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Keywords:

  • hepatosplenic γδ+ T-cell lymphomas ;
  • 2′-deoxycoformycin;
  • cytotoxic effects;
  • haematological response;
  • Pentostatin

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Hepatosplenic γδ+ T-cell lymphoma represents a rare neoplasm of post-thymic phenotype, characterized by an aggressive clinical course and a poor response to conventional chemotherapy. In the present study, we have examined the cytotoxic effects of the purine analogue 2′-deoxycoformycin (dCF) on cultured mononuclear cells and purified γδ+ tumour cells from bone marrow or peripheral blood of four patients with hepatosplenic γδ+ T-cell lymphoma. At a concentration of 10 µm, dCF, in the presence of 2′-deoxyadenosine (dAdo), displayed an early and selective cytotoxic effect on γδ+ tumour T cells. After 48 h of in vitro exposure to dCF, the absolute number of viable CD3+/γδ+ tumour T cells was reduced by more than 90% in all samples with respect to control cultures, with absolute counts of viable CD3+/αβ+ lymphocytes being reduced only by 6–40% of the initial cell input. Analysis of cultures after 5 d of exposure to dCF plus dAdo revealed the persistence of normal CD3+/αβ+ T cells, which accounted, however, for only 20–25% of the initial cell input. Accordingly, the combination of dCF (10–100 µm) plus dAdo was able to induce a dose-dependent inhibition of clonogenic growth and [3H]-thymidine incorporation in purified CD3+/CD4/CD8γδ+ tumour cells. We also report that one patient with hepatosplenic γδ+ T-cell lymphoma in terminal leukaemic phase showed a striking haematological response to single-agent dCF given as fourth-line treatment. In particular, the selective clearance of γδ+ tumour T cells in peripheral blood and bone marrow was observed starting after the second course of treatment. Our results suggest that dCF may represent a potentially active drug for the management of this aggressive form of T-cell lymphoma.

Peripheral T-cell lymphomas (PTCLs) expressing the γδ T-cell receptor (TCR) ( Gaulard et al, 1990 ) represent the neoplastic counterparts of a minor subpopulation of tissue-restricted γδ+ T lymphocytes mainly involved in immune surveillance processes occurring at the epidermal and epithelial linings ( Haas, 1993; De Libero, 1997). γδ+ T cells usually account for about 1–15% of peripheral blood lymphocytes but show a preferential location within the splenic red pulp and the gastrointestinal tract, where they may represent 30% of the whole T-cell population ( Falini et al, 1989 ; Bordessoule et al, 1990 ).

Among the rare neoplasms arising from the γδ+ T cells, the hepatosplenic γδ+ T-cell lymphoma represents a distinct clinicopathological entity recently included in current lymphoma classifications ( Harris et al, 1994 ; Jaffe et al, 1999 ). Hepatosplenic γδ+ T-cell lymphoma is characterized by an extranodal presentation with marked hepatosplenomegaly and a typical growth pattern of tumour cells predominantly involving hepatic sinusoids, splenic red pulp and bone marrow sinuses ( Gaulard et al, 1986 ; Farcet et al, 1990 ; Wong et al, 1995 ; Cooke et al, 1996 ; Salhany et al, 1997 ). Tumour cells display a typical immunophenotype (TCRαβ, TCRγδ+, CD3+, CD2+, CD5, CD4, CD8) with frequent expression of CD56 and of the cytotoxic granules-associated protein, TIA-1, but not, usually, of perforin and granzyme B, reflecting therefore the antigenic profile of non-activated cytotoxic T cells ( Farcet et al, 1990 ; Wong et al, 1995 ; Cooke et al, 1996 ; Salhany et al, 1997 ; Chan, 1999).

Hepatosplenic γδ+ T-cell lymphomas are clinically aggressive, with early relapses despite an initial response to multiagent chemotherapy and a median survival of 12–29 months as reported by different series ( Farcet et al, 1990 ; Wong et al, 1995 ; Cooke et al, 1996 ; Salhany et al, 1997 ). Relapses are usually associated with disease dissemination to lymph nodes and bone marrow ( Farcet et al, 1990 ; Wong et al, 1995 ; Cooke et al, 1996 ; Salhany et al, 1997 ), including a terminal leukaemic phase with blast-like transformation ( Mastovich et al, 1994 ; Salhany et al, 1997 ). Given the unsatisfactory results of current chemotherapy regimens and the very poor outcome of these patients, the identification of new agents displaying cytotoxic activity on tumour cells of γδ+ T-cell lymphomas appears of great relevance.

