Risks of Developing Epstein-Barr Virus–Related Lymphoproliferative Disorders After T-Cell–Depleted Marrow Transplants

CAMPATH-1M (rat IgM) and CAMPATH-1G (rat IgG2b) monoclonal antibodies were produced by the Therapeutic Antibody Centre and supplied to BMT centers for a variety of physician-initiated trials to prevent GVHD and/or transplant rejection.20 CAMPATH-1M was used with human complement in vitro for depletion of T cells to prevent GVHD. CAMPATH-1G was used in three different ways: (1) in vitro (without complement) to opsonize donor T cells, marking them for destruction in vivo; (2) in vivo before the transplant to deplete recipient T cells to prevent rejection; and (3) in vivo at or just after the transplant to deplete donor cells and prevent GVHD. Combinations of these modes of antibody treatment were used in different trials as described more fully in Hale et al.20 

Informed consent was obtained for participation in study protocols and submission of data to the CAMPATH registry according to the normal procedure at each center.

A database is maintained with details and outcome of all BMTs using these antibodies. We believe it is comprehensive, because it correlates with the antibody distribution records. Between July 1982 and May 1996, a total of 2,578 transplants were recorded, and all of them were reviewed to identify any suspect cases of BLPD. The collaborating transplant centers were contacted with a written questionnaire to obtain confirmation of the suspect cases and to discover whether there were any others that had not been reported. Replies were received from 46 of 54 centers, representing 97% of the patients in the database. Eight new cases were identified from the survey, but 4 of them occurred since May 1996 and were therefore not included in this analysis because we do not yet have details of all patients transplanted in this period (approximately 3 to 400 patients). The results were analyzed by the method of T-cell depletion and the match between donor and recipient. Statistical comparisons were performed using the χ² test, log rank survival analysis, or Mann-Whitney U test, as appropriate.

A total of 20 possible cases of BLPD were identified (Table1). The distribution with respect to patient's original disease, age, year of transplant, donor or recipient gender, and occurrence of graft rejection or graft failure was unremarkable. All cases occurred within the first year posttransplant (which is typical), with the exception of case no. 2, a patient who died at 17 months posttransplant of secondary lymphoma. In this case, there was no positive diagnosis of EBV, and it was considered possible that the lymphoma represented progression of the original malignancy (chronic myeloid leukemia [CML]). However, it has been included as a case of possible BLPD for the purpose of this analysis. Most of the cases occurred before the era of therapy with donor lymphocyte infusions and progression was generally very rapid. Sometimes the diagnosis was only made postmortem. However, case no. 20 has been successfully treated with donor T-cell infusions.

The frequency of BLPD was analyzed according to the relationship between donor and recipient and according to the method of T-cell depletion (Table 2). Transplants from HLA-identical siblings were considered in one group and all other transplants (mismatched family donors and unrelated donors) in another. Most patients received marrow depleted of T and B cells with CAMPATH-1 antibodies either in vitro (marrow was treated with CAMPATH-M or CAMPATH-1G before infusion) or in vivo (the patient was treated with CAMPATH-1G on and/or after the day of transplant). Many patients also received CAMPATH-1G and occasionally other monoclonal antibodies as part of the conditioning regime to prevent rejection. The number of patients who received antibody therapy for rejection prophylaxis is shown in Table 2. Some of the patients who received lymphocyte-depleted bone marrow also received infusions of small numbers of donor lymphocytes (T-cell addback), sometimes at the time of transplantation23 and sometimes during the following 3 months.24 The dose was typically in the range of 10⁴ to 10⁶ per kilogram. These patients have been analyzed separately.

Smaller groups of patients had no T-cell depletion or received bone marrow depleted of T cells by E-rosetting25 or by an alternative methodology (12 with T-cell–specific antibodies plus rabbit complement, 2 with elutriation, and 2 with CD34 selection). In all these cases, CAMPATH-1G was used as part of the conditioning regimen.

There was no case of BLPD in the 144 nondepleted transplants. Among 20 patients transplanted from mismatched or unrelated donors with E-rosette depletion, there were 5 cases of BLPD (actuarial risk, 29% ± 16% at 2 years). In 2,401 transplants in which donor T cells were depleted with CAMPATH-1M or CAMPATH-1G, there were 15 patients who suffered from BLPD and/or secondary lymphoma (actuarial risk, 1.1% ± 0.4% at 2 years). There was no significant difference between sibling and nonsibling donors, and we could not detect an association with the use of antibody for rejection prophylaxis or any particular antibody protocol. There were no cases of BLPD in the subset of 303 patients who received T-cell addback, but this is still not a large enough number to be significantly different from the patients with complete T-cell depletion.

The median age of the E-rosette cases (7 years) was less than that of the CAMPATH cases (36 years), but this simply reflects the difference in the populations treated by the two methods.

Diagnosis of BLPD was earlier among patients transplanted from unrelated donors or mismatched family donors (median, day 85) compared with HLA-identical sibling donors (median, day 238;P < .001, Mann-Whitney test). This difference is still significant if we exclude the doubtful case no. 2 (P < .005) or analyze days of survival rather than days to diagnosis (P < .001) or exclude the E-rosette patients (P < .005). Among the nonsibling donors, diagnosis was also earlier in the patients who received E-rosette–treated marrow (median, day 59) compared with CAMPATH-1–treated marrow (median, day 116;P < .003). However, this difference is no longer significant if we compare survival times. Because all the E-rosette patients were children treated at a single center, we surmise that BLPD might have been diagnosed sooner than in the disparate group of CAMPATH-1 patients observed at many different centers.

