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1 (2004) 33, & 2004 Nature Publishing Group All rights reserved /04 $ Viral infections Influence of cytomegalovirus (CMV) sero-positivity on CMV infection, lymphocyte recovery and non-cmv infections following T-cell-depleted allogeneic stem cell transplantation: a comparison between two T-cell depletion regimens S Chakrabarti 1,2, DW Milligan 2, J Brown 1, H Osman 2, IB Vipond 1, DH Pamphilon 1 and DI Marks 1 1 Adult Bone Marrow Transplant Unit and Public Health Laboratories, Bristol Royal Hospital for Sick Children, Bristol, UK; and 2 Departments of Haematology and Virology, Birmingham Heartlands Hospital, Birmingham, UK Summary: We compared the incidence and outcome of preemptively treated cytomegalovirus (CMV) infection, lymphocyte recovery and non-cmv infections between two different TCD modalities, one employing CD34 þ selection and T- cell add-back (TCAB), preceded by Campath-1H in vivo (CD34 þ /TCAB group, n ¼ 29), and the other using grafts incubated with Campath-1H in vitro (Campath-1H in vitro group, n ¼ 32). The probabilities of CMV reactivation and recurrence were 67 and 83.6% in the CD34 þ /TCAB group and 42.9 and 20% in the Campath-1H group (P ¼ 0.07 and 0.02). Donor seropositivity reduced CMV reactivation in the Campath-1H group, but not in the CD34 þ /TCAB group. The durations of positive PCR/antigenemia positivity and antiviral therapy were also significantly longer in the CD34 þ /TCAB group. However, only two patients developed CMV disease in each group. The mean absolute lymphocyte counts ( 10 9 /l) at 30 days (0.27 vs 0.4, P ¼ 0.03) and 100 days (0.77 vs 1.4, P ¼ 0.01) were significantly lower in the CD34 þ /TCAB group along with a higher incidence of non-cmv infections in CMV at-risk patients, but not in the CMV low-risk group. These findings suggest that the modality of TCD should be tailored according to the CMV risk status, and CMV sero-positive patients should receive a less extensively T- cell-depleted graft and a CMV sero-positive graft if possible. (2004) 33, doi: /sj.bmt Published online 1 December 2003 Keywords: T-cell depletion; cytomegalovirus; Campath- 1H; CD34 þ selection Correspondence: Dr S Chakrabarti, Department of Haematology, Queen Elizabeth Hospital, Birmingham B15 2TH, UK; suparno@doctors.org.uk Received 12 June 2003; accepted 22 August 2003; published online 1 December 2003 Cytomegalovirus (CMV) infection continues to be a major concern in allogeneic stem cell transplant recipients, despite the development of effective preventative treatment strategies. This is because of two reasons. Firstly, a shift in the onset of CMV disease to late post transplant period due to the early use of prophylactic antiviral therapy 1 3 and failure of antiviral therapy to improve the outcome of progressive CMV disease. 4,5 A better understanding of the kinetics of CMV-specific T-cell reconstitution has underlined the importance of CMV-specific immunity in preventing CMV reactivation and its progression. 1,6,7 Secondly, although the incidence of CMV disease has been reduced by prophylactic or preemptive therapy, CMV sero-positivity seems to affect adversely the overall transplant outcome independent of CMV reactivation and treatment. This is particularly relevant for T-cell-depleted allogeneic transplantation T-cell depletion (TCD) of the graft, which is the most effective method for prevention of GVHD, has been reported to be an important risk factor for CMV reactivation and CMV disease This is most probably due to a delayed recovery of CMV-specific T cells. However, the incidences of CMV infection and disease have varied depending on the method of TCD. We analysed the impact of two different regimens of TCD on the incidence and outcome of preemptively treated CMV infection, lymphocyte recovery and non-cmv infections in relation to recipient and donor sero-positivity. Patients and methods We evaluated 29 allograft recipients who received a CD34 þ selected graft with a T-cell add-back (CD34 þ / TCAB group) in the Adult BMT unit at Bristol Royal Hospital for Sick Children 17 and 32 patients who received Campath-1H-treated PBSC graft (Campath-1H in vitro group) at Birmingham Heartlands Hospital. 18 All were transplanted for haematological malignancies. Conditioning treatment Conditioning regimens consisted of cyclophosphamide 60 mg/kg for 2 days or etoposide 60 mg/kg single dose