The agent 2′-deoxycoformycin (dCF; Pentostatin) is a potent inhibitor of adenosine deaminase (ADA) ( Agarwal et al, 1977 ), which displays, in the presence of 2′-deoxyadenosine (dAdo), a striking cytotoxic activity against different types of malignant T cells ( Carson et al, 1978 ; Yu et al, 1980 ; Russell et al, 1986 ; Piga et al, 1989 ; Brogden & Sorkin, 1993) . Even though the mechanism underlying the cytotoxic effect of dCF is not yet fully understood, it appears mainly to correlate with the accumulation of deoxyadenosine triphosphate ( Carson et al, 1978 ; Cohen et al, 1978 ; Seto et al, 1985 ). Induction of DNA strand breaks, alterations in DNA repair processes, inhibition of RNA transcription and triggering of apoptotic death have been also shown to occur in different target cells exposed to the drug ( Brogden & Sorkin, 1993 ; Cheson & Grever, 1997). Although dCF has a well-documented clinical activity against different malignancies of mature T-cell phenotype ( Brogden & Sorkin, 1993 ; Catovsky, 1996; Cheson, 1998), no data have been so far reported as to the cytotoxic effects of dCF on neoplastic γδ+ T cells.

In the present study, we show that dCF exerts a potent cytotoxic and antiproliferative effect in vitro and in vivo on tumour cells from patients with aggressive γδ+ T-cell lymphomas. Our results suggest that dCF may represent a potentially active new agent for the treatment of these rare T-cell malignancies.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Patients

We analysed four patients with hepatosplenic γδ+ T-cell lymphoma, who were observed at our Institution. Diagnoses were based on previously established criteria ( Farcet et al, 1990 ; Cooke et al, 1996 ). Clinical characteristics of patients under study including age, sex, disease status at time of sample collection, sites of involvement and clinical outcome are summarized in Table I. In cases 2, 3 and 4, fresh tumour cells were analysed, whereas in case 1 cryopreserved cells were studied. Survival was calculated starting from the time of first diagnosis.

Based on the failure of previous lines of therapy (Table I) and on the refusal of further aggressive treatments, patient 3 was treated on a compassionate basis with dCF (Nipent), administered as a weekly bolus intravenous (i.v.) injection of 4 mg/m2 for three courses. An additional course of dCF was administered after a 14-d interval after course 3. Peripheral blood samples were collected weekly during dCF therapy and subjected to immunophenotypic analysis by a direct whole blood lysis technique.

Cell isolation and purification

Mononuclear cells from heparinized bone marrow (cases 1 and 2) and peripheral blood (cases 3 and 4) samples obtained during diagnostic procedures were separated upon centrifugation over a Ficoll–Hypaque (Pharmacia, Uppsala, Sweden) gradient, washed twice with Hank's balanced salt solution (HBSS) and utilized for immunophenotyping and tissue culture studies. For experiments involving [3H]-thymidine ([3H]-TdR) incorporation and clonogenic assays, tumour T cells from patients 2, 3 and 4 were further purified by removing CD4+ and CD8+ cells with anti-CD4 and anti-CD8 immunomagnetic beads (Dynabeads; Dynal, Oslo, Norway), as previously described ( Gattei et al, 1999 ). The resulting cell preparations contained more than 95% of tumour T cells (γδ+, CD3+, CD4, CD8), as assessed by two-colour flow cytometry.

