New approaches using nonmyeloablative-conditioning regimens have been developed to cause minimal procedure-related toxicity. Such novel therapeutic options are being explored with good preliminary results concerning feasibility and engraftment. However many aspects remain under-evaluated, and few data are available about viral and nonviral infections after these highly immunosuppressive regimens. We present our preliminary data on 21 patients receiving a highly immunosuppressive conditioning strategy, focusing on early infectious complications. Early viral infections before day 45, especially CMV, occurred at a high rate (65%). Furthermore, 33% of patients presented with late bacterial infections (predominately gram negative) although they were not neutropenic compared to conventional conditioning regimens. Although there is presently real interest in these new conditioning regimens which result in reduced immediate transplant-related mortality, it is important that investigators be aware of these pitfalls which may secondarily increase transplant toxicity. Further studies are needed to confirm these findings. Bone Marrow Transplantation (2000) 26, 251–255.
Allogeneic bone marrow transplantation (allo-BMT) has been successfully used to treat patients with hematological malignancies.1 Due to the increased risk of conditioning regimen-related toxicities and graft-versus-host disease (GVHD), this procedure is generally limited to young patients with good performance status.23 Despite its hazardous immediate and late complications, allo-BMT offers an important antileukemic and antitumoral advantage due to alloreactivity against host tumor cells.45 The immune reactions between donor-derived immunocompetent T lymphocytes and host-type tumor cells have been well established to be the major antitumor agent in allo-BMT.4 Evidence supporting the so-called graft-versus-leukemia (GVL) effect includes the higher risk of relapse following T cell-depleted or syngeneic transplants.67 Furthermore, donor lymphocyte infusions (DLI) may re-induce remissions in many patients relapsing after allo-BMT.8
Recently, new approaches using nonmyeloablative-conditioning regimens have been developed to obtain minimal procedure-related toxicity.910 These approaches aim at extending the use of allo-BMT to patients who are not eligible for high-dose chemotherapy or total body irradiation by increasing the immunosuppressive aspect of the preparation. Such novel therapeutic options are being explored at the moment with good preliminary results concerning feasibility and engraftment.910
We present our preliminary data in 21 patients receiving a so-called mini-allotransplant, focusing essentially on early infectious complications, which we found to be particularly increased.
Patients and methods
From January 1998 to October 1999, 21 patients (11 males and 10 females) with hematological malignancies and solid tumors were treated with a combination of fludarabine, busulfan and ATG.10 Patient characteristics are summarized in Table 1. In brief, the median age was 52 years (range, 29 to 59 years). The median time to transplantation from diagnosis was 24 months (range, 3 to 93 months). Most of the 21 patients would not have been considered eligible for conventional allo-BMT in our institution due to age or poor performance status: 12 patients (57%) had a Karnofsky score below 50%. Two patients with acute myeloid leukemia (AML) had previously had a solid tumor. Prior to allo-BMT, two patients had received an autologous peripheral blood stem cells transplant in the course of their disease (one non-Hodgkin's lymphoma (NHL) and one chronic myeloid leukemia (CML)). Twelve patients (57%) were refractory to salvage chemotherapy (5 ST, 1 ALL, 3 NHL, 1 HD and 2 CML) and five high-risk patients (24%) in first or second complete remission (five AML). Two MDS (9.5%) were not previously treated and two CML (9.5%) were in chronic phase with no cytogenetic response to interferon. All patients were treated as inpatients in private rooms.
