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JMD 2000, Vol. 2, No. 1
Copyright © 2000 American Society for Investigative Pathology & Association for Molecular Pathology

Hepatosplenic and Subcutaneous Panniculitis-Like {gamma}/{delta} T Cell Lymphomas Are Derived from Different V{delta} Subsets of {gamma}/{delta} T Lymphocytes

Grzegorz K. Przybylski*, Hong Wu{dagger}, William R. Macon{ddagger}, Janet Finan{dagger}, Debra G. B. Leonard{dagger}, Raymond E. Felgar{ddagger}, Joseph A. DiGiuseppe§, Peter C. Nowell{dagger}, Steven H. Swerdlow, Marshall E. Kadin||, Mariusz A. Wasik{dagger} and Kevin E. Salhany{dagger}

From the Institute of Human Genetics, * Poznan, Poland; the University of Pennsylvania, {dagger} Philadelphia, Pennsylvania; Vanderbilt University, {ddagger} Nashville, Tennessee; the Johns Hopkins University, § Baltimore, Maryland; the University of Pittsburgh, Pittsburgh, Pennsylvania; and Harvard University, || Boston, Massachusetts


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Gamma/delta T cell lymphomas ({gamma}/{delta} TCL) represent rare, often aggressive types of T cell malignancy that are clinically and pathologically diverse. Most {gamma}/{delta} TCL occur as a hepatosplenic or subcutaneous type. To date, analysis of the T cell receptor {delta} (TCR{delta}) gene repertoire of hepatosplenic {gamma}/{delta} TCL ({gamma}/{delta} HSTCL) and subcutaneous panniculitis-like {gamma}/{delta} TCL ({gamma}/{delta} SPTCL) has been reported only in a limited number of cases. In this study we analyzed 11 {gamma}/{delta} HSTCL and 4 {gamma}/{delta} SPTCL by polymerase chain reaction and immunostaining to determine their usage of the V{delta} subtypes (V{delta}1–6). It is noteworthy that 10 of 11 {gamma}/{delta} HSTCL expressed the V{delta}1 gene. The remaining case also expressed T cell receptor {delta} (TCR{delta}) as determined by flow cytometry and TCR{delta} rearrangement in Southern blot. However, the V{delta} gene expressed by this lymphoma could not be determined, which suggests usage of an as yet unidentified V{delta} gene. In striking contrast to the {gamma}/{delta} HSTCL, all 4 {gamma}/{delta} SPTCL expressed the V{delta}2 gene. Our data demonstrate that {gamma}/{delta} HSTCL are preferentially derived from the V{delta}1 subset of {gamma}/{delta} T lymphocytes, whereas {gamma}/{delta} SPTCL are preferentially derived from the V{delta}2 subset. The pattern of V{delta} gene expression in HSTCL and SPTCL corresponds to the respective, predominant {gamma}/{delta} T cell subsets normally found in the spleen and skin. This finding suggests that {gamma}/{delta} TCL are derived from normal {gamma}/{delta} T lymphocytes which reside in the affected tissues. Furthermore, the selective, lymphoma type-specific V{delta} gene segment usage may provide a molecular tool to distinguish better among various types of {gamma}/{delta} TCL lymphoma particularly in the clinically advanced, widely disseminated cases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Similar to normal T lymphocytes, T cell lymphomas (TCL) may express two different types of T cell receptor (TCR), {alpha}/ß or {gamma}/{delta}. Four TCR genes ({alpha}, ß, {gamma}, and {delta}) are composed in their germline configuration of noncontiguous segments of variable (V), diversity (D), joining (J), and constant (C) regions. During T cell differentiation, somatic VDJ rearrangements occur and thereby generate variability of the TCR.1 Only complete in-frame TCR gene rearrangements, consisting of V, D, and J regions, may form a functional TCR. Incomplete rearrangements between two D regions (D-D), between V and D region (V-D), and between D and J region (D-J) are nonfunctional.The majority of normal T lymphocytes express the {alpha}/ß heterodimer; however, approximately 5% of the T cells express the {gamma}/{delta} heterodimer.2 In contrast to {alpha}/ß T lymphocytes, development of {gamma}/{delta} T cells is not dependent on expression of major histocompatibility complex (MHC) I or MHC II molecules.3, 4 Unlike {alpha}/ß T cells, which develop almost exclusively in thymus, {gamma}/{delta} T cells can be generated in extrathymic sites such as intestinal epithelium, skin, spleen, and fetal liver.5, 6, 7, 8 The exact function of {gamma}/{delta} T lymphocytes has not been fully elucidated, but some studies suggest a role for these cells in early immune responses to infections, autoimmune disorders, and cancer immune surveillance.9 {gamma}/{delta} T cells share some features with CD8+ {alpha}/ß T lymphocytes and with natural killer (NK) cells. They show MHC-dependent and MHC-independent cytotoxity, produce lymphokines, and exhibit NK-like lytic activity.