Immunophenotyping and flow cytometry

Bone marrow and peripheral blood from all of the patients were analysed by direct two-colour immunofluorescence and flow cytometry, as previously described ( Gattei et al, 1999 ). Briefly, mononuclear cells were washed in HBSS, suspended in binding buffer (2·0 mg/ml purified human immunoglobulin, 0·2% BSA and 0·2% sodium azide in HBSS) and labelled for 30 min at 4°C with fluorescein isothiocyanate (FITC)-conjugated and phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs). Cells were then washed twice in binding buffer and analysed by flow cytometry. The following mAbs were used in this study: anti-TCRαβ/WT31,TCRγδ/11F2, CD2/Leu-5b, CD3/Leu-4, CD4/Leu-3a, CD5/Leu-1, CD7/Leu-9, CD8/Leu-2a, CD11b/Mac-1, CD11c/gp150,95, CD14/Leu-M3, CD19/Leu-12, CD22/Leu-14, CD33/Leu-M9, CD38/Leu-17, HLA-DR, CD45/HLe-1, CD56/Leu-19, CD57/HNK-1, anti-kappa F(ab)′2, anti-lambda F(ab)′2 (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA); anti-TIA-1, anti-TdT (pool HTdT-1, -3, -4) (Coulter-Immunotech, Instrumentation Laboratory, Milan, Italy); anti-CD16 (Pharmingen, San Diego, CA, USA). Non-specific binding of antibodies was assessed by labelling cells with irrelevant isotype-matched FITC- and PE-labelled mouse control immunoglobulins (Igs) (Coulter-Immunotech; Becton Dickinson). Viable antibody-labelled cells were identified according to their forward and right-angle scattering, electronically gated and analysed on a FACScalibur flow cytometer by means of the cellquest software (Becton Dickinson).

Cell cultures

Mononuclear cells from bone marrow or peripheral blood of all patients were seeded in triplicate at a density of 1·0 × 106 cells/ml into six-well (5 ml/well) tissue culture plates (Falcon, Becton Dickinson, Oxnard, CA, USA) in Iscove's modified Dulbecco's medium (IMDM; Biochrom KG, Berlin, Germany) supplemented with 10% heat-inactivated dialysed fetal calf serum (FCS; Biochrom KG), 0·1% (w/v) l-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells from all patients were cultured in complete medium alone and in the presence of 10 µm dCF (Nipent), 10 µm dAdo (Sigma-Aldrich, Milan, Italy) and a combination of 10 µm dCF and 10 µm dAdo. After 2 and 5 d of culture, cells were harvested, washed in HBSS, incubated with a combination of anti-CD3–PE, anti-TCRαβ–FITC and anti-TCRγδ–FITC mAbs (Becton-Dickinson) and analysed by flow cytometry. Aliquots from each cultured sample were utilized for determination of the absolute cell counts and cell viability by the trypan blue-exclusion test.

Clonogenic assays and [3H]-TdR incorporation

Clonogenic assays were performed as previously described in detail ( Pinto et al, 1992 ; Gattei et al, 1997 ). Briefly, γδ+ CD4/CD8 purified tumour T cells (2·0 × 105 cells/ml) were cultured in 1 ml of IMDM containing 0·8% methylcellulose (Fluka Biochemika, Buchs, Switzerland) and 15% of dialysed FCS, in the presence of a combination of dCF (10, 25, 50 and 100 µm) plus dAdo (10 µm). Control cultures contained medium only and dAdo (10 µm) alone. Cells were cultured in 100-µl aliquots (6–8 replicates) in 96-well flat-bottomed microplates. After 14 d of incubation, aggregates ≥ 40 cells were scored as colonies. To ascertain further the purity of the tumour cells grown in the clonogenic assay, colony cells from control or dAdo-exposed plates were pooled and double stained with anti-CD3–PE and anti-TCRαβ–FITC or anti-TCRγδ–FITC mAbs.

For [3H]-TdR incorporation studies, purified γδ+ tumour T cells were preincubated for 6 h in the presence of a combination of dCF (10, 25, 50 and 100 µm) plus two different concentrations of dAdo (1·0 and 10 µm). Control cultures were preincubated for 6 h in medium only and with dCF and dAdo alone. After preincubation, cells were washed twice with HBSS and seeded in triplicate at a concentration of 2·0 × 105 cells/well into 96-well U-bottomed microplates (Falcon). Cells were cultured for 72 h and pulsed with 18·5 kBq/well of [3H]-thymidine (specific activity, 925 GBq/mmol; Amersham International, Amersham Place, UK) for the final 12 h of culture, harvested onto glass fibre membranes and counted in a liquid scintillation β-counter (Tri-Carb 1600TR; Canberra-Packard, Meriden, CT, USA).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Clinical findings

Clinical characteristics, disease status at the time of sample collection, details of therapy and outcomes of the patients under study are summarized in Table I. All patients displayed bone marrow involvement with γδ+ tumour cells (ranging from 28% to > 80% of mononuclear cells) at the time of sample collection (presentation in cases 1 and 4, relapse in cases 2, 3). In cases 3 and 4, an overt leukaemic involvement with γδ+ tumour cells (67–81% of circulating mononuclear cells) was also present.

