Introduction

Allogeneic stem cell transplantation (alloSCT) is a curative treatment modality for many patients with malignant and non-malignant diseases. Graft-versus-host disease (GVHD) remains the main complication after conventional myeloablative as well as after non-myeloablative conditioning.^{1, 2, 3, 4, 5, 6} Post-transplant immune suppression and techniques to deplete the graft and/or the patient for T lymphocytes have been applied for many years. Anti-CD52 antibodies, directed against the CD52 antigen expressed on human T- and B-lymphocytes, monocytes and dendritic cells, have been successfully used for T-cell depletion. Initially rat antibodies Campath-1M and Campath-1G, and later the humanized form Campath-1H (alemtuzumab), have been administered in different in vivo and in vitro treatment protocols.^{7, 8, 9, 10, 11, 12} The main technical advantage to using Campath in vitro is its easy applicability, compared to the time-consuming procedure of CD34 selection using an affinity-column. At 30 min after addition of the Campath antibody to the graft, the stem cells are infused into the patient. A drawback of using Campath is impaired immune reconstitution. Recently the use of in vivo administration of high-dose alemtuzumab in combination with fludarabine and melphalan as a non-myeloablative conditioning regimen has been extensively investigated. Despite the significant decrease in the incidence of GVHD, poor immune reconstitution and an increased incidence of infectious complications was observed.^{13, 14} Immune incompetence is probably caused by infusion of high-dose alemtuzumab since pharmacokinetic studies have shown that, in contrast to Campath-1G with an earlier reported half-life of approximately 12–13 h, alemtuzumab has a half-life of 15–21 days in the setting of the transplantation protocol.¹⁵

In vitro T-cell depletion with Campath 'in the bag' has predominantly been used after myeloablative conditioning. Only limited information is available on long-term results of in vitro usage of Campath for T-cell depletion. In this report, we describe our single centre long-term experience with in vitro T-cell depletion of the stem cell graft by Campath in 73 patients with a hematological malignancy receiving a conventional myeloablative alloSCT from an HLA identical family donor.

Patients and methods

Patients

Between January 1998 and December 2004, 73 adults with hematological malignancies received a first alloSCT after standard cyclophosphamide/TBI conditioning, using peripheral blood stem cells from an HLA-identical sibling donor, at the Leiden University Medical Center. The transplant protocol was approved by the institutional Ethics Committee. The main clinical characteristics of the 73 patients are shown in Table 1. All patients were nursed in HEPA-filtered rooms.

Table 1 - Patient and transplantation characteristics.

Full table

Donors

All patients were transplanted with stem cells from a fully HLA-matched sibling donor. Donor stem cells were mobilized with filgrastim (Amgen, Thousand Oaks, CA, USA), 10 g/kg/day s.c. for 4 or 5 days. On day 4, donors received two injections of filgrastim. Apheresis was performed on day 5, using a continuous flow blood cell separator (Cobe Spectra; Gambro BCT; Lakewood, CO, USA). A minimum dose of 5 10⁶ CD34⁺ cells/kg of the recipient body weight was targeted for transplantation. After filtering and centrifugation, the buffy coat was harvested. T-cell depletion of the stem cell product was performed by incubation with alemtuzumab (20 mg) for 30 min at room temperature under continuous gentle agitation.⁷ The number of CD34⁺ cells was analyzed by flow cytometry.

Transplantation

Patients were conditioned with a standard myeloablative regimen. They received cyclophosphamide (Cy) at 60 mg/kg/day i.v. administered for 2 consecutive days (days -6 and -5) and single dose TBI (9 Gy; 25 cGy/min with lung- and eye-shielding) at day -1. Immediately following incubation with alemtuzumab, the stem cell product was infused intravenously on day 0. No post-transplant GVHD prophylaxis, cytomegalovirus (CMV) prophylaxis or hematopoietic growth factors were administered. All patients received supportive care, that is antimicrobial prophylaxis, hydration, blood component support, parenteral nutrition, according to institutional protocols.

Post-transplantation analysis

Neutrophil engraftment was defined as the first of 2 consecutive days with an absolute neutrophil count >0.5 10⁹/l following a post-transplant nadir. Platelet engraftment was defined as the first of 3 consecutive days with a platelet count of >20 10⁹/l without transfusion. Graft failure was defined as the failure to reach an absolute number of neutrophils of 0.5 10⁹/l at day 60.

The presence of CMV lower matrix protein pp65-positive leukocytes in peripheral blood was sought weekly until 3 months after transplantation. Positive CMV antigenemia was defined as the presence of positively stained leukocytes.¹⁶ Since July 2002, CMV reactivation was detected with a quantitive CMV-DNA PCR.¹⁷ Patients with CMV reactivation were treated with (val-)ganciclovir for 2 weeks. Cytomegalovirus disease was diagnosed on the basis of an inflammatory process considered to be due to CMV confirmed by immediate-early-antigen assay or CMV cultures.

