Extensive molecular mapping of TCRα/δ- and TCRß-involved chromosomal translocations reveals distinct mechanisms of oncogene activation in T-ALL.

Chromosomal translocations involving the TCR loci represent one of the most recurrent oncogenic hallmarks of T-cell acute lymphoblastic leukemia (T-ALL) and are generally believed to result from illegitimate V(D)J recombination events. However, molecular characterization and evaluation of the extent of recombinase involvement at the TCR-oncogene junction has not been fully evaluated. In the present study, screening for TCRß and TCRα/δ translocations by FISH and ligation-mediated PCR in 280 T-ALLs allowed the identification of 4 previously unreported TCR-translocated oncogene partners: GNAG, LEF1, NKX2-4, and IL2RB. Molecular mapping of genomic junctions from TCR translocations showed that the majority of oncogenic partner breakpoints are not recombinase mediated and that the regulatory elements predominantly used to drive oncogene expression differ markedly in TCRß (which are exclusively enhancer driven) and TCRα/δ (which use an enhancer-independent cryptic internal promoter) translocations. Our data also imply that oncogene activation takes place at a very immature stage of thymic development, when Dδ2-Dδ3/Dδ3-Jδ1 and Dß-Jß rearrangements occur, whereas the bulk leukemic maturation arrest occurs at a much later (cortical) stage. These observations have implications for T-ALL therapy, because the preleukemic early thymic clonogenic population needs to be eradicated and its disappearance monitored.

ATM-deficient thymic lymphoma is associated with aberrant tcrd rearrangement and gene amplification.

Ataxia telangiectasia mutated (ATM) deficiency predisposes humans and mice to T lineage lymphomas with recurrent chromosome 14 translocations involving the T cell receptor alpha/delta (Tcra/d) locus. Such translocations have been thought to result from aberrant repair of DNA double-strand breaks (DSBs) during Tcra locus V(D)J recombination, and to require the Tcra enhancer (Ealpha) for Tcra rearrangement or expression of the translocated oncogene. We now show that, in addition to the known chromosome 14 translocation, ATM-deficient mouse thymic lymphomas routinely contain a centromeric fragment of chromosome 14 that spans up to the 5' boundary of the Tcra/d locus, at which position a 500-kb or larger region centromeric to Tcra/d is routinely amplified. In addition, they routinely contain a large deletion of the telomeric end of one copy of chromosome 12. In contrast to prior expectations, the recurrent translocations and amplifications involve V(D)J recombination-initiated breaks in the Tcrd locus, as opposed to the Tcra locus, and arise independently of the Ealpha. Overall, our studies reveal previously unexpected mechanisms that contribute to the oncogenic transformation of ATM-deficient T lineage cells.

Gamma-delta T-cell lymphomas.

Peripheral T-cell lymphomas (TCLs) are uncommon neoplasms, accounting for about 12% of all lymphoid tumors worldwide. TCLs in which gammadelta T-cell receptors are expressed (gammadelta TCLs) are extremely aggressive and rare (<1% of lymphoid neoplasms). gammadelta TCLs originate from gammadelta T cells, a small subset of peripheral T cells with direct antigen recognition capability acting at the interface between innate and adaptive immunity. Two distinct gammadelta TCL entities are recognized: hepatosplenic T-cell lymphoma (HSTL) and primary cutaneous gammadelta T-cell lymphoma (PCGD-TCL). HSTL is a well-characterized extranodal lymphoma that has a disguised onset, secondary to intrasinusoidal infiltration of the spleen, liver and bone marrow, has a rapidly progressive course that is poorly responsive to chemotherapy, and often ensues in the setting of immune system suppression. PCGD-TCL can present with prominent epidermal involvement or with a panniculitis-like clinical picture that can be complicated by a concurrent hemophagocytic syndrome; the disease shows biological and phenotypic overlap with other extranodal gammadelta TCLs that involve the respiratory or gastrointestinal tract mucosa. The regular application of phenotypic and molecular techniques is crucial for the diagnosis of gammadelta TCLs. In this Review, we discuss the clinical and biological features, the diagnostic challenges and the therapeutic perspectives of HSTL and PCGD-TCL.