Characterization of tumour cells from patients with γδ+ T-cell lymphomas

The expression of TCRγδ enabled the selective analysis of tumour cell immunophenotypes by discriminating residual TCRαβ+ normal T lymphocytes ( Mastovich et al, 1994 ; Salhany et al, 1997 ). Dual-colour immunofluorescence analysis indicated that tumour cells expressed in all instances a typical CD3+, TCRαβ, TCRγδ+, CD5, CD4, CD8 immunophenotype. Malignant cells always expressed CD2 and stained positively for the TIA-1 antigen but not for granzyme B. CD7 was only expressed on tumour cells from patient 4, whereas CD56 was detected on neoplastic T cells (γδ+) from patients 1 and 2. In all cases, tumour cells expressed CD45, but conversely did not react with mAbs recognizing TdT, CD57, CD11c, CD11b, pan-B (CD19, CD22) and myelomonocytic antigens (CD14, CD33).

In vitro effects of dCF on tumour cells from γδ+T-cell lymphomas

Bone marrow or peripheral blood mononuclear cells from all of the patients were cultured (1·0 × 106 cells/ml) in the presence of dAdo (10 µm), dCF (10 µm) and a combination of dCF plus dAdo. Two and five days after seeding, cells were harvested, counted and stained with a combination of anti-CD3, anti-TCRαβ and anti-TCRγδ mAbs. Results shown in Table II indicate that dCF, in the presence of dAdo, exerted an early and selective cytotoxic effect on CD3+/γδ+ tumour T cells from all of the patients. After 2 d of culture with dCF plus dAdo, tumour T cells co-expressing CD3 and TCRγδ were no longer detected (cases 1, 2 and 3) or had decreased by more than 90% (case 4) with respect to control cultures (Table II). Accordingly, the percentage of residual normal T cells (CD3+/αβ+) showed a consensual relative increase in all cultures (Table II). Results from a representative experiment assaying the cytotoxic effects of a 48-h exposure to dCF plus dAdo on peripheral blood cells from patient 3 are shown in Fig 1. Analysis of absolute counts of viable CD3+/γδ+ tumour T cells after a 48-h exposure to dCF plus dAdo revealed a reduction of more than 90% in all samples with respect to control cultures, with absolute counts of viable CD3+/αβ+ lymphocytes being reduced by 6% to 40% of the initial cell input ( Fig 2). In all instances, dCF alone or dAdo alone did not exert any significant cytotoxic effect on either tumour (CD3+/γδ+) or normal (CD3+/αβ+) T cells, whose relative percentages remained substantially unaffected with respect to control cultures (Table II). Flow cytometric analysis of cultures after 5 d of exposure to dCF plus dAdo revealed the persistence of normal CD3+/αβ+ T cells, which then accounted, however, for only 20–25% of the initial cell input (data not shown). The kinetics of dCF plus dAdo cytotoxicity towards normal resting T cells was therefore similar to those previously reported ( Kefford & Fox, 1982; Brox et al, 1984 ).

image

Figure 1. Immunophenotypic analysis of bone marrow mononuclear cells from patient 3 cultured from 2 d in medium only (control; left, upper and lower panels) and in the presence of dCF (10 µm) plus dAdo (10 µm) (dCF + dAdo; right, upper and lower panels). Cells were double stained with anti-CD3–PE and anti-TCRαβ–FITC or anti-TCRγδ–FITC mAbs as described in the Materials and methods and were analysed on a FACScalibur flow cytometer by forward and right-angle scatter gating on the lymphocyte region. The relative percentages of CD3+/TCRαβ+ cells (residual normal T cells) and CD3+/TCRγδ+ cells (tumour cells) are indicated in the upper right quadrant of each dot plot.

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image

Figure 2. Cytotoxic effects of dCF on TCRγδ+ tumour cells from patients with γδ+ T-cell lymphomas. Mononuclear cells were cultured for 2 d in the absence (medium) and the presence of dCF (10 µm) plus dAdo (10 µm), harvested, counted and subjected to immunophenotypic analysis as described in the footnote to Table II. Figures indicate absolute numbers of viable CD3+/TCRαβ+ cells (residual normal T cells) and CD3+/TCRγδ+cells (tumour cells).