{gamma}/{delta} TCL represent a rare type of T cell malignancy. They comprise less than 10% of peripheral T cell lymphomas10 and occur mostly at extranodal sites in hepatosplenic, subcutaneous, or intestinal form. Hepatosplenic {gamma}/{delta} TCL ({gamma}/{delta} HSTCL) is recognized as a provisional subset of peripheral T cell lymphoma in the Revised European-American Classification of Lymphoid Neoplasms (REAL),11 although a few identified cases of {alpha}/ß HSTCL appear to have similar clinicopathological characteristics. Histologically, {gamma}/{delta} HSTCL is characterized by a mixture of small to medium-sized atypical lymphocytes. To date only about 40 cases of {gamma}/{delta} HSTCL have been reported.10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 These lymphomas frequently show two nonrandom chromosomal abnormalities, isochromosome 7q [i(7)(q10)] and trisomy 8 (8+).19, 20 Affected individuals are usually young males. Patients commonly present with B symptoms and hepatosplenomegaly, but not lymphadenopathy. The disease usually follows an aggressive course with poor response to chemotherapy and short time of survival.

Subcutaneous panniculitis-like TCL (SPTCL) is an uncommon form of cutaneous lymphoma, involving mainly subcutis and often mimicking lobular panniculitis.24 SPTCL also has been proposed as a provisional subset of peripheral T cell lymphoma in the REAL classification.11 It is sometimes associated with aggressive clinical behavior and poor prognosis, particularly when accompanied by a hemophagocytic syndrome.24, 25, 26 Based on TCR expression, SPTCL can be divided into {alpha}/ß and {gamma}/{delta} SPTCL subsets, which are not recognized as distinct entities in the REAL classification. To date only a few {gamma}/{delta} SPTCL cases have been reported.26, 27, 28, 29, 30, 31

The TCR{delta} gene consists of at least six V{delta} gene segments.32, 33 A detailed analysis revealed that over 95% of the {gamma}/{delta} T cells express either V{delta}1 or V{delta}2 gene.2, 9 Interestingly, normal {gamma}/{delta} T lymphocytes present in spleen, thymus, and intestinal epithelium predominantly express the V{delta}1 gene, whereas the majority of {gamma}/{delta} T cells in peripheral blood, tonsils, and skin express the V{delta}2 gene.2 The reason for this dichotomy in the V{delta} gene usage repertoire remains unclear. V{delta} gene usage by {gamma}/{delta} TCL has not been studied so far in the great detail, and the small number of the reported cases does not allow any definitive conclusions in regard to V{delta} usage by the specific subtype of {gamma}/{delta} TCL. In this study we analyzed the V{delta} usage in 11 hepatosplenic and 4 subcutaneous {gamma}/{delta} TCL using polymerase chain reaction (PCR) and flow cytometry. Preferential usage of V{delta}1 gene was found in {gamma}/{delta} HSTCL (10/11 cases) and of V{delta}2 gene in {gamma}/{delta} SPTCL (4/4 cases). Biological and diagnostic implications of this finding are discussed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patient Samples
We investigated 15 patients with {gamma}/{delta} TCL of hepatosplenic (11 cases) and subcutaneous (4 cases) type in this study. The cases were derived from files of the participating institutions: the University of Pennsylvania, Vanderbilt University, Johns Hopkins University, the University of Pittsburgh, and Harvard University. Data on cases 1–3, 12, and 13 including V{delta} subtype expression were previously reported in part.20, 26

