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Am J Pathol. Feb 2000; 156(2): 661–669.
PMCID: PMC1850038

Frequent T and B Cell Oligoclones in Histologically and Immunophenotypically Characterized Angioimmunoblastic Lymphadenopathy

Abstract

The identification of clonal rearrangements of T cell receptor (TCR) genes is central to the diagnosis of T cell lymphomas. However, in angioimmunoblastic lymphadenopathy (AILD), first described as a nonneoplastic proliferation associated with immunodeficiency, the heterogeneity of TCR and IgH gene rearrangements suggest that some cases may harbor multiple lymphoid clones. In this study we have isolated DNA from archival paraffin biopsy material from 22 cases of AILD identified on the basis of classical histological and immunohistochemical features with the aim of establishing the occurrence of clones and oligoclones, the frequency of TCR and immunoglobulin heavy chain (IgH) variable (v) gene use, and the relationship of these findings to the presence of Epstein-Barr virus. DNA extracted from the biopsies was amplified using the polymerase chain reaction (PCR) and sequenced to detect functional and nonfunctional gene rearrangements. Epstein-Barr virus-encoded short RNA species (EBERs) were detected using in situ hybridization combined with immunochemistry to identify the phenotype of the Epstein-Barr virus-infected cells. Fifty-seven clonal products were found in 20/22 patients: TCRγ clonal products were identified in 16/22, TCRβ 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 TCRγ, TCRβ, 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 TCRγ 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.

Early reports of AILD stressed the nonneoplastic nature of this disease, 1,2 noting that neither the cytology nor the pattern of infiltration seen in biopsies or autopsies was that of a neoplasm. The disease was thought to be due to abnormal immune regulation, the majority of patients dying of infections, often with opportunistic organisms. 2 An association with lymphoma was, however, noted by Lukes and Tindle, 3 who reported the development of immunoblastic lymphoma in 3 of their 32 patients. Subsequently, there has been controversy over the separation of AILD from AILD with immunoblastic lymphoma (IBL) based on histological features such as atypical clear cells, which has contributed to the variation in the reported incidence of lymphoma in AILD from 0 to 35%. 4-6 In addition, both groups show clonality or lack of clonality. 4,7 Frizzera et al 8 has proposed that three AILD-related disorders can be recognized as AILD, AILD-like dysplasia, and AILD-like peripheral T cell lymphoma (PTCL) based on a combination of morphology, phenotype, clonality, and karyotype. However, since karyotypic abnormalities 9 and clonal rearrangements of TCR and/or immunoglobulin (Ig) genes may occur in most cases, irrespective of morphology, this proposal is difficult to sustain.

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 clones 11 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 TCRβ, TCRγ, 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.

Materials and Methods

Patients

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 [triangle] . 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.

Table 1.
Clinical Details of AILD Cases

In addition, 18 formalin-fixed, paraffin-embedded tissue biopsies diagnosed as reactive were collected from the SUHT files and included as polyclonal controls for the study. These biopsies included 13 lymph nodes, 2 spleens, and 3 tonsils. In addition, DNA isolated from blood collected from four healthy normal donors was also included as a control in all PCR assays.

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(TCRβ), 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 TCRγ, TCRβ, and IgH Gene Rearrangements

DNA was isolated from paraffin-embedded biopsy material as described previously. 21 TCRγ PCR analysis was adapted from McCarthy et al 22 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. TCRβ PCR analysis was performed using VBJB2 and DB1JB2 primer combinations. 24 TCRγ and -β PCR samples were subjected to 30 cycles of PCR (93°C for 1 minute, 52°C (TCRβ) or 55°C (TCRγ) 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 TCRγ products and from 55 to 100 bp for TCRβ 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 TCRβ PCR amplification for gene scanning analysis was included in the study due to the reported increased detection rate of TCRβ 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 TCRγ and TCRβ loci. In these experiments, TCRγ rearrangements were detected by PCR in 16/17 (94%) cases using VJ and VJP primers. TCRβ 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, TCRβ 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 TCRγ 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 TCRγ, TCRβ, 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 TCRβ products from TCRβ 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.

Results

Histology and Immunohistochemistry

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 TCRβ. 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. TCRβ 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.

TCRβ 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 TCRβ scan analysis.