The pattern of clonal immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangements in Chinese adult acute lymphoblastic leukemia patients.

We have studied forty Chinese adult ALL patients at newly diagnosis, using standard primers and protocols of BIOMED-2 multiplex PCR, to determine the feasibility of Ig and TCR gene rearrangements as diagnostic and patient-specific MRD-RQ-PCR targets for molecular monitoring. Clonal IGH, IGK, IGL, TCRB, TCRG and TCRD rearrangements were found in 86%, 22%, 9%, 19%, 77%, 55% of adult patients with B-lineage ALL, respectively. While in T-ALL, clonal IGH, TCRB, TCRG and TCRD rearrangements were detected in 6%, 83%, 78%, 33% of patients. Several specialties in the pattern of Ig/TCR gene rearrangements in Chinese adult ALL patients in comparison with those reported for children and adult patients in other countries have been noted. These results are useful for further MRD-RQ-PCR detection and quantification for all patients.

T-cell receptor gamma and delta gene rearrangements and junctional region characteristics in south Indian patients with T-cell acute lymphoblastic leukemia.

Clonal T-cell receptor (TCR) gamma and delta gene rearrangements were studied in 40 T-ALL cases (pediatrics, 29; adults, 11) using PCR with homo-heteroduplex analysis. At least one clonal TCRG or TCRD rearrangement was detected in 34 (85%) cases. TCR gamma (TCRG) rearrangement was detected in 25 (62.5%) cases that included 16 (55%) pediatrics and 9 (81.8%) adults. TCR delta (TCRD) rearrangement was detected in 14/40 (35%) cases, which included 12 (41%) pediatrics and 2 (18%) adults. The frequency of VgammaI-Jgamma1.3/2.3 was significantly more in adults than pediatrics (81.8% vs. 41.3%, P=0.02). In TCRD, Vdelta1-Jdelta1 was rearranged in 10 (25%) cases. The surface membrane CD3 positive cases are significantly associated with absence of TCRD rearrangements (surface membrane CD3+ TCRdelta- 84% vs. surface membrane CD3- TCRdelta- 48%, P value=0.03). Junctional region sequence analyzed with 10 cases each, of TCRG and TCRD, revealed an average junctional region of 7.4 nucleotides (range 2-18 nucleotides) in TCRG and 27 nucleotides (range 14-42 nucleotides) in TCRD-complete rearrangements. In TCRG, trimming at the ends of Vgamma and Jgamma germline nucleotides resulted in deletion, on an average of 9.2 nucleotides. In TCRD, deletion of nucleotides of the Vdelta and Jdelta gene segments on an average was 3.5 nucleotides. The junctional region of TCRD is more diverse than TCRG; nevertheless, the frequency of TCRG was more than that of TCRD and hence we rely more on TCRG clonal markers to quantitate the minimal residual disease in T-ALL.

DHPLC based fraction collection of TCR-gamma rearrangements in childhood ALL: direct sequencing of products amplified by a single or a multiplex PCR approach.

Clonal T-cell receptor gamma (TCR-gamma) rearrangements are frequently used for detection of minimal residual disease (MRD) in childhood acute lymphoblastic leukemia. In approximately 70-80% of cases PCR amplified clonal rearrangements can be sequenced directly. The remaining 20-30% are rearranged on both alleles for the same target and disables direct sequencing. Here we describe a novel HPLC based method for identification and characterisation of TCR-gamma rearrangements either by a single or a multiplex PCR approach. The latter one amplifies several Vgamma segments in two distinct reactions either with a Jgamma1.3/2.3 or a Jgamma1.1/2.1 specific primer. The clonality status was evaluated on a high resolution micropellicular DNASep matrix (WAVE, Transgenomic) at different temperatures. From 331 samples analysed, 151 samples were positive for VgammaI-Jgamma1.3/2.3 including 51 biclonal rearrangements. For characterisation of these biclonal products or for products generated by multiplex-PCR, a second HPLC run was performed utilising a tandem arranged fraction collector. From clearly separated biclonal/biallelic products, several collected fractions were air-dried and afterwards sequenced directly with the appropriate Jgamma primer. We conclude from our results that HPLC is a fast and reliable method for identification of TCR-gamma rearrangements. The fraction collection simplifies the characterisation of single alleles within biclonal or biallelic rearrangements or within multiplex PCR products. The target identification process prior to routine MRD analysis will be shortened due to a simplified screening and sequencing strategy.