The conditioning regimen was slightly adapted from the schedule reported by Slavin et al,10 with fludarabine (Fludara; Schering, Lys-Lez Lannoy, France) 30 mg/m2 for 6 consecutive days (administered intravenously over 30 min from day −10 to −5), oral busulfan 4 mg/kg/day for 2 consecutive days (days −6 to −5) and anti-T-lymphocyte globulin (Thymoglobuline; Imtix-Pasteur Merieux, Lyon, France) 2.5 mg/kg/day for 4 consecutive days (administered intravenously over 6 to 8 h from day −4 to −1). Supportive care followed our practice for many years, and included antibacterial prophylaxis with intravenous vancomycin at 2 g daily starting at day −2 (vancomycin was stopped as soon as the absolute neutrophil count (ANC) exceeded 0.5 × 109/l) and oral gut decontamination with colimicin and neomycin. Pneumocystis carinii prophylaxis included trimethoprim/sulfamethoxazole (10 mg/kg/day trimeth- oprim) administered pre-transplantation (days −10 to −1) and as soon as the ANC exceeded 0.5 × 109/l (7 mg/kg/day) twice weekly. Prophylaxis against herpes simplex virus included intravenous acyclovir 250 mg × 3/day starting on day +2 to day +9. Antifungal prophylaxis included amphotericin B 10 mg total dose per day starting on day +1 during initial hospitalization and three times per week for 3 months in the out-patient clinic. Broad-spectrum antibiotics were begun for a fever greater than 38.5°C or clinical signs of infection.
Patients received filgrastim (Neupogen; Amgen, Neuilly-sur-Seine, France) at 5 μg/kg/day from day +1 post-transplantation until achievement of an ANC greater than 0.5 × 109/l. Hemoglobin was maintained at a level of 7 g/dl and the platelet count was maintained at 10 × 109/l with leukocyte-depleted and irradiated blood products. All patients received heparin at a dose of 1 mg/kg/day from day −10 until ANC reached 0.5 × 109/l, to prevent veno-occlusive disease (VOD).
GVHD prophylaxis consisted of cyclosporin A (CsA) at a dose of 3 mg/kg/day by continuous intravenous infusion changed to oral at day +21, or as soon as tolerated. CsA doses were adapted to serum levels and renal function.
Patients were assessed systematically twice per week for CMV infection by antigenemia assay (CINAkit; Argene Biosoft, Varilhes, France). This method uses a monoclonal antibody pool which recognizes the lower matrix structural phosphoprotein pp65; in a positive specimen, results are reported as number of antigen positive cells and the patient was considered positive when at least two infected cells out of 2 × 105 leukocytes were present, when pre-emptive therapy, ganciclovir, was commenced. All donor–recipient pairs were CMV seropositive prior to transplantation.
All donors were HLA-compatible siblings. Eleven were men and 10 were women. Eleven donor–recipient pairs were sex-mismatched. Chimerism status was assessed regularly by FISH in the sex-mismatched pairs, and VNTR in all other pairs. We chose to collect bone marrow rather than peripheral blood stem cells due to the increased rate of chronic GVHD associated with PBSC.1112 The median number of CD34+ cells infused was 2.3 × 106/kg (range, 1.1 to 3.3 × 106/kg).
The conditioning regimen was well tolerated except for the first day of ATG when most patients experienced mild fever and hypotension. Twenty patients survived beyond day 30, but one patient (UPN 972) who had refractory HD died 7 days after receiving her donor cells. She developed progressive pneumonia and died of multi-organ failure. Her death could not be attributed to conditioning regimen toxicity. All patients maintained a good oral intake throughout the procedure. Only one patient (UPN 951) developed moderate VOD, and no other patients showed evidence of hepatic abnormalities.
Except for the patient who died on day +7, all patients achieved an ANC greater than 0.5 × 109/l at a median of 13 days post-transplantation (range, 9 to 21 days). Platelet counts of 20.0 × 109/l or greater without transfusions were reached at a median of 19 days (range, 13 to 130 days). Median initial hospitalization duration was 36 days (range, 15 to 76 days).
Acute GVHD (one grade 1 and five grade 2) involving skin or gut occurred in six patients (28%). One patient (UPN 988) developed grade 3 cutaneous and hepatic acute GVHD which responded to steroid therapy alone. Acute GVHD occurred at a median of 45 days (range, 15 to 65 days). UPN 982, with no spontaneous GVHD and with rapidly developing leukemia, received a donor leukocyte infusion (DLI of 1 × 107 CD3+ cells/kg). He developed grade 3 acute GVHD 26 days later (day 72 post-transplantation).