2 198 and fractionated total body irradiation (TBI) at 1440 cgy in eight doses. Patients not receiving TBI received cyclophosphamide and busulphan 16 mg/kg over 4 days in four daily divided doses. All patients in the CD34 þ / TCAB group received intravenous Campath-1H at a dose of 0.2 mg/kg/day from day 9 to day 5 as an antirejection measure. T-cell depletion The detailed methods of TCD have been described previously. In brief, for the Campath-1H in vitro group, 20 mg (n ¼ 22) or 10 mg (n ¼ 10) Campath-1H was added to the final product and incubated at 251C in an agitator for 30 min before infusion. In the CD34 þ /TCAB group, selection of CD34 þ cells was carried out by the Clinimacs procedure (Miltenyi Biotech). A TCAB was prepared from a sample of the initial bone marrow or PBSC harvest or the negative fraction residue of the CD34 selection procedure. The calculated cell volume containing the desired CD3 þ T-cell dose was either added directly to the selected CD34 þ cells before infusion or infused alongside the selected CD34 þ cells. GVHD prophylaxis Patients received intravenous cyclosporine A at 3 mg/kg in two divided doses from day 1 until they were able to tolerate oral cyclosporine. In the absence of GVHD, oral cyclosporine was generally tapered between day þ 100 and 180 post transplantation. However, the duration of immunosuppression was modified according to the disease status at the time of transplant, with patients at a higher risk of relapse having early discontinuation of their immunosuppression. The dose of cyclosporine A was titrated to maintain plasma levels between 125 and 225 ng/l. CMV surveillance Pretransplant serum samples from all patients and donors were tested for serological evidence of past infection with CMV by an ELISA assay. CMV sero-positive recipients (R) or those receiving grafts from CMV sero-positive donors (D) were defined as being at risk for CMV disease. Among the patients at risk of CMV disease, CMV seropositive recipients receiving graft from a CMV sero-positive (R þ /D þ ) or sero-negative donor (R þ /D ) were defined as high risk (CMV-HR) and CMV sero-negative patients receiving transplant from a sero-positive donor (R /D þ ) were termed as intermediate risk (CMV-IR). CMV seronegative patients receiving grafts from CMV sero-negative donors were considered as low risk for CMV disease (CMV-LR). CMV-LR patients received CMV-negative blood products, while CMV-HR and CMV-IR patients were given unscreened blood products. In the CD34 þ /TCAB group, all CMV at-risk patients received intravenous acyclovir at 500 mg/m 2 thrice daily from day 1 to day þ 35, followed by oral acyclovir at 400 mg t.i.d. until day 100. The Campath-1H in vitro group did not receive any CMV prophylaxis. The CMV at-risk patients were screened every week for CMV reactivation by a nested polymerase chain reaction (PCR)-based assay in the CD34 þ /TCAB group and by a non-nested assay (Roche, Amplicor) in the Campath-1H in vitro group as described previously. 19 The patients were screened every week from transplant to 100 days post transplant. Generally, those patients who had CMV infection before 100 days or had GVHD were screened for 180 days or beyond. Patients with persistent infection or disease and/or GVHD were screened beyond this period at the discretion of the individual centre. Preemptive antiviral therapy The patients received antiviral therapy if two consecutive qualitative PCR assays were positive. Ganciclovir 5 mg/kg b.d. or foscarnet mg/kg in three divided doses was employed as the first-line therapy. Dose adjustments were made for impairment of renal function. One patient in the Campath-1H in vitro group received cidofovir as the firstline therapy as part of a local study protocol. 20 Either of these drugs or cidofovir at 5 mg/kg weekly for 2 weeks and fortnightly thereafter was used for secondary preemptive therapy. Treatment was stopped if two consecutive PCR assays were negative. Treatment was switched from one drug to the other if there was clinically significant nonoverlapping toxicity. This was also instituted if there was persistent infection after 2 3 weeks treatment or if the infection recurred within 2 weeks after obtaining two PCR-negative results. Treatment of CMV disease CMV disease was treated with either ganciclovir 5 mg/kg b.d. or foscarnet 180 mg/kg in three divided doses or a combination of both. Intravenous immunoglobulin was also added to the antiviral regimen. Supportive care All patients were nursed in single rooms with highefficiency particulate air (HEPA) filters. Antimicrobial prophylaxis consisted of oral itraconazole suspension, ciprofloxacin and i.v. and then oral acyclovir (see above) from the beginning of conditioning treatment until 100 days post transplant for the CD34 þ /TCAB group and fluconazole, oral acyclovir and ciprofloxacin until engraftment for the Campath-1H in vitro group. Oral cotrimoxazole was initiated when the neutrophil count was greater than /l. Definitions and statistical methods CMV infection was defined as PCR positivity on two consecutive assays, without clinical signs or symptoms. Recurrence of CMV infection was defined as CMV infection occurring after two negative PCR assays following treatment of the initial episode of infection. CMV

