripheral T cell lymphoma (PTCL). Rather, they suggest
that AILD as defined by histology and immunohistochemistry is either a
heterogeneous entity or represents a lymphoproliferation associated
with immunodeficiency in which clonal T cell or B cell proliferation
may occur.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Clonal rearrangements of TCR genes are a useful additional diagnostic index of T cell lymphoma but the interpretation of clonality in AILD is complicated by the heterogeneity of TCR and IgH gene rearrangements.7-16 In addition, clonal rearrangements have been shown to regress or appear during the course of the disease, suggesting that these cases may harbor multiple clones11 similar to those found in cases with acquired immunosuppression.13,16 This conclusion is supported by cytogenetic studies that reveal a characteristic chromosome pattern consistent with the frequent presence of karyotypically unrelated abnormal clones, which may also appear and regress in a high proportion of AILD cases.9,17 Given the heterogeneous nature of AILD it is reasonable to propose that a relationship between reactive and clonal T and B cell lymphoproliferations exist in this entity.
Clonal studies in AILD have depended on the identification of rearranged alleles by Southern blot analyses or detection of clonal products after polymerase chain reaction (PCR) amplification. The detection of gene rearrangements do not necessarily imply that they are functional indicators of lymphocyte lineage, as nonfunctional rearrangements may occur in both T and B cells during differentiation. Cross-lineage IgH rearrangements also occur in T cell lymphoma and leukemia,18 with a higher incidence occurring in lymphoblastic T cell malignancies (10–15%) compared to mature T lymphocyte neoplasms (5%).19 This problem can be overcome by sequence analyses of PCR products to reveal the functional or nonfunctional nature of a specific TCR of IgH gene rearrangements in these cases.
We have undertaken a retrospective molecular study of T and B cell
populations in 22 archival cases of AILD using PCR amplification of DNA
isolated from formalin-fixed biopsy tissue to detect clonal TCRß,
TCR, and IgH gene rearrangements for the purpose of establishing i)
occurrence of clonal/oligoclonal TCR and IgH rearrangements among the
group; ii) the presence of functional rearrangements as established by
sequence analysis to investigate T cell lineages associated with each
case, evidence for preferential V gene family use by these cell
populations, and presence of B cell clones; and iii) the role, if any,
of EB virus in the etiology of these lymphoproliferations, especially
in those cases where functional IgH rearrangements have been
identified.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Twenty cases diagnosed as AILD in the histopathology files of
Southampton University Hospitals NHS Trust (SUHT) between 1990 and 1995
were entered into the study. Two additional cases were referred by one
of us (K. McC.). The patients ranged in age from 45 to 75 years and the
male:female ratio was 13:9. Clinical details of patients in the study
are given in Table 1
. Biopsies were
derived from a variety of lymph node sites and fixed in 10% formalin.
All biopsies were reviewed and the histological diagnosis confirmed by
immunohistochemistry. One patient (case 17) had a splenic biopsy 3
months postpresentation.
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Histology and Immunohistochemistry
Paraffin sections were cut at 5 µm and stained with hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), Giemsa, and reticulin using the Gordon and Sweet method. Immunohistochemistry was performed using the streptavidin-biotin peroxidase complex technique on 5-µm sections mounted on silane-coated slides. The antibodies used included CD3, BF1(TCRß), and 1F8(CD21) on sections pre-treated with pronase, CD8, UCHL1(CD45RO), and CD79a on sections pre-treated in a microwave with citrate buffer, CD4 on sections pre-treated in a pressure cooker in EDTA, and L26(CD20) on sections with no pretreatment.
Epstein-Barr Virus (EBV)
Formalin-fixed, paraffin-embedded sections were prepared from all biopsies and used for in situ hybridization (EBV polyprobe kit, Novacastra Laboratories, UK) to detect EBV-encoded short RNA species (EBER). EBV-positive controls consisting of a known EBV-positive case and negative controls were assayed in the same run. Double staining for CD3, CD45RO, CD20, and EBER was performed on 5-µm sections using the streptavidin-biotin complex technique. The peroxidase label was identified using 3-amino-9-ethylcarbazole (AEC), giving a red reaction product. In situ hybridization for EBERs was then performed using the Novacastra EBV probe kit following the manufacturer’s protocol.