The different events are summarized in Table 2. In brief, 65% of the patients (n = 13 out of 20 evaluable patients) developed early cytomegalovirus (CMV 1) blood infection (before day 45) at a median time of 31 days post-transplantation (range, 20 to 43 days). Interestingly, all these patients developed CMV infection prior to any acute GVHD, with no clinical signs or symptoms attributable to CMV infection. Five developed acute GVHD after the onset of CMV 1 infection. Eight patients with CMV 1 infection never had acute GVHD. Using the antigenemia assay, there was a median of four infected leukocytes (range, 2 to 30 leukocytes). All patients were treated with ganciclovir (Cymevan; Roche, Neuilly-sur-Seine, France) at a standard dose of 5 mg/kg twice daily for 14 days. None of the patients received maintenance therapy with ganciclovir. For all patients, antigenemia was cleared at a median time of 7 days after the onset of treatment. Five patients among these 13 developed a new CMV blood infection (CMV 2) at a median of 37 days following CMV 1 (range, 28 to 68 days) and 23 days after discontinuation of antiviral treatment (range, 14 to 54 days). Except for one patient (UPN 953), all patients with CMV2 infection responded again to ganciclovir therapy. UPN 953 required foscarnet therapy due to persistence of CMV antigenemia after 7 days of ganciclovir (Foscavir; Astra France, Rueil Malmaison, France 180 mg/kg/day for 14 days) at CMV 2 infection. Foscarnet was rapidly successful in this patient.
During the period of neutropenia, patients in our series did not encounter bacterial infections. Nevertheless, seven patients (33%) developed late bacterial infections: median time for onset of bacterial infections was 124 days (range, 66 to 172 days). Blood cultures from these patients revealed six gram-negative bacteria (two Pseudomonas aeruginosa, one Pseudomonas putida, one Xanthomonas maltophilia, one Escherichia coli, one Enterobacter cloacae) and one gram-positive bacteria (Staphylococcus aureus). These seven patients had recovered full neutrophil counts at the onset of septicemia. At the time of these bacterial infections, patients were not receiving prophylactic antibiotics, but still had tunnelled lines in situ. However, these could not be formally considered as the source of infection. All seven patients responded rapidly to appropriate antimicrobial therapy. At follow-up, we encountered no documented fungal infections.
At time of follow-up, all evaluable patients were in full donor chimerism before day 60 post allo-BMT.
Response and survival
As of 4 November 1999, median follow-up was 188 days (range, 62 to 409 days). Thirteen patients remain alive, 11 of whom are disease-free by all measurable criteria, with good quality of life; the two other surviving patients have active disease. Apart from the patient who died 7 days post transplantation of multi-organ failure, six deaths were attributed to relapse or progressive disease, and one patient (UPN 984) died while in complete remission, of gastrointestinal hemorrhage related to lymphoproliferative infiltrates in the gut. Unfortunately, no adequate specimens were collected to document EBV infection. Responses and outcome for each patient are shown in Table 3.
Data from Slavin et al10 and from the MD Anderson group9 reported the feasibility of nonmyeloablative conditioning regimens in patients with hematological malignancies. The rationale for such strategies is to induce efficient and potent immunotherapy mediated by allogeneic lymphocytes and to offer patients a significant improvement in terms of quality of life, cost-effectiveness and disease-free survival. Our preliminary data are in accordance with those previously published for low-toxicity regimens: no grade 3 or 4 toxicity was observed. Furthermore, engraftment of donor cells and hematologic recovery in our cohort seems to be similar to larger series using conventional myeloablative conditioning regimens. Nevertheless, we were surprised in our series by the high rate of CMV infections occurring in patients with no or little GVHD. This differs from our experience in patients treated with standard conditioning regimens.