3 disease was defined as demonstration of CMV by culture or early antigen detection tests in bronchoalveolar lavage specimen in the presence of recent respiratory signs and symptoms with pulmonary infiltrates on radiology, or histological evidence of CMV infection from other visceral sites. CMV infection and disease were defined as late if they occurred after 100 days. CMV-related death was defined as definite if death occurred within 6 weeks of the diagnosis of CMV disease, unrelated to the underlying disease or another aetiology. Short of tissue diagnosis, death in a patient with a persistently positive PCR with no other obvious cause and presence of clinical signs or symptoms, which could be explained by CMV disease, was considered possible CMV-related death. Disease status and engraftment were defined as in previous studies. 17,18 Acute and chronic GVHD were defined according to standard criteria. 21,22 Acute GVHD was evaluable in all patients beyond day 0 and chronic GVHD in survivors beyond 100 days. Nonrelapse mortality was defined as death from any cause in patients in remission. Univariate P-values and odds ratios were calculated from 2 2 contingency tables using Fisher s exact modification as necessary. Continuous variables were compared using Mann Whitney s nonparametric method. Cumulative probabilities of CMV reactivation, CMV recurrence, engraftment and nonrelapse mortality were analysed by censoring for competing risk factors by the Kaplan Meier method, and the difference between the groups was compared using log rank w 2 test. Table 1 Characteristics of T-cell-depleted transplant recipients Campath-1H in vitro (n ¼ 32) Age (years), median (range) 36 (21 57) 40 (22 60) Gender (male/female) 18/14 20/9 Disease AML/ALL 12/9 10/5 CML/CLL 10/0 7/1 MM/NHL 1/0 4/2 High-risk disease Donor type Related Unrelated 8 13 CD34+ cells/kg in the graft ( 10 6 ) Conditioning Cyclo/TBI VP16/TBI 9 5 Bu cyclo 1 2 Days to neutrophil /l, median (95% CI) * Days to neutrophil /l, 14 ( ) 13 ( ) median (95% CI) CMV at-risk Acute GVHD grade Chronic extensive GVHD 4 7 Non-CMV infections 8 12 AML, acute myeloid leukaemia; ALL, acute lymphocytic leukaemia; MDS, myelodysplastic syndrome; CML, chronic myeloid leukaemia; CLL, chronic lymphocytic leukaemia; NHL, non-hodgkin s disease; TBI, total body irradiation; bu, busulphan; cyclo, cyclophosphamide. *The difference between the two groups is significant at Po Results Patient characteristics Detailed characteristics of this patient cohort are shown in Tables 1 and 2. In brief, 32 patients received Campath-1H in vitro treated graft and 29 received CD34 þ /TCAB graft. The age distribution was similar and so were the underlying diagnoses. In all, 13 (44.8%) patients in the CD34 þ /TCAB group and eight (25%) in the Campath-1H in vitro group received an unrelated donor (UD) graft. All patients in the Campath-1H in vitro group and 23/29 patients in the CD34 þ /TCAB group received a PBSC graft, the bone marrow graft recipients in the latter being from UD only. The CD34 þ cell content in the graft was similar in the two groups. In the CD34 þ /TCAB group, TCAB to the graft was a median of /kg. Except for one patient where a higher TCAB was planned ( /kg), the majority received a TCAB of /kg (range /kg). The targeted TCAB dose was achieved in all the patients. The CD3 þ T-cell dose infused in the Campath-1H in vitro group was a median of /kg (22 454) following incubation with Campath-1H with less than one log depletion of T cells in vitro. There was no difference in the graft composition between the CMV risk groups in either cohort. Neutrophil engraftment was prompt in both groups, although platelet engraftment was delayed in the CD34 þ / Table 2 Characteristics of T-cell-depleted transplant recipients at risk of CMV infection Campath-1H in vitro (n ¼ 14) (n ¼ 20) Age (years), median (range) 39 (25 57) 41 (22 60) Gender (male/female) 9/5 12/8 Disease AML/ALL 3/5 6/3 CML/CLL 6/0 5/1 MM/NHL 0/0 4/1 High-risk disease 3 9 Donor type* Related Unrelated 2 8 Days to platelet /l, 11 ( ) 29 ( ) median (95% CI)* Days to neutrophil / 14 ( ) 13 ( ) l, median (95% CI) Acute GVHD grade Chronic extensive GVHD 2 5 Non-CMV infections* 1 8 Bacterial 0 1 Viral 1 5 Fungal 0 4 AML, acute myeloid leukaemia; ALL, acute lymphocytic leukaemia; MDS, myelodysplastic syndrome; CML, chronic myeloid leukaemia; CLL, chronic lymphocytic leukaemia; NHL, non-hodgkin s disease. *The difference between the two groups is significant at Po0.05.