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In a different experiment, we wished to determine the effects of dCF on the clonogenic growth of purified tumour cells from patients 3 and 4. The results showed that dCF, in the presence of dAdo, induced a dose-dependent inhibition of tumour cells' clonogenic capacity, which was reduced by 70% (patient 3) to 67% (patient 4) at the lowest dCF concentration of 10 µm( Fig 3A and B). Higher dCF concentrations resulted in a reduction of tumour cells' clonogenic growth by up to 90% with respect to control plates (medium only) ( Fig 3A and B). Inhibition of clonogenic growth by dCF alone and dAdo alone never exceeded 20% at the highest concentrations of 50 µm and 10 µm respectively (data not shown and Fig 3A and B). Immunophenotypic analysis of colony cells grown in control plates and dishes exposed to dAdo revealed that > 90% of colony cells co-expressed CD3 and TCRγδ, confirming the purity of the tumour cell populations tested in the clonogenic assays ( Fig 3C and data not shown).

image

Figure 3. Inhibition of clonogenic growth of tumour cells from γδ+ T-cell lymphomas by dCF. Purified tumour cells (2·0 × 105 cells/ml) were seeded in complete medium [IMDM with 20% FCS containing 0·8% methylcellulose, in the presence of increasing concentrations dCF (10, 25, 50 and 100 µm) plus dAdo (10 µm)]. Control cultures contained medium only (CNT) and dAdo (10 µm) alone. After 14 d, aggregates composed of more than 40 cells were scored as colonies. Data are expressed as the mean number of colonies/well ± s.e.m. of triplicate cultures. (A) Patient 3; (B) patient 4; (C) phenotypic analysis of colony cells grown in control plates from patient 4. Colony cells grown in control dishes were pooled and double stained with anti-CD3–PE and anti-TCRαβ–FITC or anti-TCRγδ–FITC mAbs. Cells were then analysed on a FACScalibur flow cytometer. The relative percentages of CD3+/TCRαβ+ cells (residual normal T cells) and CD3+/TCRγδ+ (tumour cells) are indicated in the upper right quadrant of each dot plot.

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To evaluate the effects of a short-term exposure to dCF on spontaneous tumour cell proliferation, purified CD4/CD8/γδ+ primary tumour cells from cases 2, 3 and 4 were incubated for 6 h with increasing concentrations of dCF (10, 25, 50 and 100 µm) in the presence of dAdo (1·0 and 10 µm), washed, seeded into microwell plates, cultured for 72 h and pulsed with of [3H]-TdR for the final 12 h of culture. As shown in Fig 4, dCF, in the presence of dAdo (1·0 µm and 10 µm), induced a significant and dose-dependent inhibition of spontaneous proliferation of primary tumour cells from patients with γδ+ T-cell lymphomas. At a concentration as low as 10 µm, dCF, plus dAdo (10 µm), induced a greater than 70% inhibition of [3H]-TdR incorporation in all of the tested tumour cell samples ( Fig 4). Inhibition of tumour cell proliferation in the presence of dCF alone (10–100 µm) or dAdo alone (1·0 and 10 µm) never exceeded 15%.

image

Figure 4. Effects of a short-term exposure to dCF on the proliferation of tumour cells from γδ+ T-cell lymphomas. Purified tumour cells were preincubated for 6 h in the presence of increasing concentrations (10, 25, 50 and 100 µm) of dCF alone (open circles), a combination of dCF (10, 25, 50 and 100 µm) and two different concentrations of dAdo (1·0 µm, closed squares, and 10 µm, closed triangles), washed and seeded at a concentration of 2·0 × 105 cells/well into 96-well U-bottomed microplates. Control cultures contained medium only and dAdo alone. After 72 h of incubation, cells were pulsed with 18·5 kBq/well of [3H]-thymidine for the final 12 h of culture, harvested onto glass fibre membranes and counted in a liquid scintillation β-counter. Data are expressed as percentage of control [3H]-thymidine incorporation (mean c.p.m. ± s.e.m. of triplicate cultures).