Cytogenetic Analysis and Fluorescence in Situ Hybridization (FISH)
Metaphase cytogenetic analysis was performed by standard trypsin Giemsa banding using unstimulated cell cultures of spleen or bone marrow cells. Slides for FISH were prepared from spleen or peripheral blood mononuclear cells according to a standard method. In brief, liquid nitrogen-stored, dimethyl sulfoxide-preserved frozen cells from three patients were cultured overnight, cytospun onto slides at a concentration of 104 cells/slide, air-dried, fixed in Carnoy’s for 20 minutes, and air-dried overnight. The cytospins were analyzed with VYSIS (Downers Grove, IL) CEP 8 probe to enumerate chromosome 8 centromeres or combination of CEP 7 and LSI D7S486 probes to detect simultaneously chromosome 7 centromere and band 7q31 on the chromosome’s long arm. The staining was performed as recommended by the probe manufacturer.

Immunophenotype Analysis
Flow cytometry and frozen section or paraffin immunohistochemistry were used for immunophenotyping of the lymphomas. All cases were studied by flow cytometry and/or frozen section immunohistochemistry with a set of standard anti-T cell and anti-NK cell antibodies as previously described.20, 26 To confirm the {gamma}{delta} phenotype, we used antibodies to the {alpha}/ß and {gamma}/{delta} TCR (Endogen, Woburn, MA).20, 26 V{delta} subtype expression was also determined by flow cytometry or frozen section immunohistochemistry using commercially available monoclonal antibodies specific for different V regions of the TCR{delta} chain (V{delta}1 and V{delta}2, Endogen; V{delta}3, Immunotech, Westbrook, ME).

Southern Blot Analysis
Southern blot analysis for TCR{delta} gene rearrangements was performed on genomic DNA extracted from frozen tumor in five cases of {gamma}/{delta} HSTCL. The DNA was treated with restriction enzymes EcoRI, HindIII and BamHI, transferred to a nylon membrane and hybridized to a TCR{delta} gene probe TCRDJ1 (Dako Corp., Carpinteria, CA), which corresponds to the J{delta}1 exon and its 3' flanking region. A nonoverlapping 3.0-kb probe, which corresponds to the J{delta}2 exon and its 5' flanking region (pjk 3.0s, kindly provided by Dr. Carlo Croce, Philadelphia, PA)34 was also used in one case that did not show a TCR{delta} rearrangement using the TCRDJ1 probe.

PCR
PCR of 50 µl total volume was performed in a Trio-Thermoblock (Biometra, Goettingen, Germany) with 0.1 µg of genomic DNA, 10 pmol of each primer, 5 nmol each dATP, dCTP, dGTP, dTTP (Perkin Elmer-Cetus, Norwalk, CT), 1.5 U Taq polymerase, and PCR Buffer (Perkin Elmer-Cetus) including 10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 1.5 mmol/L MgCl2, and 0,001% (w/v) gelatin. After 3' denaturation at 94°C, 35 PCR cycles were performed, each cycle consisting of denaturation at 94°C, annealing at 60°C, and extension at 72° (for 1 minute each), followed by a final 7-minute extension at 72°C. Oligonucleotide primers used for PCR were previously described26, 35, 36, 37 and are listed in Table 1Go . Their specificity was confirmed by sequencing of their products36, 37 (and data not shown) and nested PCR which yielded products of expected molecular weight (data not shown). To exclude DNA contamination, negative controls were included, and to avoid false negative results an internal positive control was run in each reaction. Amplification of recombinase activating gene (RAG1) served as such a positive control. PCR products were visualized by 2% agarose gel electrophoresis containing ethidium bromide. Under these experimental conditions, lymphoma-derived, clonal TCR{delta} gene rearrangements, which are present in the large proportion of cells in the samples, are seen as distinct, well-defined bands in the gel. Because the incidence of a specific V{delta} rearrangement is low in normal reactive polyclonal T cells, TCR{delta} genes from reactive T cells present in the samples are amplified, but not visible as distinct bands.