4 200 TCAB group. However, this was mostly in UD bone marrow graft recipients. There was no difference in the incidence of acute or chronic GVHD between the two groups (Tables 1 and 2). CMV infection Overall, 13/29 patients in the CD34 þ /TCAB group developed a CMV infection compared to 6/32 patients in the Campath-1H in vitro group (P ¼ 0.05). The median time to first CMV infection was 19 days (range 1 55) post transplant in the CD34 þ /TCAB group compared to 52 days (range 9 75) (P ¼ 0.02) in the Campath-1H in vitro group. There was no impact of age, donor status, underlying disease, donor type, CD34 þ cell dose or GVHD on the incidence or time to CMV infection. Within the CD34 þ /TCAB group, TCAB did not influence CMV reactivation. Recipient donor sero-status (Table 3) The probability of developing a CMV infection among CMV-HR and CMV-IR patients was 67% (95% CI 47 87) in the CD34 þ /TCAB group and 42.9% (95% CI ) in the Campath-1H group (P ¼ 0.07; Figure 1). However, CMV infection was restricted entirely to the CMV-HR group. None of the CMV-IR (R /D þ ) or CMV-LR patients developed CMV infection in either group. The probability of reactivating CMV in CMV-HR patients was 92% (13/15, 95% CI ) in the CD34 þ / TCAB group, compared to 50% (6/12, 95% CI 23 77) in the Campath-1H in vitro group (P ¼ 0.002; Figure 1a). This was entirely due to the effect of donor sero-positivity in the Campath-1H in vitro group (Table 3). While almost all Campath-1H in vitro (n ¼ 32) CD34+/ TCAB P-value R+/D No. of patients 2 9 CMV infection CMV recurrence CMV disease R+/D+ No. of patients 10 6 CMV infection CMV recurrence CMV disease CMV infection, recurrence and disease according to sero- Table 3 status R /D+ No. of patients 2 5 CMV infection/recurrence/disease 0/0/0 0/0/0 1.0 R /D No. of patients 18 9 CMV infection/recurrence/disease 0/0/0 0/0/0 1.0 R, recipient; D, donor. R þ /D patients reactivated in both groups, only 4/10 R þ /D þ patients in the Campath-1H in vitro group reactivated, compared to 5/6 patients in the CD34 þ / TCAB group (P ¼ 0.08). This difference was not due to more patients receiving UD grafts in the CD34 þ /TCAB group as 2/2 UD transplant recipients reactivated CMV in the Campath-1H in vitro group, compared to 5/8 in the CD34 þ /TCAB group (P ¼ 1.0). Recurrence of CMV infection Among evaluable patients, the probability of recurrence of CMV infection was 83.6% (10/12 patients, 95% CI ) in CD34 þ /TCAB group and 20% (1/5 patients, 95% CI 0 54) in the Campath-1H in vitro group (P ¼ 0.02; Figure 1b). Of the CMV-HR patients evaluable beyond 100 days, 9/13 patients in the CD34 þ /TCAB group (probability 80%, 95% CI ) experienced a late CMV reactivation compared to 1/11 (probability 9.1%, 95% CI 0 27) in the Campath-1H in vitro group (P ¼ 0.001). Recipient or donor sero-positivity had no impact on recurrence of CMV infection in the CD34 þ / a Probability of CMV reactivation b Probability of CMV reactivation P = Campath-1H in-vitro Days post-transplant P = 0.02 Campath-1H in-vitro Days post-transplant Figure 1 Probabilities of CMV infection (a) and recurrence (b) among CMV high-risk patients.