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Clinical activity of dCF in a patient with hepatosplenic γδ+ T-cell lymphoma

Given the failure of previous lines of therapy (Table I) and based on the refusal of further multiagent chemotherapy, patient 3. was treated on a compassionate basis with i.v. dCF (Nipent), administered as a weekly bolus injection of 4 mg/m2 for three courses. At the start of therapy, the patient displayed an overt leukaemic phase with circulating atypical large- and medium-sized lymphoid cells, a leucocyte count of 14·8 × 109/l, with 64% lymphocytes (79% of which expressed CD3 and TCRγδ and 16% of residual normal CD3+/αβ+ T cells; Fig 5), 9·8% neutrophils, haemoglobin of 12·0 g/dl and a platelet count of 97·0 × 109/l. Bone marrow analysis showed massive infiltration (approximately 90% of bone marrow cells) with atypical large- and medium-sized lymphocytes co-expressing CD3 and TCRγδ.

image

Figure 5. Changes in circulating γδ+ tumour T cells of patient 3 during therapy with 2′-deoxycoformycin. Peripheral blood samples were obtained before therapy and 1 week after each course of intravenous 2′-deoxycoformycin (4 mg/m2) and were analysed by flow cytometry with a direct whole blood lysis technique. Samples were double stained with anti-CD3–PE and anti-TCRαβ–FITC or anti-TCRγδ–FITC mAbs and analysed on a FCAScalibur flow cytometer by forward and right-angle scatter gating on the lymphocyte region. The relative percentages of CD3+/TCRαβ+ cells (residual normal T cells) and CD3+/TCRγδ+ (tumour cells) are indicated in the upper right quadrant of each dot plot.

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After an initial rise to 38·3 × 109/l (with 68% atypical lymphocytes and 15·8% neutrophils) 1 week after the first dCF course, a stepwise reduction of total leucocyte counts took place 7 d after the second administration of dCF, eventually leading to a leucocyte count of 4·7 × 109/l (50% lymphocytes, 30% neutrophils) and 74·0 × 109/l platelets 1 week after the third course of dCF. An additional course of dCF was then administered after a 14-d interval from course 3. One week after the last course of dCF (i.e. 6 weeks after starting of dCF), the patient presented with a total leucocyte count of 4·2 × 109/l, 38% lymphocytes, 40% neutrophils, 120 × 109/l platelets and a haemoglobin of 13·7 g/dl. Morphologically identifiable atypical lymphoid cells accounted for less than 1–2% of circulating lymphocytes, whereas bone marrow examination showed a slightly hypoplastic marrow with 25% morphologically normal small-sized lymphocytes and less than 3% of atypical lymphoid cells.

Flow cytometry analysis documented the progressive clearance of neoplastic T cells from peripheral blood ( Fig 5). The relative proportion of CD3+/γδ+ tumour cells (79% of circulating lymphoid cells before therapy) was reduced to 19% and 8% 1 week after the second and third treatment courses respectively. One week after the last course of dCF, CD3+/γδ+ cells accounted for less than 5% of peripheral T lymphocytes ( Fig 5). Concurrently, the relative percentage of normal T cells (CD3+/αβ+) in peripheral blood showed a stepwise increase from 16% (before treatment) to 79% (1 week after the fourth dCF course) ( Fig 5). The patient also displayed a > 50% reduction of hepatomegaly.

The haematological response persisted up to 4 weeks after the last administration of dCF, when the patient was referred for allogeneic bone marrow transplantation. The patient died 2 months afterwards as a result of transplant-related complications.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Hepatosplenic γδ+ T-cell lymphomas display an aggressive clinical course and are associated with a poor outcome and short survival due to the lack of durable remission after conventional multiagent chemotherapy ( Farcet et al, 1990 ; Wong et al, 1995 ; Cooke et al, 1996 ; Salhany et al, 1997 ). It is therefore important to identify new agents that display anti-tumour activity in this subset of T-cell neoplasms, with the hope that their inclusion in chemotherapy regimes may modify the overall poor results of current treatments.