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  		Table 1. List of Primers

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Multiparameter Analysis of the {gamma}/{delta} TCL
All cases were evaluated independently by at least two hematopathologists. According to the REAL classification, 11 cases were classified as {gamma}/{delta} HSTCL and 4 cases as {gamma}/{delta} SPTCL. Typical histological findings from two representative cases are shown in Figure 1Go . Detailed clinical features of all cases are summarized in Table 2Go . Thirteen of the patients were male and only two were female; patient age ranged from 18 to 68 years (median, 42 years). Most patients presented with B symptoms, anemia, thrombocytopenia, and/or leukopenia. Three {gamma}/{delta} SPTCL patients developed severe hemophagocytic syndrome. All patients followed an aggressive clinical course with short survival of 1 to 36 months (median, 10 months). Immunophenotyping data of the cases are presented in Table 3Go . All lymphomas expressed T-cell-associated markers, mainly CD3, CD2, and/or CD7. Most were negative for CD4 and CD8. The {gamma}/{delta} T cell phenotype was confirmed in all cases by positive staining with anti-TCR{gamma}/{delta} antibody and/or negative staining with anti-TCR{alpha}/ß antibody. Although case 4 showed staining with anti-TCRß in a small subset of cells, these probably represented reactive T cells, because most cells were TCR{gamma}/{delta}+. Lymphoma cells usually expressed at least one of NK-associated marker, chiefly CD56. Interestingly, whereas the majority of {gamma}/{delta} HSTCL expressed also CD16 and, to a lesser degree, CD11c, {gamma}/{delta} SPTCL expressed only CD56.



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  		Figure 1. Histology of representative cases of {gamma}/{delta} HSTCL (A and B, Patient 5 in Table 2Go ) and {gamma}/{delta} SPTCL (C and D, Patient 12). A: Diffuse involvement of splenic parenchyma. B: Relatively monomorphic population of medium-size lymphocytes with round to oval nuclei, small nucleoli, and a moderate amount of pale cytoplasm. C: Lobular panniculitis-like pattern. D: Predominance of pleomorphic, small lymphocytes with irregular and hyperchromatic nuclei and scant cytoplasm.

 

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  		Table 2. Clinical Features of Hepatosplenic and Subcutaneous {gamma}/{delta} T Cell Lymphomas

 

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  		Table 3. Immunophenotype of Hepatosplenic and Subcutaneous {gamma}/{delta} T Cell Lymphomas

 
We were also able to perform cytogenetic analysis in three cases of {gamma}/{delta} HSTCL (cases 2, 4, and 5). By FISH all three cases have shown cytogenetic abnormalities characteristic for HSTCL: isochromosome 7q and trisomy 819, 20, 38, 39 (Figure 2)Go . Standard metaphase cytogenetic analysis demonstrated an abnormal karyotype in case 2 including the presence of isochromosome 7q and trisomy 8 (46, X, -Y, i(7)(q10), +8, del(10)(q?22) and failed to show any abnormalities in the remaining two cases.



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  		Figure 2. FISH analysis of {gamma}/{delta} HSTCL (Patient 2). A: CEP 8 probe to enumerate chromosome 8 centromeres showed the presence of trisomy 8 in 20% of the analyzed cells. B: Combination of CEP 7 probe (green staining) and LSI D7S486 probe (red staining) to detect simultaneously chromosome 7 centromere and band 7q31 on the chromosome’s long arm showed the presence of isochrome 7 in 28% of the analyzed cells. A positive cell in metaphase is seen in the center of this photograph. Similar results showing the presence of both trisomy 8 and isochromosome 7 were obtained in patients 4 and 5 (not shown).

 
TCR{delta} Gene Rearrangements in {gamma}/{delta} T Cell Lymphomas
Southern blot analysis using the TCRDJ1 probe and the pjk 3.0s probe revealed clonal TCR{delta} gene rearrangements in all 5 {gamma}/{delta} HSTCL cases investigated (cases 1–5; see Table 4Go ). In three cases two rearranged bands were visible, indicating rearrangement of both TCR{delta} alleles. In case 4 the presence of a rearranged and a germline band in Southern blot probably represented a monoallelic rearrangement or, less likely, contribution of the germline sequence by the non-lymphoma cells combined with deletion of one rearranged allele in the lymphoma cells. Finally, in case 2, a single rearranged band was observed but the germline band was absent, suggesting deletion of the second allele. Such deletion occurs on rearrangement of the surrounding TCR{alpha} gene; however, immunophenotyping indicated that the TCR{alpha} gene was not expressing functional protein in this case.