5 Table 4 Duration of CMV infection and antiviral therapy Campath-1H in vitro (n ¼ 32) Mean7s.d. (days) Mean7s.d. (days) P-value Duration of infection First episode Recurrence Duration of antiviral therapy First episode Recurrence s.d., standard deviation. Absolute lymphocyte count recovery and CMV sero- Table 5 status Campath-1H in vitro (n ¼ 32) Mean7s.d. (days) Mean7s.d. (days) P-value CMV at-risk ALC at 30 days ALC at 100 days CMV low-risk ALC at 30 days ALC at 100 days s.d., standard deviation. 201 TCAB group (Table 3). However, recurrence of CMV infection tended to be less frequent in the Campath-1H in vitro among the R þ /D þ subgroup (1/4 vs 3/5 in the CD34 þ /TCAB group, P ¼ 0.2). There was no impact of patient age, UD status or disease status or GVHD on the recurrence of CMV infection. Preemptive therapy (Table 4) The CD34 þ /TCAB group experienced a longer persistence of CMV infection during the first episode of reactivation and during recurrence as well. This resulted in a significantly longer duration of antiviral therapy in this group. Seven patients in the CD34 þ /TCAB group required a change in the first-line therapy and six of them received cidofovir during CMV recurrence. Only one patient in the Campath-1H in vitro group required a change in the firstline therapy. CMV disease and death In the CD34 þ /TCAB group, one UD graft recipient with chronic GVHD developed CMV pneumonia while on steroids and succumbed to it. Another patient, also a UD transplant recipient, developed fatal pneumonitis early after transplant, and CMV was detected from respiratory specimens. However, the diagnosis of CMV pneumonia was not confirmed in this patient. In the Campath-1H in vitro group, both patients who developed CMV disease failed to comply with CMV monitoring. One of them was a UD graft recipient who presented with CMV pneumonitis after failing to adhere to weekly monitoring and succumbed to it. Another was a sibling graft recipient, who presented with CMV colitis and improved following treatment with GCV. ALC recovery in the CMV low-risk cohort was similar in the two groups. There was no difference in the ALC recovery between the UD and related donor graft recipients in either group. Although the overall incidences of symptomatic non- CMV viral and fungal infections were similar between the two groups, there was a significantly higher incidence of both viral and fungal infections in the CMV at-risk patients in the CD34 þ /TCAB group (Table 2), irrespective of the donor status. Two of the eight patients developed infections with more than one pathogen. Five patients developed viral infections (adeno, three; influenza A, one; rotavirus, one), one patient developed post-engraftment bacterial infection and four patients developed fungal pneumonia, diagnosed on clinical and radiological grounds. In the CMV at-risk cohort of the CD34 þ /TCAB group, all but one patient with non-cmv infections experienced CMV reactivation and received antiviral therapy. However, the onset of the non-cmv infections did not correlate with the onset or duration of antiviral therapy. The CMV at-risk patients in the Campath-1H in vitro group did not experience any postengraftment bacterial or fungal infections. Only one patient developed symptomatic herpes simplex infection. Nonrelapse mortality Nonrelapse mortality (NRM) tended to be slightly but not significantly higher in the CD34 þ /TCAB group for the CMV at-risk patients with a probability of 40% (6/20 died, 95% CI 15 65), compared to 22% (3/14 died, 95% CI 2 44) in the Campath-1H in vitro group (P ¼ 0.2), but not in the CMV low-risk patients (2/9 died, probability 25.9%, 95% CI 0 51 in the CD34 þ /TCAB group vs 3/18 died, probability 16.7%, 95% CI 0 36). The causes of nonrelapse death are detailed in Table 6. Immune reconstitution and non-cmv infections The absolute lymphocyte count (ALC) tended to be higher in the Campath-1H in vitro group compared to the CD34 þ /TCAB group at both 30 days ( /mm 3 vs /mm 3, P ¼ 0.07) and 100 days ( /mm 3 vs /mm 3, P ¼ 0.08). This difference was entirely due to the significantly delayed ALC recovery in the CMV atrisk patients in the CD34 þ /TCAB group (Table 5). The Discussion The incidence of both CMV reactivation and disease is reported to be increased following TCD transplantation Protection against CMV is related to the speed of recovery of CMV-specific T-cell immunity, both CD4 þ and CD8 þ subsets. 1,6,7 This is generally delayed in TCD graft recipients in comparison to those receiving unmanipulated grafts and is