In the present study, we have demonstrated that dCF exhibits a potent in vitro cytotoxic effect on primary tumour cells from patients with hepatosplenic γδ+ T-cell lymphomas. Our results indicate that dCF, at the lowest concentration of 10 µm, in the presence of dAdo was able to kill 95–99% of CD3+/TCRγδ+ tumour T cells, while displaying a far more limited cytotoxic effect (4–40% cell killing) on residual (CD3+/TCRαβ+) normal T lymphocytes. The combination of dCF (10 µm) and dAdo also produced a greater than 60% reduction in the clonogenic capacity of tumour cells, whereas a short-term exposure (6 h) to the drug resulted in a more than 70% inhibition of their spontaneous proliferation, as assessed by [3H]-TdR incorporation. At higher concentrations of dCF (50–100 µm), tumour cell proliferation and clonogenic growth were inhibited by more than 90% and 70–80% respectively.

The cytotoxic activity of dCF against γδ+ tumour T cells was confirmed by its ability to induce a striking haematological response in a pretreated patient with hepatosplenic γδ+ T-cell lymphoma in terminal leukaemic phase. Three courses of intravenous dCF (4 mg/m2) were administered on a weekly basis, followed by a final course after a 14-d interval. At the end of dCF treatment, an almost complete clearance, i.e. from 80% to less than 5%, of peripheral blood and bone marrow tumour cells was achieved, along with a normalization of the haemogram and a significant reduction of the hepatomegaly.

The mechanism underlying the potent growth inhibitory activity of dCF towards γδ+ tumour T cells remains to be established. No specific data are available as to ADA expression in neoplastic γδ+ T cells, but the clinical activity of dCF in B-cell tumours ( Catovsky, 1996; Cheson & Grever, 1997; Cheson, 1998) suggests that intracellular ADA levels may not represent the only determinants on which dCF can exert its cytotoxic activity. It has been proposed that the response to dCF may relate to the cellular expression of a complex formed by ADA and the dipeptidyl peptidase IV(DPPIV)/CD26 acting as an ADA-binding protein ( Morimoto & Schlossman, 1998). In this regard, it is of interest that tumour cells of hepatosplenic γδ+ T-cell lymphomas have been shown to express high levels of surface CD26 and DPPIV activity ( Ruiz et al, 1998 ). Specific studies, including evaluation of purine enzyme patterns in γδ+ lymphocytes, are warranted to unveil the mechanism of dCF activity on γδ+ tumour T cells.

Several clinical trials have demonstrated that dCF is an active agent for the treatment of different types of T-cell malignancies, including PTCLs, cutaneous T-cell lymphomas, T-prolymphocytic leukaemia and large granular lymphocyte leukaemia ( Cummings et al, 1991 ; Dearden et al, 1991 ; Mercieca et al, 1994 ; Monfardini et al, 1996 ; Ho et al, 1999 ). In these studies, however, the type of TCR expressed by tumour cells, i.e. αβ or γδ, was usually not reported. Therefore, apart from the present results, no additional data on the anti-tumour activity of dCF towards malignant γδ+ T cells are available. Interestingly, however, an haematological response to 2-chlorodeoxyadenosine (2-CDA) was recently described in a patient with relapsed γδ+ T-cell lymphoma ( Lin et al, 1999 ). It appears therefore that related purine analogues such as 2-CDA and, as shown here, dCF may share anti-tumour activity in neoplasms derived from γδ+ T cells.

Although the weekly schedule of dCF displays a consistent clinical activity in several T-cell malignancies ( Dearden et al, 1991 ; Mercieca et al, 1994 ; < Catovsky, 1996; 36Monfardini et al, 1996; Cheson, 1998; Ho et al, 1999 ), it may not necessarily represent the optimal strategy for treating aggressive diseases such as γδ+ T-cell lymphomas. In this regard, a 5-d schedule of dCF (2 mg/m2) at 28-d intervals was recently evaluated in relapsed–refractory chronic lymphocytic leukaemia ( Johnson et al, 1998 ). More aggressive schedules and/or inclusion in combination chemotherapy regimens could be therefore explored for better exploiting the anti-tumour activity of dCF in γδ+ T-cell malignancies.

In conclusion, we have shown that dCF may be a promising drug which warrants further exploration in aggressive γδ+ T-cell lymphomas.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This work has been supported by a grant from the Ministero della Sanità, Ricerca Finalizzata IRCCS, Rome, Italy. The authors are grateful to Dr Rosaria De Filippi (University of Udine, Italy) for critically reviewing the manuscript.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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