Genotypic Studies
All PCR methods were first established using positive controls including 3 T cell lines and 17 clonal T cell neoplasms previously characterized by Southern blot analysis. For Southern blot analysis, high molecular weight DNA was extracted from the biopsies by routine methods, digested with 3/4 restriction enzymes, Southern blotted, and probed with 32P-labeled TCR and Ig probes as previously described.20
PCR Analysis of TCR, TCRß, and IgH Gene Rearrangements
DNA was isolated from paraffin-embedded biopsy material as
described previously.21
TCR PCR analysis was adapted
from McCarthy et al22
and used primers VG11 (detects VGI
genes, VG101 (detects VGIII and VGIV genes), VG9 (detects VGII gene),
JG12 (detects JG1.3 and JG2.3 genes) and JP12 (detects JG1.1 and JG2.1
genes). The primers were used in the following combinations: VG11,
VG101, VG9, JG12 and VG11, VG101, JP12. TCRß PCR analysis was
performed using VBJB2 and DB1JB2 primer combinations.24
TCR and -ß PCR samples were subjected to 30 cycles of PCR (93°C
for 1 minute, 52°C (TCRß) or 55°C (TCR) for 1 minute, 73°C
for 1 minute). PCR products were electrophoresed through a 10%
polyacrylamide gel. Sizes of the TCR PCR products ranged from 75 to 110
bp for TCR products and from 55 to 100 bp for TCRß products.
Clonal bands are defined as one or two narrow sharp intense bands
visible on polyacrylamide gels after electrophoresis, whereas
polyclonal PCR products appear as a smear within the appropriate size
range. Oligoclonal populations are defined by three or more distinct
bands. These definitions also apply to IgH FR2 PCR analyses.
Fluorescent TCRß PCR amplification for gene scanning analysis was included in the study due to the reported increased detection rate of TCRß in T cell leukemia/lymphoma.25 PCR amplification was performed using a seminested approach using VBcon, JBI(2), and JBII(2) primers in the first round of amplification, and VBcon (5' fluorescently labeled), JBI(1) and JBII(1) for the second round of amplification.25 Two percent of the volume of the first-round PCR products was used for the second round of amplification. Samples were subjected to 40 (first round)/30 (second round) PCR cycles (92°C for 1 minute, 50°C (first round)/56°C (second round) for 40 seconds, 72°C for 30 seconds). For analysis on the ABI automated gene sequencer, 1 µl of PCR product was mixed with 3 µl formamide and 0.5 µl of an internal size standard (Genescan-2500 Rox; Applied Biosystems, Warrington, UK) and denatured for 3 minutes at 90°C before being separated on a high resolution polyacrylamide gel and analyzed using GENESCAN 672 software. Products from clonal cell populations produce one or two sharp peaks of fluorescence corresponding to the PCR-amplified clonal rearranged allele(s), whereas DNA extracted from normal polyclonal peripheral blood produce a fluorescence spectrum composed of polyclonal PCR fragments of different sizes in a normal Gaussian distribution. Oligoclonal scan profiles are observed as three or more distinct peaks above a normal polyclonal background.
The TCR PCR methods in this study were established using DNA from
clonal T cell tumors, including 4 lymphocytic leukemias, 7
lymphoblastic lymphomas, 2 unclassified peripheral T cell lymphomas, 2
enteropathy-associated T cell lymphomas, and 1 T cell angiocentric
lymphoma, previously characterized by Southern blot analysis as clonal
for TCR and TCRß loci. In these experiments, TCR rearrangements
were detected by PCR in 16/17 (94%) cases using VJ and VJP primers.
TCRß PCR with primers for VJ and DJ detected rearrangements in 8/17
(47%) and 10/17 (59%) cases, respectively; using both primer
combinations the success rate was 71%. In comparison, TCRß gene
scanning was successful in 13/16 (81%) of cases. IgH Southern blot and
PCR analyses were completed in all cases and IgH clonal gene
rearrangements were identified in 2 cases by both techniques. Three T
cell lines (Molt4, Jurkat, and HPBALL) were also shown to be clonal by
TCR PCR analysis and gene scanning; two of the lines were shown to
be clonal with McCarthy’s VJ primers but no clonal product was found
with Molt4 due to lack of specificity of the consensus VB primer with
BV2S1.24
Sequence analysis of Molt4 and Jurkat confirmed
published sequences.26,27
In a study of frozen and
paraffin material of the same biopsy from 30 patients, concordant
results were obtained in all but one case. In that case, the DNA was
too degraded from the paraffin biopsy to produce unambiguous PCR
results.