6 202 Table 6 status Causes of nonrelapse mortality according to CMV risk Campath-1H in vitro (n ¼ 32) CMV at-risk (n ¼ 14) CMV low-risk (n ¼ 18) CMV at-risk (n ¼ 20) CMV low-risk (n ¼ 9) Infection CMV Other virus Bacterial Fungal TTP Pneumonitis Others the most likely cause for the increase in CMV infections. However, the pattern of CMV infection following TCD is not predictable and is likely to be dictated by the method and extent of TCD. We observed a distinct difference in the incidence and pattern of CMV infection between patients receiving Campath-1H-treated grafts and those receiving CD34 þ / TCAB grafts. The overall incidence of CMV infection was 67% in the CD34 þ /TCAB group and it was detected earlier during the post transplant period at a median of 19 days. This pattern of early CMV reactivation has been reported following TCD allografts by other investigators It is worth noting that high-dose acyclovir did not confer any protection against early CMV infection in the CD34 þ /TCAB group. On the other hand, CMV infections in the Campath-1H in vitro group were similar to those reported following unmanipulated graft transplants, with an incidence of 42.9% and onset at a median of 52 days. Recurrence of CMV infection and late CMV infection were also higher in the CD34 þ /TCAB group with an incidence of over 80%, compared to less than 10% in the Campath- 1H in vitro group. The duration of infection and antiviral therapy was significantly prolonged in the CD34 þ /TCAB group. The differences in the pattern of CMV infection between the two groups might have been partly due to the difference in the sensitivity of PCR assays, but both the assays were equally sensitive detecting 50 copies of CMV-DNA. This is probably more suggestive of an impact of the modality of TCD on the immune reconstitution. 23 The apparent dose of T cells infused in the Campath-1H in vitro group was much higher, but as reported previously most of the T-cell lysis due to Campath-1H is likely to occur by antibodydependent cell cytotoxicity in vivo. Hence it is difficult to ascertain the exact degree of TCD in these groups. Thus the best surrogate marker for the extent of TCD may be the speed of lymphocyte recovery. Delay in the lymphocyte recovery was documented in the CD34 þ /TCAB group both at 30 and 100 days. As both groups had a low incidence of acute and chronic GVHD and received similar post transplant immunosuppression, the delay in lymphocyte recovery could be attributed almost entirely to the TCD modality. It was interesting that the difference in immune reconstitution was contributed by a delayed recovery in the CMV at-risk group as the ALC was similar between the two TCD groups in the CMV low-risk group. This was further evident by the fact that a higher incidence of non-cmv infections was also noted in this cohort. The other factor contributing to the impaired immune reconstitution could have been a greater number of UD graft recipients 24 in the CD34 þ /TCAB group, which was not apparent on statistical analysis due to small sample size. This pattern of CMV infection and the immune reconstitution noted in the CD34 þ /TCAB group was probably due to the additional use of Campath-1H in vivo, which at least in the CMV at-risk group had adversely affected the immune recovery. Campath-1H was used in the conditioning protocol as an antirejection measure, but might have caused further TCD of the graft and delayed the early recovery due to its prolonged persistence in the circulation when used at a higher dose in vivo. 25 Campath- 1H in vivo used in a similar dosage schedule has been reported to have resulted in a high incidence of CMV infection and recurrence following nonmyeloablative conditioning. 19 In addition, the use of high-dose Campath-1H in vivo has been correlated with non-cmv viral infections In this study, the CMV at-risk group receiving CD34 þ /TCAB and Campath-1H in vivo was affected in terms of CMV infection, non-cmv infection, immune recovery and a nonsignificant trend towards higher NRM, irrespective of the donor status. In an earlier report we had demonstrated an impact of delayed early lymphocyte recovery on the survival in the CD34 þ /TCAB group. 17 This study further elucidates the possible negative impact of Campath-1H in vivo in the CMV at-risk group. A recent study 29 that employed a similar conditioning protocol with Campath-1H and TCD with CD34 þ selection, but without any TCAB, reported a much higher incidence of infection-related mortality of 63.6% and a significant delay in lymphocyte recovery irrespective of CMV sero-status. The use of TCAB with the graft in our protocol possibly had moderated the deleterious effect of Campath-1H in vivo on immune reconstitution at least in the CMV seronegative group. On the other hand, the lower dose of Campath-1H used in vitro along with the greater amount of T cells infused with the graft might have resulted in less extensive T-cell lysis in vivo, and the earlier clearance of the antibody might have hastened the immune recovery as well. The difference in the overall incidence of CMV infection between the two groups was influenced by the donor serostatus. CMV infection was equally high in the two TCD groups in R þ /D cohort, and none of the R /D þ or R /D patients experienced CMV infection. On the other hand, R þ /D þ patients in the Campath-1H group had less CMV reactivation than those in the CD34 þ /TCAB group. Thus, donor sero-positivity might have had a protective influence on CMV reactivation in CMV sero-positive patients in the Campath-1H group, but not in the CD34 þ /TCAB group. This probably highlights that the donor-derived CMV-specific immunity might reduce reactivation of the virus, if TCD is less extensive. Similar beneficial effect of donor sero-positivity has been reported