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  		Table 4. Immunophenotypic and Molecular Analysis of the TCR V{delta} Gene Subset Expression in Hepatosplenic and Subcutaneous {gamma}/{delta} T Cell Lymphoma

 
Usage of V{delta} Segments in Hepatosplenic and Subcutaneous {gamma}/{delta} T Cell Lymphomas
To determine the usage of TCR{delta} gene segments by {gamma}/{delta} T cell lymphomas, PCR was performed with V{delta}1-V{delta}6 specific 5' primers and a J{delta}1 3' primer. We also looked for incomplete TCR{delta} gene rearrangements: D{delta}2J{delta}1, frequently occurring in T-ALL,37, 40 and V{delta}2D{delta}3 and D{delta}2D{delta}3 rearrangements, which were frequently found in B-precursor ALL.41 . To each reaction control primers which amplify a 600-bp fragment of recombinase activating gene-1 (RAG1) were added to exclude false negative results. In this experimental setup, both TCR{delta} and RAG1 amplification products should be detected in the samples which contain a dominant T cell clone. In the reactive, polyclonal conditions detection of RAG1 but not TCR{delta} amplification product is expected. Because in cases 7–11 no fresh tissue was available, DNA was extracted from archival formalin-fixed, paraffin-embedded samples. Because tissue fixation frequently leads to partial DNA degradation, PCR was performed with internal V{delta}1b and V{delta}2b primers which amplify a shorter fragment (~140 bp) and RAG2 control primers, which amplify a 220-bp DNA fragment. We were also able to perform analysis of the V{delta} usage on the protein level by flow cytometry using anti-V{delta}1, -V{delta}2, and -V{delta}3 antibodies in 7 cases. Representative results for {gamma}/{delta} HSTCL PCR using fresh-frozen tissue are shown in FigureGo 3and results using formalin-fixed tissue in Figure 4Go . Representative results for {gamma}/{delta} SPTCL PCR are shown in Figure 5Go . All PCR and flow cytometry data are summarized in Table 4Go . Strikingly, 10 of 11 {gamma}/{delta} HSTCL showed a V{delta}1J{delta}1 rearrangement and in 4 of them an additional, incomplete D{delta}2J{delta}1 rearrangement of the second allele was detected. In one {gamma}/{delta} HSTCL (patient 1; see Table 4Go ) no amplification product was obtained. This case expressed the TCR{gamma}/{delta} (Table 3)Go ; however, it was negative for V{delta}1–3 expression (Table 4)Go , as determined by immunophenotyping. Furthermore, on Southern blot, it showed a rearrangement pattern different from other {gamma}/{delta} HSTCLs analyzed in this study. These findings suggest that a variable TCR{delta} gene segment other than V{delta}1-V{delta}6 might have been used in this case. In contrast to {gamma}/{delta} HSTCL, all 4 {gamma}/{delta} SPTCL showed rearrangement of the V{delta}2J{delta}1 gene segment (Figure 5Go and Table 4Go ). Case 14 showed also the D{delta}2J{delta}1 rearrangement and case 13 the V{delta}3J{delta}1 rearrangement of the second allele. Because flow cytometry analysis in case 13 showed a V{delta}2 expression, the V{delta}3J{delta}1 rearrangement was recognized to be nonfunctional.



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  		Figure 4. PCR analysis of V{delta} gene usage in archival paraffin-embedded samples of hepatosplenic {gamma}/{delta} T cell lymphoma. Lane M, molecular weight marker; lanes 1–3, Patient 7; lanes 4–6, Patient 8; lane C, negative control. PCR was performed with the J{delta}1 primer and the V{delta}1b primer (lanes 1 and 4), the V{delta}2b primer (lanes 2 and 5), and the D{delta}2 primer- (lanes 3 and 6). The upper band of ~220 bp represents RAG2 reference gene, bands of ~120 bp in lanes 1 and 4 represent a V{delta}1J{delta}1 rearrangement, and the band of ~140 bp in lane 6 represents an incomplete D{delta}2J{delta}1 rearrangement.