IgH FR2 PCR analysis was performed using a seminested PCR amplification method.28 The first-round PCR consisted of 30 cycles of amplification of 250 ng template DNA using primers FR2 and LJH followed by 20 cycles of amplification using 0.5 µl from the first round PCR using primers FR2 and VLJH. Cycling conditions for both rounds consisted of 93°C for 45 seconds, 50°C for 45 seconds, and 72°C for 110 seconds. PCR products were electrophoresed through a 5% polyacrylamide gel. Sizes of the IgH FR2 PCR products ranged from 240 to 280 bp. Our detection rate was 88% for demonstrating B cell clonality in a series of 40 immunohistologically characterized B cell neoplasms, including 24 B cell leukemias (22 B cell chronic lymphocytic leukemia, 1 B cell prolymphocytic leukemia, 1 hairy cell leukemia) and 16 B cell non-Hodgkin’s lymphoma (data not shown).
Sensitivity
DNA from known positive clonal controls were serially diluted (comprising 100, 50, 40, 30, 20, 10, 5, 2, and 1% of tumor DNA) with DNA from reactive polyclonal controls. After appropriate PCR amplification, clonal products could be detected at a level of 2% clonal DNA on a polyclonal background smear/electrophoregram for all PCR protocols.
Cloning and Sequencing of PCR Products
Amplified TCR, TCRß, and IgH FR2 PCR products were gel
electrophoresed and subsequently purified using GeneClean or
MERmaid kits (Anachem, Luten Bedfordshire, UK) before
transformation into JM109-competent cells (Promega, Madison, WI). We
preferentially sequenced clonal TCRß products from TCRß scanning,
because VB gene usage can be assigned from these products. Clones were
randomly selected for each case. In some instances, the final extension
time of the PCR cycling was extended to 1 hour to increase the cloning
efficiency of PCR products.29
Single-stranded DNA was
prepared and sequenced by the dideoxy chain termination method using
sequenase (Amersham International PLC, Little Chalfont,
Buckinghamshire, UK) and M13 primers. Sequence data analysis was
performed using GenBank, Vbase,30
and current databases
with MacVector 4.1.4 sequence analysis software for determination of V,
D, J gene usage and N region addition, as well as stop codons and frame
shifts which result in nonfunctional sequences. Clonal populations were
defined where two or more sequences with identical CDR3 sequences were
obtained from two independent PCR reactions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The lymph nodes in all cases of AILD showed loss of normal lymph node architecture with no reactive follicles and occasional burned-out germinal centers. In most biopsies the lymphoid infiltrate extended through the capsule of the node, often into the perinodal fat. Despite this, the subcapsular sinus was usually easily visible and often appeared to gape. All biopsies showed a proliferation of arborizing high endothelial venules well delineated by silver staining for reticulin and PAS staining that highlighted amorphous perivascular material. The lymphoid cells ranged from small lymphocytes and plasma cells through medium and large lymphoid cells. Clusters of clear cells of variable size and prominence were seen in all cases. Morphologically atypical cells accompanied by atypical mitoses were seen in only one case (case 19). These occurred as single cells with large, often irregular, nuclei and included multinucleated forms.
Immunohistochemistry showed the lymphoid infiltrate to comprise predominately CD3+, CD45RO+ T cells, mostly of the CD4+ subclass, expressing TCRß. The majority of these cells were small to medium-sized lymphocytes with occasional blast cells. The clusters of clear cells were always of the T cell phenotype. A diffuse infiltrate of small CD8+ lymphocytes was present in all biopsies. These were usually a minority population constituting 5 to 20% of all T cells. However, in 2 cases (cases 3 and 12) they constituted 50 to 60% of the T cells.
The B cell population identified by antibodies to CD20 and CD79a was always greatly reduced, consisting of clusters of small lymphocytes that often appeared to have been pushed to the periphery of the lymph node. Occasional B blasts, often situated at the edge of aggregates of small B cells, were present in all cases. Small aggregates of B blasts were seen in 2 cases (case 9 and 17). The atypical cells seen in case 19 were CD20-negative and some expressed membrane CD3; however, the close proximity of small T cells made it difficult to determine the phenotype of many of the atypical cells. Antibody to CD21 showed an expanded network of dendritic reticulum cells either focally or throughout the node in all cases.