Heat shock protein induced TCR gammadelta gene rearrangements in patients with oral cancer.

We analyzed the T cell receptor (TCR) gammadelta gene repertoire in peripheral blood and tumor compartment of oral cancer (OC) patients before and after stimulation with heat shock proteins (hsp), which are known ligands for gammadelta T cells. Clonal TCR gamma and delta gene rearrangements in lymphocytes from tumor compartment and peripheral blood were studied using TCR Vgamma and Vdelta gene primers in PCR followed by heteroduplex analysis. Vgamma gene segments derived from VgammaI or VgammaII gene families were most dominantly expressed in peripheral blood lymphocytes (PBL) as compared to tumor infiltrating lymphocytes (TIL) of OC patients. Of the rearranged TCR delta alleles Vdelta1-Jdelta1 and Vdelta2-Jdelta1 gene rearrangements were the most predominant in PBL and TIL of OC patients respectively. Stimulation of gammadelta T cells with hsp 60/70 demonstrated a selective clonal expansion of Vgamma9-Vdelta2 (VgammaII family) subset indicating that, this expanded population of cells could be responsible for eliciting an immune response against oral tumor cells.

IgH DJ rearrangements within T-ALL correlate with cCD79a expression, an immature/TCRgammadelta phenotype and absence of IL7Ralpha/CD127 expression.

cCD79a and IgH VDJ/DJ rearrangements are considered to be relatively specific for B lymphoid precursors. We looked for both in cCD3+, CD7+, CD19- T-ALLs classified by TCR status into alphabeta or gammadelta/immature (IM) lineages, with individualization of HOX11L2+ T-ALLs since they represent an intermediate alphabeta/gammadelta category. cCD79a was expressed at low levels in 47% of T-ALL and was most frequent in IMgamma T-ALLs. IgH rearrangements were common in gammadelta/IM (45%) and HOX11L2+ (35%) T-ALLs compared to HOX11L2-negative cases (3%; P<0.001). CD127 (IL7Ralpha) expression was also more common in the gammadelta/IM lineage but its expression was virtually mutually exclusive of IgH rearrangement. Low-level cCD79a expression alone should therefore not be interpreted as evidence of B lineage affiliation in immature leukemias. gammadelta/IM lineage T-ALLs potentially include two distinct categories: predominantly IgH+, cCD79a+, CD127- cases which retain gammadelta and B lymphoid potential and IgH-, cCD79a-, CD127+ cases with restricted T lineage potential.

Hemophagocytic syndrome and hepatosplenic gammadelta T-cell lymphoma with isochromosome 7q and 8 trisomy.

The authors describe a 15-year-old boy with hepatosplenic gammadelta T-cell lymphoma associated with hemophagocytic syndrome (HPS) along with isochromosome 7q and trisomy 8. He presented with prolonged fever, mild anemia, thrombocytopenia, and hepatosplenomegaly. Physical examination, radiography, and ultrasound tomography revealed no lymphoadenopathy. He had elevated levels of serum ferritin, interferon-gamma, soluble interleukin-2 receptor, and interleukin-6. Bone marrow aspirate showed hypercellularity with 50% lymphoblasts and erythrophagocytosis of macrophage. A cytogenetic study of bone marrow revealed an abnormal karyotype, 47,XY,I(7q),+8, in 5/30 cells. Clonal rearrangement of the genes for T-cell receptor gamma and delta chains was elucidated by polymerase chain reaction. He achieved a complete remission after intensive chemotherapy and underwent splenectomy 18 months after diagnosis. Although the patient was clinically in remission, minimal residual disease (MRD) was detected in the removed spleen by polymerase chain reaction. This might mean that this type of lymphoma is refractory, as reported previously, and might indicate that marrow ablative therapy is needed to achieve a cure. The present case illustrates the usefulness of MRD analysis, and MRD studies in this group of disorders may be helpful in the decision of whether to continue a more aggressive therapeutic approach.