7 by other groups employing antibodies with narrow specificity such as anti-cd6 antibodies. 30,31 The other notable outcome of this study has been the effective control of CMV infection with a PCR-guided preemptive treatment approach as reported previously. 32,33 The incidence of CMV disease was similar among the two TCD groups, which suggests that even though the antiviral therapy was protracted and recurrent in the CD34 þ / TCAB group, this was reasonably effective in preventing CMV disease, despite a high incidence of CMV reactivation. On the other hand, a higher incidence of fungal and non-cmv viral infections was observed in this group, which has also been described previously. 34,35 Fungal infections in patients with CMV infection are not always related to GCV-associated neutropenia. It has been described in patients without CMV reactivation, implying a role of CMV sero-positivity and occurrence of other viral infections in the predisposition to fungal infections. The negative impact of CMV sero-positivity on the outcome of TCD transplantation has been described in previous studies. This problem has been largely reported in UD graft recipients, 9,10 but also after HLA-identical sibling transplantation, 8 independent of the method of CMV prevention. Interestingly enough, CMV reactivation or disease per se did not contribute to the adverse outcome in these studies. How then CMV sero-positivity of the recipient or donor influences the outcome is not entirely clear. A relationship between CMV sero-positivity and GVHD had been suggested in one study on HLA-identical sibling donors, where partial TCD was employed. 8 Otherwise, the detrimental effect of CMV sero-positivity was evident only if the graft is extensively T-cell depleted, particularly with in vivo T-cell antibodies like Campath-1 or anti-thymocyte globulin. 9,10 There could be two possible hypotheses for these findings. First, CMV infection might affect the immune reconstitution, which increases the propensity to develop other opportunistic infections, while progression of CMV infection is effectively checked by preemptive or prophylactic therapy aided by sensitive detection assays. Recently, direct infection of dendritic cells by human CMV has been shown to impair the antigen presentation by the affected dendritic cells, 36 suggesting a direct cause effect relationship between CMV and impaired immune response. Second, long-term use of GCV can predispose the recipient to fungal or bacterial infections and delay the reconstitution of CMV-specific immunity. 1,37 Extensively, TCD grafts fail to confer donor-derived CMVspecific immunity in the early post transplant period leading to protracted anti-cmv therapy to control the progression of CMV infection and this in turn might affect the later regeneration of CMV-specific immunity. The impact of CMV-specific immunity can extend beyond controlling CMV progression. An analysis of EBMT megafile demonstrated the favourable outcome of CMV sero-positive recipients if transplanted with sero-positive grafts following UD transplantation. 38 This impact of seropositive grafts was not simply related to reduction of CMV disease, but an overall reduction of transplant-related mortality. This could justify the case for tailoring the TCD regimen based on CMV risk status and the use of CMV sero-positive grafts for sero-positive recipients. Thus, this comparative study suggests that the modality and degree of TCD influences the pattern and outcome of CMV infections. Recipient and donor CMV sero-positivity is an important variable to be considered in the context of TCD. Campath-1H-treated grafts probably resulted in less extensive TCD, and donor-derived CMV-specific immunity might have had a positive impact on CMV infections following this modality of TCD. On the other hand, CMV at-risk patients receiving a CD34 þ selected graft with TCAB were probably adversely affected by the use of Campath-1H in vivo, irrespective of the donor status. These findings suggest that the modality of TCD should be tailored according to the CMV risk status and CMV seropositive patients should receive a less extensively T-celldepleted graft and a CMV sero-positive graft if possible, irrespective