 


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  		Figure 5. PCR analysis of V{delta} gene usage in subcutaneous panniculitis-like {gamma}/{delta} T cell lymphoma. A: Patient 14. B: Patient 15. Lane M, molecular weight marker; lanes 1–6, PCR products obtained with V{delta}1-V{delta}6 specific primers, respectively, and the J{delta}1 primer; lane C, negative control; lane 7, D{delta}2 and J{delta}1 primers, lane 8, V{delta}2 and D{delta}3 primers; lane 9, D{delta}2 and D{delta}3 primers. The upper band of ~600 bp represents RAG1 reference gene, bands of ~400 bp in lanes A2 and B2 represent a V{delta}2J{delta}1 rearrangement, and the band of ~140 bp in lane A7 represents an incomplete D{delta}2J{delta}1 rearrangement.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
T cell lymphomas expressing {gamma}/{delta} TCR represent recently recognized, rare subsets of non-Hodgkin’s lymphoma.11 The literature on {gamma}/{delta} T cell lymphomas remains sparse.10, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 Because only a few cases were analyzed in each of the published studies, any preferences in V{delta} gene usage could not have been adequately addressed. Our present report, which describes 11 cases of {gamma}/{delta} HSTCL and 4 cases of {gamma}/{delta} SPTCL, is the largest study to date on {gamma}/{delta} TCL. It is the first study focused on immunophenotypical and molecular subtyping of these rare but clinically distinct subtypes of peripheral TCL. There was an excellent concordance among results obtained by molecular and immunophenotypic approaches (Table 4)Go . Our data demonstrate that the two types of {gamma}/{delta} TCL express different subset of {gamma}/{delta} TCR. We found that 10 out of 11 {gamma}/{delta} HSTCL expressed the V{delta}1 gene, whereas all 4 {gamma}/{delta} SPTCL used the V{delta}2 gene (P = 0.001, Fisher’s exact test). This result indicates that selection of the specific V{delta} subtype is lymphoma type-dependent. The results of two previous, rather limited studies on V{delta} gene usage which evaluated a total of 6 {gamma}/{delta} HSTCL10, 15, 18 and 2 {gamma}/{delta} SPTCL,31 support our conclusion. In these studies, 4/6 {gamma}/{delta} HSTCL expressed the V{delta}1 gene, and in the remaining 2 {gamma}/{delta} HSTCL cases, an unidentified V gene was used. In contrast, both {gamma}/{delta} SPTCL expressed the V{delta}2 gene. It is well established that normal {gamma}/{delta} T lymphocytes which reside in spleen express predominantly the V{delta}1 gene, whereas most {gamma}/{delta} T lymphocytes present in subcutis express the V{delta}2 gene.42 The identified unique V{delta} gene usage patterns in {gamma}/{delta} HSTCL and {gamma}/{delta} SPTCL reflects local predominance of either V{delta}1+ or V{delta}2+ subset within their normal T cell counterparts. This strongly suggests that both {gamma}/{delta} HSTCL and {gamma}/{delta} SPTCL are derived from the local lymphoid tissue. V{delta} subtype analysis; rather limited studies on other {gamma}/{delta} T cell lymphomas also suggested preferences in V{delta} usage. Two out of three precursor T cell lymphomas involving lymph nodes expressed the V{delta}1 (the third expressed an unidentified V{delta} gene),43 3/3 nasal {gamma}/{delta} TCL expressed the V{delta}2 gene,31 2/3 gastro-intestinal {gamma}/{delta} TCL expressed the V{delta}3, and 1 expressed the V{delta}2 gene.31 In a large study on {gamma}/{delta} T cell acute lymphoblastic leukemia, the vast majority (26/30) of cases, similarly to {gamma}/{delta} HSTCL, expressed the V{delta}1 gene, 2 cases used the V{delta}2 gene, one case used the V{delta}3 gene and 2 cases used the V{alpha} gene rearranged to the J{delta}1 segment.44 The V{delta} gene expression pattern in TCR {gamma}/{delta}+ T-ALL resembled that of TCR {gamma}/{delta}+ thymocytes and differed markedly from that of peripheral blood {gamma}/{delta}+ T cells. These data support the conclusion that T-ALL is a malignant counterpart of thymocytes rather than peripheral blood {gamma}/{delta}+ T cells.