Allelic exclusion at the TCR delta locus and commitment to gamma delta lineage: different modalities apply to distinct human gamma delta subsets.

Expression of a beta-chain, as a pre-TCR, in T cell precursors prevents further rearrangements on the alternate beta allele through a strict allelic exclusion process and enables precursors to undergo differentiation. However, whether allelic exclusion applies to the TCR delta locus is unknown and the role of the gamma delta TCR in gamma delta lineage commitment is still unclear. Through the analysis of the rearrangement status of the TCR gamma, delta, and beta loci in human gamma delta T cell clones, expressing either the TCR V delta 1 or V delta 2 variable regions, we show that the rate of partial rearrangements at the delta locus is consistent with an allelic exclusion process. The overrepresentation of clones with two functional TCR gamma chains indicates that a gamma delta TCR selection process is required for the commitment of T cell precursors to the gamma delta lineage. Finally, while complete TCR beta rearrangements were observed in several V delta 2 T cell clones, these were seldom found in V delta 1 cells. This suggests a competitive alpha beta/gamma delta lineage commitment in the former subset and a precommitment to the gamma delta lineage in the latter. We propose that these distinct behaviors are related to the developmental stage at which rearrangements occur, as suggested by the patterns of accessibility to recombination sites that characterize the V delta 1 and V delta 2 subsets.

Age-related patterns of immunoglobulin and T-cell receptor gene rearrangements in precursor-B-ALL: implications for detection of minimal residual disease.

Detailed Southern blot and PCR analysis of Ig heavy (IGH), Ig kappa (IGK), T-cell receptor delta (TCRD), and TCR gamma (TCRG) genes were performed in 289 children with precursor-B-ALL in order to determine age-related Ig/TCR patterns and their implications for detection of minimal residual disease (MRD). Overall, IGH, IGK, TCRD, and TCRG gene rearrangements were detected in 98, 62, 90, and 58% of patients, respectively. The frequency of IGH and TCRD rearrangements was independent of rearrangements in one of the other three loci, whereas Ig kappa deleting element and TCRG rearrangements preferentially coincided. Southern blot analysis showed that oligoclonality of IGH, IGK, and TCRD was interrelated, that is, oligoclonality in one locus was related with a higher chance of oligoclonality in another locus. Combined Southern blot and PCR analysis revealed that Ig/TCR patterns were age related: children younger than 3 years or older than 10 years showed a higher prevalence of incomplete IGH rearrangements and a lower prevalence of IGK deletions, TCRG rearrangements, and TCRD rearrangements than children between 3 and 10 years. In addition, IGH oligoclonality was more frequent in the younger and older children. These age-related differences probably reflect ALL subsets with different cellular origin and differences in the duration of the preleukemic phase between the initial and final leukemogenetic hit. The more immature Ig/TCR gene rearrangement pattern in children younger than 3 years or older than 10 years resulted in relatively low numbers of potential MRD-PCR targets per patient, particularly if only monoclonal rearrangements were taken into account. These data provide insight into the immunobiological characteristics of Ig/TCR gene rearrangements in childhood precursor-B-ALL and form a useful basis for designing improved strategies for the identification and selection of MRD-PCR targets.

Polymerase chain reaction on cerebrospinal fluid cells in suspected leptomeningeal involvement in childhood acute lymphoblastic leukemia: comparison to cytomorphological analysis.