AILD Study Group

Twenty-five TCRγ clonal bands were found in 17 cases (cases 1–13, 15, 16, 18, and 20) including 3 oligoclonal bands in case 8 (Table 2) [triangle] . 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.

Table 2.
EBV and Genotype of AILD Cases

TCRβ clonal bands, assessed by electrophoresis for VJ and DJ primers, were found in 6 and 4 cases, respectively (Table 2) [triangle] . In case 12, two clonal bands were detected by VJ as well by DJ primers. In contrast, TCRβ 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 [triangle] .

Figure 1.
Electrophoretic profiles of fluorescent TCRβ products from one positive control, one polyclonal control and six cases. Relative fluorescence intensities (ordinate) are plotted as a function of PCR fragment size (abscissa). Cases are numbered according ...

IgH clonal products were found in 6 cases after DNA amplification with FR2 primers. In 5 cases (cases 6, 11, 13, 19, 20), one clonal band was detected, whereas in the remaining case (case 18), two clonal bands were detected. In an additional 3 cases (cases 1, 10, 15) oligoclonal bands were observed. In total, IgH clonal and oligoclonal bands were found in 9/22 cases (Table 2 [triangle] , Figure 2 [triangle] ).

Figure 2.
Polyacrylamide gels showing IgH FR2/JH PCR products. Lanes M: pBR322 HaeIII molecular weight marker (basepairs indicated on right side). Lane 1: positive clonal control; lane 2: polyclonal control; lane 3: negative control; lane 4: case 6; lane 5: case ...

In case 17 with a follow-up splenectomy 3 years later, DNA analysis revealed a clonal IgH FR2 rearrangement not present in the original biopsy. Furthermore, the original TCR gene rearrangement was also present. The spleen biopsy histology was consistent with follicle center cell lymphoma, grade I.

Sequence Analysis

Sequences were obtained from 44 clonal/oligoclonal products from 20 cases; the deduced amino acid sequences are summarized in Table 3 [triangle] . TCRγ and TCRβ 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 TCRγ in cases 1, 2, 8, and 9, TCRβ 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 TCRγ sequences (cases 3, 4, 8, 9, 11, 12), 10 in-frame TCRβ 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) [triangle] . Nonfunctional TCRγ and TCRβ 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).

Table 3.
Sequence Analysis

TCRγ and -β gene usage is tabulated in Table 3 [triangle] . The majority of TCRγ 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 TCRβ 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) [triangle] . A unexpected BD2 rearrangement to TCR BJ1S1 gene was also identified in a sequence from case 18 (Table 3) [triangle] .

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) [triangle] . 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 Casali 31 (data not shown).

EBV

In situ hybridization for EBERs showed positive cells in 15 of 20 cases (Table 3) [triangle] . 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.

Discussion

Although AILD was first described as a nonneoplastic proliferation associated with immunodeficiency, it is clear that the authors of the REAL classification 32 regard this entity “as a T cell lymphoma because most cases show clonal rearrangements of T cell receptor genes.” Nevertheless, the situation is more complex, and in a recent review, Frizzera collated the findings of nine studies comprising specimens from 107 patients with AILD on which DNA analysis had been performed by Southern blot analysis. 8 Clonality was detected in specimens from 78 of 107 patients (73%) at presentation and clonal rearrangements of the TCRβ gene were found in 68 of 84 specimens (81%). Ig genes were rearranged in 6 of 84 specimens (7%) and with TCR genes in 10 of 84 specimens (12%) giving a total of 16/84 specimens (19%) with Ig rearrangements. Although these data imply heterogeneity of cell lineages among clonal populations in AILD with a higher incidence of IgH clonal rearrangements than that observed in other mature T cell neoplasms, 19 they do not allow unequivocal lineage assignment in individual cases, as it is well documented that cross lineage rearrangements occur in lymphoma and leukemia. 18-19

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 TCRγ, TCRβ (gene scanning), and IgH primers, comparable with other published studies using PCR. 23,24 A combination of TCRβ 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.