CMV infection is the most common viral infection and a major cause of morbidity and mortality after allo-BMT.13 Among our 20 patients who survived beyond day 30 post transplantation, we observed an incidence of 65% of early CMV infection occurring a median of 31 days following transplantation (range, 20 to 43 days). The median number of positive cells (CMV antigenemia (Ag) at diagnosis was 4 (range, 2 to 30 cells). A second episode of blood CMV infection was seen in 38% (n = 5) of patients with early infection at a median of 72 days after allo-BMT (range, 49 to 93 days). Median number of positive cells was three (range, 2–88 cells) at this time.
In a large study by Meyers et al,13 evidence of CMV infection was present in 51% of 545 patients studied between 1979 and 1982. CMV infection occurred among 36% of seronegative patients and 69% of seropositive patients. In the same study, it was found that the occurrence of acute GVHD significantly increased risk of CMV infection (average relative rate, 1.8). This close relationship between acute GVHD and CMV infection was not apparent in our series, where all CMV infections developed prior to any acute GVHD. When pre-emptive antiviral treatment with ganciclovir ends, the incidence of CMV recurrence could be expected to be as high as 16%.14 Nevertheless, in our series, pre-emptive therapy did not seem to reduce the incidence of CMV recurrence (38%) as could be expected. Our follow-up is not long enough to evaluate the impact of this high rate of CMV recurrence on the occurrence of CMV disease and survival.
In the pre-antiviral era, the median time of onset of CMV disease was 50 to 60 days after allo-BMT,15 but with pre-emptive therapy the disease occurred most often late in the first year.16 Furthermore, CMV Ag proved to be a sensitive, specific and rapid technique for early diagnosis of CMV infection after allo-BMT.14 This technique seems to reflect viral load in the systemic circulation, and high numbers of CMV antigen-positive cells are associated with considerable transplant-related mortality.17 In comparison to classical series, the Ag results (median, four cells) observed in our cohort do not seem to correlate with CMV-related mortality.
Relative to viral infections, bacterial infections predominate in the early recovery phase post allo-BMT and bacterial pathogens account for more than 90% of the first infections during neutropenia. Our data suggest a different profile since no bacterial infections were documented during neutropenia, while we observed seven late severe septicemia (35%) episodes occurring at a median of 124 days (range, 66 to 172 days), compared to the 65% of early CMV infections occurring at a median of 30 days following transplantation. All these bacterial infections occurred when patients had full neutrophil recovery.
Our results suggest that nonmyeloablative-conditioning regimens can effectively decrease early severe bacterial infections: this can be explained by the short duration of neutropenia following such regimens. On the other hand, these regimens seem to be associated with frequent CMV infection and recurrence, requiring multiple and prolonged hospitalizations. Moreover, combined in vivo/ex vivo T cell depletion influences the occurrence of active CMV infection and disease,18 and early active CMV infection in T cell-depleted patients is significant when compared with published data.1920 Conditioning regimens containing ATG result in depletion of lymphocytes. Our data suggest such regimens play a major role in the onset of early CMV infection. Fludarabine has been demonstrated to inhibit the mixed lymphocyte reaction in vitro. Added to ATG, the combination may lead to a prolonged state of profound immune depression. Further studies with a larger number of patients are necessary to determine if these results can be confirmed. We consider it important to investigate other nonmyeloablative conditioning regimens and to assess the role of ATG in order to determine whether other agents or methods would provide optimal short- and long-term immune recovery allowing an acceptable infection risk.
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Mohty, M., Faucher, C., Vey, N. et al. High rate of secondary viral and bacterial infections in patients undergoing allogeneic bone marrow mini-transplantation. Bone Marrow Transplant 26, 251–255 (2000). https://doi.org/10.1038/sj.bmt.1702509
- allogeneic bone marrow transplantation
- nonmyeloablative conditioning
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