The leptomeningeal involvement of central nervous system is defined in the most centers by the presence of blast cells in the CSF or the presence of cranial-nerve palsies. Sometimes, cytology does not allow clear distinction between lymphoblasts and normal cells, and auxiliary methods to the precise identification of leukemic cells in cerebrospinal fluid is necessary. We analyzed CSF from 11 consecutive patients, in whom a differential diagnosis of leptomeningeal involvement was made, including 4 patients at diagnosis and 7 patients during the treatment by cytomorphological analysis and PCR and automatic sequencing. Six patients were considered with leptomeningeal involvement by conventional analysis: unequivocal cytomorphological involvement was considered in 5 patients, and in one it was assumed to be due to cranial-nerve palsy, with no blast cells detected in cerebrospinal fluid. In 2 it was considered suspicious and in 3 negative. PCR and sequencing analysis showed involvement in 6 patients; 5 of the 6 patients were considered to have leptomeningeal involvement based on clinical and cytomorphological criteria, and, in one of the patients, it was suspicious. Our data suggest that the use of PCR and sequencing can be useful in confirming CNS leukemia and eliminating other conditions when used together with the cytomorphological analysis.

Peripheral blood T-cell clonality in mycosis fungoides and nonlymphoma controls.

In mycosis fungoides (MF), T-cell clonality is reported in about 90% of skin and 40% of blood samples. However, identity of blood and cutaneous T-cell clone and prognostic relevance of blood T-cell clonality remain controversial. By PCR/fluorescence fragment analysis with estimation of clonal fragment lengths and relative peak heights, we objectively identified T-cell clonality unrelated to malignant lymphoproliferation in healthy donors (5/38), autoimmune dermatoses (3/8), and nonlymphoma skin cancer (9/39). This T-cell expansion of undetermined significance (TEXUS) was also found in 8/64 MF patients. Dissemination of neoplastic cells into blood, as identified by identical clonal fragment lengths in blood and skin, was detected in 23/64 MF patients. When monitoring for progression at TNM stage for a mean of 45.7 months, univariate analysis identified age of >60 years and detection of a related blood T-cell clone to be of prognostic relevance, whereas detection of TEXUS, sex, TNM stage at initial diagnosis, and detection of a cutaneous T-cell clone were irrelevant. Although multivariate analysis was not possible, further stratification clearly indicated an age of >60 years to be the predominating prognostic factor. In conclusion, investigation of T-cell clonality in skin and blood samples at the initial diagnosis cannot predict the clinical course of MF and the occurrence of TEXUS should be considered when assessing blood T-cell clonality.

PCR detection of clonal IgH and TCR gene rearrangements at the end of induction as a non-remission criterion in children with ALL: comparison with standard morphologic analysis and risk group classification.

BACKGROUND: The initial response to induction therapy is currently considered one of the most important prognostic factors in acute lymphoblastic leukemias (ALL). A series of methods for the detection of submicroscopic levels of residual disease in patients with ALL mainly based on PCR and immunophenotyping has been developed, demonstrating that the presence of high levels of residual disease at the end of induction therapy is an important, independent prognostic factor. We determined the usefulness of PCR detection of minimal residual disease using consensus primers as a non-remission criterion. PROCEDURE: Bone marrow samples obtained from 49 children with ALL were analyzed at diagnosis and at the end of induction therapy for the detection of clonal IgH, TCRdelta, and TCRgamma rearrangements by PCR. The results were compared with those obtained by standard morphologic analysis and risk group classification. RESULTS: Patients who had clonality detected at the end of induction showed a significantly higher recurrence rate and lower event-free survival than those without detected clonality (24.9% vs. 89.7%) (P < 0.0001). Multivariate analysis revealed that detection of clonality at the end of induction was the most important, independent prognostic factor when associated with age, number of white blood cells, and immunophenotyping. CONCLUSIONS: PCR detection of clonality using consensus primers is a relatively simple technique that is able to identify patients with a high chance of recurrence, and shows a higher sensitivity and a better prognostic value than standard morphologic analysis and risk group classification, defining a new remission criterion. However, further multicentric prospective studies using this technique employing a larger number of cases are necessary to confirm these findings.

PCR-heteroduplex analysis of TCR gamma, delta and TAL-1 deletions in T-acute lymphoblastic leukemias: implications in the detection of minimal residual disease.