The percentage of residual T cells in bone marrow treated by CAMPATH-1M and complement was estimated by E-rosette analysis or flow cytometry and reported for a large proportion of the patients (828/1,267). For HLA-matched siblings, the median total nucleated cell dose was 2.1 × 10⁸/kg and the median proportion of residual T cells was 0.4%, giving a dose of approximately 8 × 10⁵ T cells/kg infused. For other donors, the median total nucleated cell dose was 3.6 × 10⁸/kg and the median proportion of residual T cells was 0.2%, giving a dose of approximately 7 × 10⁵ T cells/kg infused. It is very likely that these figures are an overestimate of the number of functional donor T cells, because lymphocytes coated with CAMPATH-1M antibody that escaped lysis in vitro may still be lysed when they encounter additional complement in vivo. By the same token, it is not possible to measure the extent of cell lysis with CAMPATH-1G, because complement was not added in vitro and much of the cell lysis would occur after infusion of the marrow. Residual numbers of T cells were not reported for the bone marrow depleted by the E-rosette method.

The risks of BLPD after lymphocyte depletion with CAMPATH-1 are not substantially different from those reported for conventional BMTs in which there was no T-cell depletion. For example, Zutter et al5 report 5 cases among 1,868 HLA-identical sibling transplants (0.45%) and 3 among 386 HLA-mismatched transplants (1.4%). However, the frequency of BLPD seen by CAMPATH users is substantially lower than that described in many other protocols of more specific T-cell depletion, whether using E-rosettes or other monoclonal antibodies where actuarial risks up to and exceeding 20% have been reported.4,10,12,15,26 

A group of 20 patients who received marrow depleted by albumin gradient and E-rosette treatment were reported by one center to the CAMPATH users registry. These patients all received CAMPATH-1G as part of the conditioning regimen and were included in the 65 patients previously reported in Gerritsen et al.12 We observed that they suffered an exceptionally high incidence of BLPD (29%). This group was not directly comparable with the rest of the CAMPATH-1–depleted patients for several reasons: (1) the average age was significantly lower (all were children), (2) all were transplanted from unrelated donors (18) or parents (2), and (3) a unique conditioning regime was used including a CD11a (LFA-1) antibody as well as CAMPATH-1G.12 It is not possible to directly compare the levels of T-cell or B-cell depletion achieved by the different methods in the clinical cases, because data were not reported for the E-rosette–depleted marrow and a significant amount of cell lysis may occur in vivo with the CAMPATH-1 antibodies, especially CAMPATH-1G. However, there are numerous published data that show that both methods can give a profound degree of T-cell depletion when measured by functional analysis such as limiting dilution. For example, Frame et al27 found 99.4% depletion of T cells by CAMPATH-1M plus complement, compared with 99.8% by soybean agglutinin plus E-rosettes. Jabado et al26 compared the depletion measured by immunophenotyping with limiting dilution analysis. By staining with anti-CD3, they found a mean of 3.1 × 10⁵ T cells/kg infused after CAMPATH-1M depletion and 1.6 × 10⁵ after E-rosette depletion. However, the number of viable T cells estimated by limiting dilution analysis was more than 10-fold lower for the CAMPATH-1M–depleted samples.

The most conclusive evidence for the extent of T-cell depletion achieved with CAMPATH-1M or CAMPATH-1G is the effective prevention of GVHD that is achieved in the absence of posttransplant immunosuppression in both HLA-matched and mismatched transplants.19-21 It is conceivable that a few T cells are spared, particularly if they are CD52⁻,28 and perhaps these are sufficient to control any emerging B cells. More likely, we think that the CAMPATH-1 will have depleted the donor B cells and thus removed both a potential reservoir of latent virus and its immediate target.

If donor B cells are preserved (eg, by E-rosette depletion), but CAMPATH-1G was administered to the recipient (to prevent rejection), we anticipate the highest risk; indeed, this is borne out by other reports.12,26 

It has often been remarked that the risk of BLPD is higher for mismatched transplants than for transplants from HLA-identical donors.4,10,12 It would not be surprising if mismatched patients were at greater risk due to higher levels of immunosuppression and relatively impaired recovery of T-cell immunity. However, this effect was not seen in the CAMPATH-1–depleted transplants. Possibly the depletion of donor B cells by CAMPATH-1 is sufficient to overcome the other risk factors (eg, see Gerritsen et al12). Most of the patients also received CAMPATH-1G in vivo for prevention of rejection. This would have depleted any residual recipient B cells as well, and we cannot exclude a beneficial effect by this mechanism. However, it seems less likely, because we saw no association between the incidence of BLPD and the use (or not) of CAMPATH-1G in vivo.

T-cell depletion is currently enjoying a renaissance of interest after the success of transplants with peripheral blood stem cells. The ability to infuse large numbers of stem cells may overcome any problems of graft rejection or delayed engraftment that were previously seen with T-cell–depleted bone marrow. The possibilities of storing cryopreserved donor T cells or harvesting fresh ones may enable effective treatment of relapse and of viral infections including cytomegalovirus and EBV.29 We suggest that the recipient EBV status should be checked before transplantation and that a negative result should be taken into account in deciding future management. However, if donor B cells can be removed from the transplant along with the T cells, the risks of developing lymphoproliferative disease would appear to be substantially reduced.

The authors greatly appreciate the contributions of many BMT teams who contributed data for this report. The participating centers are listed in Hale et al.20 We thank the reviewers A and B for their constructive comments on this paper.