A detailed immunophenotypic analysis, performed in {gamma}/{delta} HSTCL and {gamma}/{delta} SPTCL showed a similar pattern of T-cell associated antigens (CD3+, CD2+ CD7+, CD5-, CD4-, CD8-). All lymphomas expressed also NK-associated antigens, but some differences were observed. Five out of six {gamma}/{delta} HSTCL tested expressed both CD16 and CD56, whereas all 3 {gamma}/{delta} SPTCL tested expressed only CD56 and, finally, 1 {gamma}/{delta} HSTCL, which used an unidentified V{delta} gene, expressed neither CD16 nor CD56 but did express CD11c antigen. Furthermore, in contrast to {gamma}/{delta} SPTCL, {alpha}/ß SPTCL do not express CD56.26 Taken together, the above data suggest a relationship between the type of TCR and a pattern of expression of NK cell-associated markers among various types of hepatosplenic and subcutaneous TCL. However, the number of the TCL cases analyzed by us and others is still too small to draw any definitive conclusions in this regard.

It is often difficult to diagnose TCL on histological grounds alone, especially the cases involving skin. Molecular analysis has proven to be very useful in this respect. Detection of a clonally rearranged TCR{gamma} gene often allows to distinguish T cell lymphoma from benign, reactive T cell proliferation or B cell lymphoma highly enriched in reactive T lymphocytes.45, 46 Our study indicates that analysis of V{delta} gene usage may be helpful in diagnosis and proper classification of {gamma}/{delta} TCL. The observed dichotomy in the V{delta} gene usage between {gamma}/{delta} HSTCL and {gamma}/{delta} SPTCL indicates that analysis of expression of the V{delta} gene subtype by either molecular or immunological method may permit better discrimination among different types of {gamma}/{delta} TCL, particularly in the clinically advanced, generalized cases with multi-organ involvement. Furthermore, because V{delta} gene rearrangements show an extensive diversity of the joining site, lymphoma-specific probes could be developed to monitor minimal residual disease in {gamma}/{delta} TCL.18

In summary, our results indicate that hepatosplenic and subcutaneous panniculitis-like {gamma}/{delta} T cell lymphomas are derived from different V{delta} subsets of {gamma}/{delta} T lymphocytes. Whereas {gamma}/{delta} HSTCL belong usually to the V{delta}1 subset, {gamma}/{delta} SPTCL represent the V{delta}2 subset. The exact properties of either normal or malignant V{delta}1+ and V{delta}2+ {gamma}/{delta} T cells leading to this different, tissue-specific expression of the V{delta} subsets have not been determined. Whether the restricted, highly lymphoma type-specific V{delta} gene expression in {gamma}/{delta} HSTCL and {gamma}/{delta} SPTCL plays a role in the pathogenesis of these lymphomas also remains to be determined.



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  		Figure 3. PCR analysis of V{delta} gene usage in hepatosplenic {gamma}/{delta} T cell lymphoma. A: Patient 2. B: Patient 3. Lane M, molecular weight marker; lanes 1–6, PCR products obtained with V{delta}1-V{delta}6 specific primers, respectively, and J{delta}1 primer; lane C, negative control; lane 7, D{delta}2J{delta}1, lane 8, V{delta}2D{delta}3; lane 9, D{delta}2D{delta}3. The upper band of ~600 bp represents RAG1 reference gene, bands of ~400 bp in lanes A1 and B1 represent a V{delta}1J{delta}1 rearrangement, band of ~140 bp in lane B7 represents an incomplete D{delta}2J{delta}1 rearrangement.

 

   Footnotes
 
Address reprint requests to Mariusz A. Wasik, M.D., 7.106 Founders Building, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 3400 Spruce Street, Philadelphia, PA 19104. E-mail: wasik{at}mail.med.upenn.edu

Supported in part by grants from the Committee for Scientific Research (KBN PO5A 01516 to G. K. P.) and the National Cancer Institute (CA76627 to M. A. W.).

W. Macon’s current address: Mayo Clinic, Rochester, MN.

R. Felgar’s current address: University of Rochester, Rochester, NY.

J. DiGiuseppe’s current address: Hartford Hospital, Hartford, CT.

K. Salhany is deceased.

Accepted for publication December 3, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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