The histology of the follow-up splenectomy on case 17 showed a follicle center cell lymphoma, grade I.
Genotypic Findings
Control Tissue
In 18 biopsies with reactive histology and 4 peripheral bloods, polyclonal products were found in all reactions. TCRß sequence analysis of 34 sequences from 2 peripheral bloods and 2 reactive tonsils revealed different sequences within the same case and between the four cases.
TCRß PCR products from 8 clonal T cell cases (2 lymphocytic leukemias, 4 lymphoblastic lymphomas, and 2 PTCL), used for verification of PCR methods (see Methods), were sequenced. In each case a clonal sequence was found, corresponding in size to the clonal peak on TCRß scan analysis.
AILD Study Group
Twenty-five TCR clonal bands were found in 17 cases (cases
1–13, 15, 16, 18, and 20) including 3 oligoclonal bands in case 8
(Table 2)
. With the primer pair VGJG, 19
clonal bands were detected in 15 cases, with 2 or more clonal bands
being detected in cases 1, 2, 8, 10, and 12. In cases 6, 7, and 13,
clonal products were identified with the VGJGP primer pair combination.
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. In case 12, two
clonal bands were detected by VJ as well by DJ primers. In contrast,
TCRß clonal bands defined by scanning of fluorescent products after
PCR amplification were found in 10/22 cases; 1 clonal and 2 clonal
peaks were identified in 4 cases (cases 7, 10, 13, 16) and 6 cases
(cases 8, 11, 15, 17–19), respectively. In addition, oligoclonal peaks
were observed in cases 1 and 21. Representative scans are shown in
Figure 1
.
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, Figure 2
).
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Sequence Analysis
Sequences were obtained from 44 clonal/oligoclonal products from
20 cases; the deduced amino acid sequences are summarized in Table 3
. TCR and TCRß products were
sequenced from 12 and 11 cases, respectively, and IgH products were
sequenced from 8 cases. Multiple sequences per locus were identified in
9 cases including TCR in cases 1, 2, 8, and 9, TCRß in cases 7,
10, and 15, and IgH in cases 10, 11, 15, and 18. Twenty-eight
functional in-frame sequences were identified including 7 in-frame
TCR sequences (cases 3, 4, 8, 9, 11, 12), 10 in-frame TCRß
sequences (cases 7, 10, 11, 14–19) and 11 in-frame IgH sequences
(cases 6, 10, 11, 13, 15, 18–20). Both functional TCR and IgH
sequences were found in cases 11 and 18 (Table 3)
. Nonfunctional TCR
and TCRß sequences were found in 8 cases (cases 1, 2, and 5–10) and
5 cases (cases 7, 8, 10, 13, 15) respectively. Nonfunctional IgH
sequences were found in 2 cases (cases 11, 15; Table
4).
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and -ß gene usage is tabulated in Table 3
. The majority of
TCR gene recombinations used GVI and GJ1S3/J2S3 genes (including 6
functional and 8 nonfunctional sequences); in 2 cases GVI was
rearranged to J2S1 gene and in the remaining case GVI was rearranged to
GJ1S1. In cases with functional TCRß rearrangements TCRBV2S1 genes
were used in 4 cases, whereas in the remaining cases BV4S1, BV5S1,
BV6S7, BV13S3, and BV17S1 were used. TCRBV4S1 and BV17S1 were used in 3
cases with nonfunctional sequences. A variety of TCRB-J gene segments
and both BD genes were used, although in case 16 the D segment could
not be unambiguously assigned, and in cases 7 and 10 no D gene segment
could be assigned (Table 3)
. A unexpected BD2 rearrangement to TCR
BJ1S1 gene was also identified in a sequence from case 18 (Table 3)
.
The majority of cases with IgH rearrangements used VH3 and VH4 genes
and a wide range of D segment genes with a predominance of the JH4
genes (Table 3)
. In these cases the incidence of somatic mutations was
assessed by comparing the VH sequences with the closest germ line
sequences in VBase.30
Three clones from 3 cases harbored
unmutated V genes, whereas in 8 clones from 8 cases there were varying
degrees of replacement and silent mutations ranging from 0.6 to 10.1%.
Case 11 had significant clustering of replacement amino acids in CDR2,
indicative of antigen selection based on the method of Chang and
Casali31
(data not shown).