TCRβ and/or TCRγ clonal bands defined as one/two bands on gel electrophoresis or as TCRβ clonal peaks on scans, were detected in 20/22 patients, an incidence of 91%. TCRγ clonal bands were observed 17 patients (77%). TCRβ 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, TCRβ 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 TCRγ, TCRβ, 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

TCRγ 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 TCRγ was functionally rearranged in 6 cases and was the only functional gene in 5 cases, all predominantly infiltrated with T cells expressing CD3 and TCRβ. 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 TCRγ rearrangements in TCRβ-expressing cells where, for technical reasons, we were unable to detect TCRβ clonal rearrangements, since in-frame TCRγ rearrangements have been described in TCRAB thymocytes at low frequency. 35

TCRβ PCR products from 11 cases were sequenced and functional and nonfunctional TCRβ 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 TCRβ protocols in this study, as McCarthy’s primers lack homology with BV families V2, V4, and V8. 24 In three cases functional TCRβ gene rearrangements were detected in the absence of TCRγ gene rearrangements, contrary to expectation where nonfunctional TCRγ genes are usually associated with functional TCRβ gene rearrangements. 37 This finding may also result from lack of primer homology for all TCRγ 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 al 41 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 tonsils 44 (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.”

Acknowledgments

This work was supported from the Leukaemia Research Fund.

Footnotes

Address reprint requests to Dr. J. L. Smith, Molecular Immunology Group, Wessex Immunology Service, Southampton University Hospitals NHS Trust, Southampton SO16 6YD, UK. E-mail: .moc.liamtoh@6jhtims

Supported by a grant from the Leukaemia Research Fund (number 9538).