Assessment and grading of acute and chronic GVHD (cGVHD) were made using the Glucksberg and Shulman criteria, respectively.^{18, 19} Patients with engraftment and surviving over 90 days were evaluable for cGVHD. Chimerism analysis was performed on bone marrow samples at fixed time points (months +3, 6, 9, 12, 15, 18, 21, 24). In sex-mismatched combinations chimerism was studied by fluorescent in situ hybridization for X and Y chromosomes. Sex-matched donor/patient combinations, chimerism was determined on post-Ficoll bone marrow using a STR-PCR based protocol.²⁰ Complete donor chimerism was defined as 100% donor chimerism at 3 months after alloSCT. Toxicity was graded according to the World Health Organization toxicity scale. Complete (CR) and partial responses (PR) were determined at fixed time points by standard disease-specific criteria as defined by the International Bone Marrow Transplant Registry. Data were analyzed as of 1 May 2005. Transplant-related mortality (TRM) was defined as death in continuous complete remission. Disease relapse was defined as hematological recurrence of the disease, except for CML where cytogenetic reappearance of the Philadelphia chromosome was also classified as relapse. Event-free survival (EFS) was defined as survival in continuous complete remission.

Statistical analysis

At the time of analysis the median follow-up was 51 months (range, 3–69). The time-to-event data (OS, EFS and TRM) were compared by the Kaplan–Meier method with the log-rank test.

Results

Patient and transplantation characteristics

Seventy-three patients were enrolled in this study with no selection according to disease type. Patient characteristics are detailed in Table 1. Acute leukemia and chronic myeloid leukemia were the most common indications. Patients with acute leukemia in first remission, or with chronic myeloid leukemia in first chronic phase were considered to be standard-risk (n=53) and all other patients were classified as high-risk (n=20). The median age at transplantation was 44 years (range, 19–61). The median number of CD34⁺ cells in the stem cell graft was 7.5 10⁶ CD34⁺ cells/kg of the recipient body weight (range, 0.5–18).

Engraftment and chimerism

Neutrophil engraftment (ANC >0.5 10⁹/l) occurred at a median of 16 days (range, 8–29) after stem cell infusion (Table 2). No graft failures were observed. The median day to reach 20 10⁹/l platelets was 11 days (range, 5–75). Chimerism studies on bone marrow cells demonstrated complete donor chimerism in 29 of 69 evaluable patients at 3 months after alloSCT. The other 40 patients showed mixed chimerism with a high percentage donor cells. The median % donor cells at 3 months after alloSCT was 99% (range, 15–100).

Table 2 - Engraftment, chimerism, CMV viremia and incidence of GVHD after alloSCT.

Full table

Cytomegalovirus reactivation

Twenty-eight donor–recipient pairs were CMV-seronegative before transplantation. No CMV infections were observed in these patients. After transplantation, 24 of the remaining 45 patients at-risk showed CMV reactivation (53%). All patients with CMV reactivation were treated with (val)ganciclovir. No CMV disease was observed in these patients.

Graft-versus-host disease after allogeneic stem cell transplantation

Twenty-four patients (33%) did not experience aGVHD after alloSCT (Table 2). In addition, severe grade III–IV aGVHD was not observed in any patient. Thirty-three patients experienced grade 1 GVHD of the skin, which resolved after local therapy. The incidence of grade II aGVHD was 22%. Chronic GVHD after alloSCT was not seen in 38 of 69 evaluable patients. Eighteen patients experienced limited cGVHD, resolving after therapy. Thirteen patients experienced extensive cGVHD after alloSCT. At follow-up only two of these 13 patients are alive.

Donor lymphocyte infusions

Donor lymphocyte infusions (DLI) was administered to patients with a hematological or molecular relapse after alloSCT (25 patients). Since 2004, we also administered DLI in patients (n=11) who showed mixed chimerism after alloSCT. One patient received DLI for treatment of persistent CMV reactivations despite adequate antiviral therapy. One patient who failed to have a response to DLI received in vitro expanded leukemia reactive cytotoxic T cells produced under GMP conditions.²¹ The dose DLI administered depended on the disease and the protocol, varying between 1 10⁶–5 10⁷ CD3⁺ cells/kg.

Outcome

Thirty-nine patients were alive at follow-up. Twenty-seven patients developed hematological disease relapse. As expected, a higher incidence of relapse was observed in patients with acute leukemia (14 of 34 patients) and multiple myeloma (six of 11 patients) compared to patients with CML (four of 16 patients). The median time to relapse was 6.9 months after alloSCT (range, 1.7–23 months). Figure 1 shows the cumulative incidence of relapse. The main causes of death were relapse (n=16), infection (n=8) and GVHD (n=4). Eight patients died due to infectious complications, consisting of bacterial (n=2), viral (n=2) and fungal (n=4) infections. Three of four patients who died due to GVHD had developed severe GVHD after administration of DLI.