EBV
In situ hybridization for EBERs showed positive cells
in 15 of 20 cases (Table 3)
. In two of these, only occasional labeled
nuclei were identified. In 11 cases, 1 to 2% of nuclei were labeled;
these were of intermediate to large size. In case 12, 25 to 30% of the
nuclei were positive. In all positive cases, double staining identified
some EBER-positive cells expressing CD20. Occasional EBER-positive
cells expressing CD3 and/or CD45RO were also seen. However, the
majority of cells expressing EBERs appeared to be of indeterminate
phenotype. Case 12 had 50 to 60% CD8+ T cells. The follicle center
cell lymphoma in case 17 was EBV-negative.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study we have investigated cell lineages in 22 cases of AILD by
PCR amplification of DNA isolated from paraffin-embedded biopsy
specimens and sequence analysis of PCR products. PCR amplification,
unlike Southern blot analyses, can be applied to poor quality DNA for
amplification of products <300 bp but suffers from the disadvantage of
limited primer homology annealing to all V gene sequences, which can
lead to false negative and discordant results using different primer
sets for a chosen chromosome locus. For this purpose the methods used
in this study were first characterized by comparison with Southern blot
analysis and by analysis of immunohistologically characterized tumors,
with success rates exceeding 80% for TCR, TCRß (gene scanning),
and IgH primers, comparable with other published studies using
PCR.23,24
A combination of TCRß protocols using two
different VJ primer sets to identify full recombinations and one DJ
primer set to identify partial gene recombinations was used to identify
the majority of T cell rearrangements.
TCRß and/or TCR clonal bands defined as one/two bands on gel
electrophoresis or as TCRß clonal peaks on scans, were detected in
20/22 patients, an incidence of 91%. TCR clonal bands were observed
17 patients (77%). TCRß gene scanning revealed clonal peaks in 10
cases and oligoclonal peaks in a further two cases, a total of 60% of
cases and was more successful than McCarthy’s VJ
primers,24
that detected clonal bands in 6 cases.
Incomplete DJ rearrangement were detected in 4 cases. Using all of the
combinations of primers and techniques, TCRß clonal rearrangements
were detected in 16/22 cases; if oligoclonal rearrangements are
included, the incidence is 18/22 (82%), compatible with Frizzera et
al.8
However, results with the IgH FR2 primer combination
revealed a higher incidence of Ig clonal rearrangements than that
previously observed.8
IgH rearrangements were identified
in 9/22 cases, an incidence of 41%, including 3 cases (14%) with
oligoclonal bands.
Forty-four PCR products from 20 cases were sequenced for lineage
assignment including TCR, TCRß, and IgH products from 12, 11, and
8 cases, respectively. Functional and nonfunctional sequences were
determined by alignment to germline sequences to indicate in-frame
rearrangements, frame shifts or the acquisition of stop codons.
Functional sequences were found in 17 cases and more than one
functional sequence was found in 6 cases. Nonfunctional sequences were
associated with other functional TCR or IgH rearrangements in 8 cases.
Nonfunctional rearrangements occur during T and B cell maturation in
association with a functional allele and the failure in 3 cases to find
a functional sequence probably reflects the limitation of PCR analysis
to detect all possible rearrangements.33
TCR sequences were found in 13 cases, where the majority of
recombinations used GVI and GJ1S3/J2S3 genes reflecting common GV and
GJ gene usage in T cell lymphomas.34
It is interesting to
note that TCR was functionally rearranged in 6 cases and was the
only functional gene in 5 cases, all predominantly infiltrated with T
cells expressing CD3 and TCRß. Whether this finding represents TCRGD
clones in a predominant population of TCRAB positive cells is difficult
to assess as antibodies to TCRD could not be applied successfully to
paraffin biopsy specimens. However, similar cases have been described
as TCR silent PTCL by Theodorou et al.34
Alternatively,
such cases may represent in-frame TCR rearrangements in
TCRß-expressing cells where, for technical reasons, we were unable to
detect TCRß clonal rearrangements, since in-frame TCR
rearrangements have been described in TCRAB thymocytes at low
frequency.35
TCRß PCR products from 11 cases were sequenced and functional and
nonfunctional TCRß rearrangements were found in 9 and 4 cases
respectively. BV4S1 and BV2S1 genes were predominantly used in 3 and 4
cases respectively, while the remaining cases used BV5S1, BV6S1, BV13S3
and BV17S1. These data suggest an association in AILD that has not been
observed in previous studies of T cell leukemias and
lymphomas.36
These data also explain the discrepancy
between TCRß protocols in this study, as McCarthy’s primers lack