References

1. Lennert K: Pathologisch-histologische klassifizierung der malignen Lymphome. Stacher A eds. Leukaemien und Maligne Lymphome. 1973, :pp 181-194 Urban and Schwarzenberg, Munich
2. Frizzera G, Moran EM, Rappaport H: Angio-immunoblastic lymphadenopathy: diagnosis and clinical course. Am J Med 1975, 59:803-818 [PubMed]
3. Lukes RJ, Tindle BH: Immunoblastic lymphadenopathy: a hyperimmune entity resembling Hodgkin’s disease. N Engl J Med 1975, 294:1-8 [PubMed]
4. Nathwani BN, Rappaport H, Moran EM, Pangalis GA, Kim H: Malignant lymphoma arising in angio-immunoblastic lymphadenopathy. Cancer 1978, 41:578-606 [PubMed]
5. Frizzera G: Atypical lymphoproliferative disorders. Knowles DM eds. Neoplastic Hematopathology. 1992, :pp 459-466 Williams and Wilkins, Baltimore
6. Lennert K, Feller AC: Histopathology of Non-Hodgkin’s lymphomas (based on the updated Kiel classification), 2nd ed. Springer Verlag, Berlin, 1992, pp 196–210
7. Weiss LM, Strickler JO, Dorfman RF, Horning SJ, Warnke RA, Sklar J: Clonal T-cell population in angioimmunoblastic lymphadenopathy, and angioimmunoblastic-like lymphoma. Am J Pathol 1986, 122:392-397 [PMC free article] [PubMed]
8. Frizzera G, Kameko Y, Sakurai M: Angioimmunoblastic lymphadenopathy and related disorders: a retrospective look in search of definitions. Leukaemia 1989, 3:1-5 [PubMed]
9. Schlegelberger B, Zhang Y, Weber-Matthiesen K, Grote W: Detection of aberrant clones in nearly all cases of angioimmunoblastic lymphadenopathy with dysproteinemia-type T-cell lymphoma by combined interphase and metaphase cytogenetics. Blood 1994, 84:2640-2648 [PubMed]
10. Feller AC, Greisser H, Schilling CV, Wacker HH, Allenbach FD, Bartels H, Kuse R, Mak TW, Lennert K: Clonal gene rearrangement patterns correlate with immunophenotype and clinical parameters in patients with angioimmunoblastic lymphadenopathy. Am J Pathol 1988, 133:549-556 [PMC free article] [PubMed]
11. Griesser H, Feller A, Lennert K, Minden M, Mak TW: Rearrangement of the β chain of the T cell antigen receptor and immunoglobulin genes in lymphoproliferative disorders. J Clin Invest 1986, 78:1179-1184 [PMC free article] [PubMed]
12. O’Connor NTJ, Crick JA, Wainscoat JS, Gatter KC, Stein H, Falini B, Mason DY: Evidence for monoclonal T lymphocyte proliferation in angioimmunoblastic lymphadenopathy. J Clin Pathol 1986, 39:1229-1232 [PMC free article] [PubMed]
13. Lipford EH, Smith HR, Pittaluga S, Jaffe ES, Steinberg AD, Cossman J: Clonality of angioimmunoblastic lymphadenopathy and implications for its evolution to malignant lymphoma. J Clin Invest 1987, 79:637-642 [PMC free article] [PubMed]
14. Ganesan TS, Dhaliwal HS, Dorreen MS, Stansfeld AG, Habeshaw JA, Lister TA: Angioimmunoblastic lymphadenopathy: a clinical, immunological and molecular study. Br J Cancer 1987, 55:437-442 [PMC free article] [PubMed]
15. Knecht H, Odermatt BF, Hayoz D, Kuhn L, Bachmann F: Polyclonal rearrangements of the T-cell receptor β-chain in fatal angioimmunoblastic lymphadenopathy. Br J Haematol 1989, 73:491-496 [PubMed]
16. Hodges E, Quin CT, Wright DH, Smith JL: Oligoclonal populations of T and B cells in a case of angioimmunoblastic T cell lymphoma predominantly infiltrated by T cells of VB5.1 family. J Clin Pathol 1997, 50:15-17 [PMC free article] [PubMed]
17. Kaneko Y, Maseki M, Sakuri M, Takayama S, Nanba K, Kikuchi M, Frizzera G: Characteristic karyotypic pattern in T-cell lymphoproliferative disorders with reactive “angioimmunoblastic lymphadenopathy with dysproteinemia-type” features. Blood 1988, 72:413-421 [PubMed]
18. Pelicci P-G, Knowles DM, Dalla-Favera R: Lymphoid tumors displaying rearrangements of both immunoglobulin and T cell receptor genes. J Exp Med 1985, 162:1015-1024 [PMC free article] [PubMed]
19. van Dongen JJM, Wolvers-Tettero ILM: Analysis of immunoglobulin and T cell receptor genes. II: Possibilities and limitations in the diagnosis and management of lymphoproliferative diseases and related disorders. Clinica Chimica Acta 1991, 198:93-174 [PubMed]
20. Smith JL, Haegert DG, Hodges E, Stacey GN, Howell WM, Wright DH, Jones DB: Phenotypic and genotypic heterogeneity of peripheral T cell lymphomas. Br J Cancer 1988, 58:723-729 [PMC free article] [PubMed]
21. Hodges E, Swindell AM, Quin CT, Lane AC, Jones DB, Sweetenham J, Smith JL: Expanded TCR Vβ5.1 family in a diffuse high-grade B cell immunoblastic lymphoma. Leukemia 1995, 9:1108-1112 [PubMed]
22. McCarthy KP, Sloane JP, Kabarowski JHS, Matutes E, Weidemann L: A simplified method of detection of clonal rearrangements of the T cell receptor γ chain gene. Diag Mol Pathol 1992, 1:173-179 [PubMed]