Detection of MRD remains one of the major goals in the treatment of acute lymphoblastic leukemia (ALL). We have used the polymerase chain reaction (PCR)-heteroduplex (HD) analysis to assess and confirm the clonal expansion of T cell receptor (TCR) gamma and delta gene rearrangements in 24 T-ALL patients at diagnosis. 52.4% revealed Vdelta1-Jdelta1; 48% Vdelta2-Ddelta3; 62.5% Vgamma1-Jgamma1 and 46% both Vdelta1-Jdelta1 and Vgamma1-Jgamma1 clonal rearrangements. 6/24 patients had TAL-1 deletion. These clonal markers were used to monitor MRD in remission/relapse bone marrow samples for periods ranging from 6 to 75 months after diagnosis. Patients who relapsed and died revealed a continuous PCR-HD positivity in their clinical remission bone marrow samples. HD analysis established identical diagnostic clone at relapse. Patients who are in long-term clinical and morphological remission achieved PCR-HD negativity in their 8-12 months bone marrow remission samples and continue to be PCR-HD negative. MRD monitored in six patients with two diagnostic PCR--HD positive clonal markers reveal an identical pattern ensuring circumvention of false positive and negative results. Thus, we conclude that PCR followed by HD analysis is a useful technique to monitor MRD in remission/relapse samples in ALL patients.

Detection of T cell receptor delta gene rearrangements in childhood B and T lineage acute lymphoblastic leukaemia by southern blot and PCR: technical comparison of two methods of analysis.

Molecular analysis of antigen receptor genes (Ig and TCR) has been useful for clonal studies in acute lymphoblastic leukaemia (ALL) patients. Rearrangements of these genes can be used to track the persistence of the leukaemic clone during the therapy. The purpose of our study was to analyse the percentage and the pattern of the rearrangements at the TCR D locus in a series of ALL patients, comparing the results obtained by Southern blot and PCR. Genomic DNA was extracted from mononuclear BM cells of 40 paediatric ALL cases, digested with different restriction enzymes and hybridized to TCRDJ1 probe to study the TCR delta locus. Amplification of the rearranged TCR delta genes was performed by PCR to define the gene segments involved. The junctional region was deduced from the sequence to obtain patient-specific primers. Among the 31 B lineage ALL samples, one or two TCR delta alleles proved to be rearranged in 53% of cases. Two different types of rearrangements were chiefly detected: Vdelta2Ddelta3 and Ddelta2Ddelta3. In T-ALL patients, the predominant rearrangement involved the Vdelta1 and the Jdelta1 gene segments.

Abnormal rearrangement within the alpha/delta T-cell receptor locus in lymphomas from Atm-deficient mice.

Atm-deficient mice (Atm(-/-)) recapitulate many aspects of the ataxia telangiectasia (AT) syndrome, including the susceptibility to tumors of lymphoid origin. To investigate the mechanism of tumorigenesis, we have examined a panel of 8 thymic lymphomas from Atm(-/-) mice. All Atm(-/-) tumors are of thymic lymphoblastoid origin, display an immature CD3(-) and CD4(+)/CD8(+) phenotype, and arise coincident with V(D)J recombination. Cytogenetically, all tumors are diploid or near diploid but exhibit multiple chromosome aberrations with an average of 4 abnormal chromosomes per tumor. All the tumors revealed chromosome 14 rearrangements precisely at the T-cell receptoralpha/delta (Tcralpha/delta) locus, suggesting the involvement of V(D)J recombination in these translocations. In addition, 11.5% of Atm(-/-) peripheral T cells showed chromosome 14 translocations, suggesting that rearrangements at the Tcralpha/delta locus occur early during tumor development in the absence of ATM. However, additional genetic aberrations are required for tumorigenesis. For example, translocations involving chromosome 12, often with chromosome 14 (more than 60%), and partial or complete trisomy of chromosome 15, with copy number increases of the c-myc oncogene were frequently observed. These observations suggest that ATM is required for normal rearrangement of the Tcralpha/delta locus but not for V(D)J recombination at other loci. The mechanisms that lead to tumorigenesis may be due to the involvement of ATM in monitoring double-stranded DNA breaks. (Blood. 2000;96:1940-1946)

Cutaneous monomorphous CD4- and CD56-positive large-cell lymphoma.