homology with BV families V2, V4, and V8.24
In three cases
functional TCRß gene rearrangements were detected in the absence of
TCR gene rearrangements, contrary to expectation where nonfunctional
TCR genes are usually associated with functional TCRß gene
rearrangements.37
This finding may also result from lack
of primer homology for all TCR rearrangements. D segment analysis
was uninformative but suggested chromosomal inversion in case 18,
direct VJ joining in cases 7 and 10, and extensive exonucleolytic
nibbling in case 16.38-40
IgH clonal and oligoclonal products from 8 cases were sequenced and functional IgH sequences were identified in all cases, including two sequences each in cases 10 and 18 and multiple sequences in case 15. Although Ig light chain restricted populations of B cells could not be detected immunohistology, these data provide unequivocal molecular evidence for the presence of one or more B cell clones in these AILD biopsies. These data also confirm and extend the historical Southern blot findings that suggest that clones of B cells may be a common finding in AILD.8 The origin of these B cell clones was assessed by alignment of sequences to germline IgH genes for the presence of somatic mutations. Evidence for significant clustering of replacement amino acids in the CD2 region was found in 1 clonal sequence (case 11) indicative of antigen selection in the germinal center and another 7 clones (cases 6, 10, 13, 15, 18, 19, and 20) had varying degrees of replacement and silent mutations (0.6–10.1%) consistent with germinal center B cells.31 Three clones from 3 cases (cases 10, 15, and 18) harbored unmutated V genes consistent with naive B cells. Serial studies using the CDR3 sequence as a clonal marker may reveal the significance of these minor B cell clones in disease progression following presentation. The association with case 17 described in this report is complex, as the IgH clonal rearrangement was not detected in the original EBV-positive biopsy and its presence was associated with an EBV-negative follicular center cell lymphoma, which may represent an independent event in a patient with T cell disease.
EBV was found in 75% of our cases by in situ hybridization for EBERs, which is lower than the 96 to 97% positivity reported previously.40,41 EBV was found in both B and T cells, but we were unable to assign a definite lineage to a high proportion of EBV-positive cells. However, the facts that i) the number of positive cells in all but two of our positive cases was greater than would be expected in reactive tissue and ii) the infected cell nuclei were larger than average suggest that there was active proliferation of EBV or EBV-infected cells in these nodes. Although Weiss et al41 have suggested that the presence of EBV+ B cells may explain the paradoxical occurrence of B cell lymphoma in a primary T cell lymphoproliferative disorder, we did not find a strong correlation between EBV and B cell clones in the majority of cases in this study or with the emergence of a B cell tumor in case 17. Nevertheless, EBV was associated with B cell oligoclones in cases 1, 10, and 15, and we cannot exclude a role for EBV in these cases. In the majority of cases, however, the presence of EBV may simply reflect the disordered immunoregulation that characterizes this disease.
In this study we have demonstrated functional T and B cell oligoclones in 6/20 (30%) cases. These data are compatible with previous cytogenetic studies of AILD and contrast with the reported frequency of unrelated clones of 0.6% in malignant lymphoma.9,17,43 Furthermore, oligoclones were not found by PCR or sequence analysis in the reactive controls or the T cell tumors used to establish the PCR methods in this study. In addition, B cell oligoclones have not been found in diffuse large B cell lymphomas or in reactive tonsils44 (C. Ottensmeier, personal communication). Our data do not support the concept of AILD as a clearly defined PTCL but offer clear experimental evidence in support of the observation of Lipford et al,13 who concluded that "AILD is a disease of proliferating lymphoid clones in which supervening malignant lymphomas may develop by a process of clonal selection."
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Acknowledgements |
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Footnotes |
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Supported by a grant from the Leukaemia Research Fund (number 9538).
Accepted for publication October 6, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain gene. Diag Mol Pathol 1992, 1:173-179[Medline]
locus in human peripheral T cell lymphomas. J Pathol 1994, 174:233-242[Medline]
ß lineage-committed thymocytes can be rescued by the 
T cell receptor (TCR) in the absence of TCRß chain. Eur J Immunol 1997, 27:2948–2958
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