23. Slack DN, McCarthy KP, Wiedemann LM, Sloane JP: Evaluation of sensitivity, specificity and reproducibility of an optimized method for detecting clonal rearrangements of immunoglobulin and T cell receptor genes in formalin-fixed, paraffin-embedded sections. Diag Mol Pathol 1993, 2:223-232 [PubMed]
24. McCarthy KP, Sloane JP, Karbarowski JHS, Matutes E, Wiedemann LM: The rapid detection of clonal T-cell proliferations in patients with lymphoid disorders. Am J Pathol 1991, 138:821-828 [PMC free article] [PubMed]
25. Kneba M, Bolz I, Linke B, Hiddemann W: Analysis of rearranged T-cell receptor β chain genes by polymerase chain reaction (PCR), DNA sequencing and automated resolution PCR fragment analysis. Blood 1995, 86:3930-3937 [PubMed]
26. Tillinghast JP, Behike MA, Loh DY: Structure and diversity of the human T-cell receptor β chain variable region genes. Science 1986, 233:879-883 [PubMed]
27. Concannon P, Pickering LA, Kung P, Hood L: Diversity and structure of human T-cell receptor β chain variable region genes. Proc Natl Acad Sci USA 1986, 83:6598-6602 [PMC free article] [PubMed]
28. Yokota S, Hansen-Hagge TE, Ludwig W-D, Reiter A, Raghavachar A, Kleihauer E, Barhram CR: Use of polymerase chain reactions to monitor minimal residual disease in acute lymphoblastic leukemia patients. Blood 1991, 77:331-339 [PubMed]
29. Li Q-B, Guy CL: Prolonged final extension time increases cloning efficiency of PCR products. Biotechniques 1996, 21:192-196 [PubMed]
30. Cook GP, Tomlinson IM: The human immunoglobulin VH repertoire. Immunol Today 1995, 16:237-247 [PubMed]
31. Chang B, Casali P: The CDR1 sequences of major proportion of human germline Ig VH genes are inherently susceptible to amino acid replacement. Immunol Today 1994, 8:367-373 [PubMed]
32. Harris NL, Jaffe ES, Stein H, Banks PM, Chan JK, Cleary ML, Delsol G, De Wolf-Peeters C, Falini B, Gatter KC, Grogan TM, Isaacson PG, Knowles DM, Mason DY, Muller-Hermelink H-K, Pileri SA, Piris MA, Ralfkiaer E, Warnke RA: A revised European-American Classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group. Blood 1994, 84:1361-1392 [PubMed]
33. McCarthy KP: Molecular diagnosis of lymphomas and associated diseases. Cancer Metast Rev 1997, 16:109-125 [PubMed]
34. Theodorou I, Raphael M, Bigorgne C, Fourcade C, Lahet C, Cochet G, Lefranc M-P, Gaulard P, Farcet J-P: Recombination patterns of TCRγ locus in human peripheral T cell lymphomas. J Pathol 1994, 174:233-242 [PubMed]
35. Livak F, Wilson A, Robson Macdonald H, Schatz DG: αβ lineage-committed thymocytes can be rescued by the γδ T cell receptor (TCR) in the absence of TCRβ chain. Eur J Immunol 1997, 27:2948–2958 [PubMed]
36. Smith JL, Lane AC, Hodges E, Reynolds WM, Howell WM, Jones DB, Janson CH: T-cell receptor variable (V) gene usage by lymphoid populations in T-cell lymphoma. J Pathol 1992, 166:109-112 [PubMed]
37. Allison JP, Lanier LL: Structure, function and serology of the T cell antigen receptor complex. Ann Rev Immunol 1987, 5:503-540 [PubMed]
38. Boehm T, Rabbitts TH: The human T cell receptor genes are targets for chromosomal abnormalities in T cell tumors. FASEB J 1989, 3:2344-2359 [PubMed]
39. Roldam EQ, Sottini A, Bettinardi A, Albertini A, Imberti L, Primi D: Different TCRBV genes generate biased patterns of V-D-J diversity in human T cells. Immunogenetics 1995, 41:91-100 [PubMed]
40. Breit TM, van Dongen JJM: Unravelling human T-cell receptor junctional region sequences. Thymus 1994, 22:177-199 [PubMed]
41. Anagnostopoulos I, Hummel M, Finn T, Tiemann M, Korbjuhn P, Dimmler C, Gatter K, Dallenbach F, Parwaresch MR, Stein H: Heterogenous Epstein-Barr virus infection patterns in peripheral T-cell lymphoma of angioimmunoblastic lymphadenopathy type. Blood 1992, 80:1804-1812 [PubMed]
42. Weiss LM, Jaffe ES, Liu XF, Chen YY, Shibata D, Medeiros LJ: Detection and localization of Epstein-Barr viral genomes in angioimmunoblastic lymphadenopathy and angioimmunoblastic lymphadenopathy-like lymphoma. Blood 1992, 79:1789-1795 [PubMed]
43. Heim S, Mitelman F: Cytogenetically unrelated clones in hematological neoplasms. Leukemia 1989, 3:6-8 [PubMed]
44. Ottensmeier CH, Thompsett AR, Zhu D, Wilkins BS, Sweetenham JW, Stevenson FK: Analysis of VH genes in follicular and diffuse lymphoma shows ongoing somatic mutation and multiple isotype transcripts in early disease with changes during disease progression. Blood 1998, 91:4292-4299 [PubMed]

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