Figure 1.

Cumulative incidence of disease relapse after alloSCT.

Full figure and legend (10K)

Figure 2 shows a TRM of 8%. At 5-years after alloSCT, OS and EFS were 48 and 43%, respectively (Figure 3).

Figure 2.

Cumulative probability of transplant-related mortality (TRM) following T-cell depleted myeloablative alloSCT with Campath 'in the bag'.

Full figure and legend (9K)

Figure 3.

Event-free survival (EFS) of T-cell depleted myeloablative alloSCT with Campath 'in the bag'.

Full figure and legend (12K)

Discussion

In this study, we report our long-term results with 'Campath in the bag' for T-cell depletion in myeloablative alloSCT with HLA-related sibling donors. T-cell depletion is the most effective method for prevention of GVHD after alloSCT. However, following T-cell depletion an increase in graft rejection, relapse and immune-incompetence has been observed. The Campath-group of antibodies has been commonly used for T-cell depletion in vivo and in vitro. Incubation of the graft with Campath results in one to two log T-cell depletion. The main T-cell depleting effect occurs, however, in vivo by a cellular effector mechanism (ADCC). Pharmacokinetic studies have demonstrated that the half-life of the humanized Campath (alemtuzumab) antibodies is much longer than the rat monoclonal antibodies. Graft failure was one of the major disadvantages associated with the use of Campath as the method for T-cell depletion in allogeneic bone marrow transplantation. No graft failures were observed in this study with 73 transplants from allogeneic mobilized peripheral blood donors. The high CD34⁺ cell dose in peripheral blood stem cell grafts compared to bone marrow grafts and the use of humanized Campath (alemtuzumab) with a longer half-life compared to rat antibodies may have facilitated engraftment, possibly due to a concomitant reduction of host T cells.^{7, 22, 23}

In our study, a low incidence of acute GVHD (grade II–IV) was observed. No severe (grade III–IV) aGVHD occurred. A strikingly high incidence of grade I aGVHD was observed (33/73 patients), which was similar to our previous experience with T-cell depletion of peripheral stem cell grafts with Campath.⁷ The incidence of grade I GVHD after transplantation with peripheral stem cells is significantly higher than after transplantation with bone marrow cells incubated with Campath and is probably due to the higher number of stem cells. All patients with grade 1 (skin) aGVHD experienced resolution with local corticosteroid therapy. Grade II GVHD was mainly confined to the skin. Chronic GVHD after alloSCT was absent in 55% of evaluable patients. Limited cGVHD has not been a major clinical problem. In 13 patients, more extensive cGVHD occurred. This incidence of cGVHD in this study is higher than data from the multi-centre study that describes an incidence of extensive cGVHD of 2%.²⁴ The difference is probably due to the longer follow-up in our study, and possibly the high numbers of CD34⁺ cells in the graft. The incidence of GVHD in our study, is as expected, significantly lower than in patients after nonmanipulated alloSCT who show an incidence of severe acute and chronic GVHD of 35 and 70%, respectively.²

The use of humanized Campath (alemtuzumab) in vivo following reduced intensity conditioning and unrelated donor myeloablative transplantation is associated with a similar decrease in the incidence of GVHD but at the cost of impaired immune reconstitution and increased infectious complications.^{25, 26} In our study, a high incidence of CMV reactivation was observed (53%). Owing to successful periodic viral surveillance and pre-emptive antiviral treatment with (val)ganciclovir, no CMV disease was observed. A low incidence of GVHD and successful pre-emptive antiviral therapy resulted in the low TRM of 8%.

In 27 patients, relapse of their disease occurred. Most relapses were observed within 9 months of alloSCT with a median of 6.9 months. Other studies have suggested that T-cell depletion does not increase risk of relapse of AML in first remission. The number of patients with AML in first remission in this study is, however, too low to confirm this observation. Early relapse of disease after T-cell depleted alloSCT remains a problem for which different strategies are being explored.^{21, 27} Currently, our policy is to treat patients with mixed chimerism 6 months after alloSCT with pre-emptive escalating DLI. This strategy is similar to the successful treatment of molecular relapse of CML after alloSCT. With the use of escalating donor lymphocyte infusions we were able to induce a complete second molecular remission in all patients with CML (n=8).

In conclusion, incubation of the graft with humanized Campath (alemtuzumab) is a simple and reliable method for prevention of GVHD following alloSCT. Owing to successful pre-emptive antiviral treatment of CMV reactivations, no increase in infectious mortality was seen, which resulted in a very low TRM (8%) in this study. This low incidence of TRM is comparable to the outcome of patients undergoing nonmyeloablative transplantation. Early disease relapse remains a major problem. New strategies including pre-emptive DLI and generation of leukemia reactive T-cells produced under GMP conditions are being explored.

References

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Acknowledgements

The authors gratefully acknowledge Monica Seltenheim and Floor Beaumont for their excellent help in acquiring patient data.