BACKGROUND: Recently, CD56 (NCAM)-positive lymphomas, such as nasal and nasal-type angiocentric NK/T cell lymphoma, aggressive NK cell leukemia/lymphoma and blastic NK cell lymphoma, were described by several authors as a unique group of lymphoma. OBJECTIVE: In this study, we intend to clarify the clinicopathological features of cutaneous CD4+ and CD56+ lymphoma. METHODS: Four patients with cutaneous CD4+ and CD56+ lymphoma were studied. RESULTS: Age at the first examination ranged from 71 to 89 years (mean = 81.2 years). One patient was female and 3 were males. The organ mainly involved at presentation was the skin. Lymphadenopathy, splenomegaly, leukemic spread and central nervous system involvement were observed as the disease progressed. The mean survival time was 12.2 months. Epstein-Barr virus was not detected within the tumor cells. CONCLUSION: This peculiar lymphoma is different from nasal and nasal-type angiocentric NK/T cell lymphoma and aggressive NK cell leukemia/lymphoma. Similar cases have been reported as blastic NK cell lymphoma/leukemia.

Hepatosplenic gamma-delta T-cell lymphoma as a late-onset posttransplant lymphoproliferative disorder in renal transplant recipients.

We report 2 cases of renal transplant recipients in whom hepatosplenic gamma-delta T-cell lymphoma (gamma-delta HSTCL) developed 5 and 10 years after transplantation. Both patients had marked hepatosplenomegaly, B symptoms (weight loss, fever, and night sweats), and abnormal peripheral blood findings, including anemia in both, thrombocytopenia and leukoerythroblastic changes in 1, and leukocytosis in the other. Markedly atypical lymphoid infiltrate of intermediate to large cells was observed in the spleen, liver, and bone marrow. The malignant cells showed typical immunophenotype of gamma-delta T cells (CD2+, CD3+, CD4-, CD8-, CD7+, gamma-delta T-cell receptor-positive, and alpha-beta T-cell receptor-negative) with clonal T-cell receptor gene rearrangement and were of the V-delta-1 subset. In addition, the cells contained a cytolytic granule-associated protein, TIA-1, and Fas ligand, indicating cytotoxic T-cell differentiation. The malignant T cells in both cases were of host tissue origin. Both cases were negative for Epstein-Barr virus genome using Southern blot analysis. The patients did not respond to reduction of immunosuppression. Despite initial response to chemotherapy, both patients died within 6 months of diagnosis. Our findings indicate that gamma-delta HSTCL can occur as a late complication in transplant recipients.

Late Epstein-Barr virus infection of a hepatosplenic gamma delta T-cell lymphoma arising in a kidney transplant recipient.

BACKGROUND AND OBJECTIVE: gd T-cell lymphomas are only exceptionally observed in transplanted patients. Aim of this study was the detailed characterization of one such case. DESIGN AND METHODS: The patient developed spontaneous splenic rupture six years after kidney transplantation. The splenic red pulp was infiltrated by medium-sized and large lymphoid cells with two or more nucleoli. At autopsy, similar lymphoid cells infiltrated the hepatic sinusoids. Histologic, immunologic and molecular studies were carried out. RESULTS: By immunohistochemistry, the atypical lymphoid cells were found to express CD3, CD45 and CD43, indicating their T-lineage origin. Approximately 99% of spleen mononuclear cells (MNC) were CD3(+), gammadelta TcR+, CD4-, CD8-, alphabeta TcR-. A clonal gammadelta TcR rearrangement (Vgamma1-Jgamma1.3/2.3-Cgamma2; Vdelta1-Ddelta2-Jdelta1) was detected. The final diagnosis was peripheral T-cell lymphoma, hepato-splenic gammadelta-type. EBV infection of spleen MNC was documented by molecular studies. However, in situ hybridization for EBER-1 (EBV-RNA) showed that only a minority of malignant lymphoid cells (5-7%) were EBV-infected. INTERPRETATION AND CONCLUSIONS: It is concluded that EBV infection was as a late event involving an already transformed gd T-cell clone.