of the donor status. References 1 Li CR, Greenberg PD, Gilbert MJ et al. Recovery of HLArestricted cytomegalovirus (CMV)-specific T-cell responses after allogeneic bone marrow transplant: correlation with CMV disease and effect of ganciclovir prophylaxis. Blood 1994; 83: Nguyen Q, Champlin R, Giralt S et al. Late cytomegalovirus pneumonia in adult allogeneic blood and marrow recipients. Clin Infect Dis 1999; 28: Einsele H, Hebart H, Kauffmann-Schneider C et al. Risk factors for treatment failures in patients receiving PCR-based pre-emptive therapy for CMV infection. Bone Marrow Transplant 2000; 25: Ljungman P. Prevention and treatment of viral infections in stem cell transplant recipients. Br J Haematol 2002; 118: Ljungman P, Deliliers GL, Platzbecker U et al. Cidofovir for cytomegalovirus infection and disease in allogeneic stem cell transplant recipients. The Infectious Diseases Working Party of the European Group for Blood and Marrow Transplantation. Blood 2001; 97: Cwynarski K, Ainsworth J, Cobbold M et al. Direct visualization of cytomegalovirus-specific T-cell reconstitution after allogeneic stem cell transplantation. Blood 2001; 97: Einsele H, Roosnek E, Rufer N et al. Infusion of cytomegalovirus (CMV)-specific T cells for the treatment of CMV infection not responding to antiviral chemotherapy. Blood 2002; 99: Broers AE, van Der Holt R, van Essen JW et al. Increased transplant-related morbidity and mortality in CMV-seropositive patients despite highly effective prevention of CMV disease after allogeneic T-cell-depleted stem cell transplantation. Blood 2000; 95: Craddock C, Szydlo RM, Dazzi F et al. Cytomegalovirus seropositivity adversely influences outcome after T-depleted unrelated donor transplant in patients with chronic myeloid leukaemia: the case for tailored graft-versus-host disease prophylaxis. Br J Haematol 2000; 112: Kroger N, Zabelina T, Kruger W et al. Patient cytomegalovirus seropositivity with or without reactivation is the most important prognostic factor for survival and treatment-related mortality in stem cell transplantation from unrelated donors using pretransplant in vivo T-cell depletion with anti-thymocyte globulin. Br J Haematol 2001; 113:

8 Meyers JD, Flournoy N, Thomas ED. Risk factors for cytomegalovirus infection after human marrow transplantation. J Infect Dis 1986; 153: Przepiorka D, Ippoliti C, Panina A et al. Ganciclovir three times per week is not adequate to prevent cytomegalovirus reactivation after T cell-depleted marrow transplantation. Bone Marrow Transplant 1994; 13: Couriel D, Canosa J, Engler H et al. Early reactivation of cytomegalovirus and high risk of interstitial pneumonitis following T-depleted BMT for adults with hematological malignancies. Bone Marrow Transplant 1996; 18: Hertenstein B, Hampl W, Bunjes D et al. In vivo/ex vivo T cell depletion for GVHD prophylaxis influences onset and course of active cytomegalovirus infection and disease after BMT. Bone Marrow Transplant 1995; 15: Martino R, Rovira M, Carreras E et al. The AlloPBSCT and Infectious/Non-infectious Complications Subcommittes of the Grupo Espanol de Trasplante Hematopoyetico (GETH). Severe infections after allogeneic peripheral blood stem cell transplantation: a matched-pair comparison of unmanipulated and CD34+ cell-selected transplantation. Haematologica 2001; 86: Hebart H, Brugger W, Grigoleit U et al. Risk for cytomegalovirus disease in patients receiving polymerase chain reactionbased pre-emptive antiviral therapy after allogeneic stem cell transplantation depends on transplantation modality. Blood 2001; 97: Chakrabarti S, Brown J, Guttridge M et al. Early lymphocyte recovery is an important determinant of outcome following allogeneic transplantation with CD34+ selected graft and limited T cell addback. Bone Marrow Transplant 2003; 32: Chakrabarti S, MacDonald D, Hale G et al. T cell depletion with Campath-1H in the bag for matched related allogeneic peripheral blood stem cell transplantation is associated with reduced graft-versus-host disease, rapid immune constitution and improved survival. Br J Haematol 2003; 121: Chakrabarti S, Mackinnon S, Chopra R et al. High incidence of cytomegalovirus infection after nonmyeloablative stem cell transplantation: potential role of Campath-1H in delaying immune reconstitution. Blood 2002; 99: Chakrabarti S, Collingham KE, Fegan CD et al. Cidofovir as primary pre-emptive therapy for post-transplant cytomegalovirus infections. Bone Marrow Transplant 2001; 28: Glucksberg H, Storb R, Fefer A et al. Clinical manifestations of graft-versus-host disease in human recipients of marrow from HL-A-matched sibling donors. Transplantation 1974; 18: Shulman HM, Sullivan KM, Weiden PL et al. Chronic graftversus-host syndrome in man. A long-term clinicopathologic study of 20 Seattle patients. Am J Med 1980; 69: Hale G. The CD52 antigen and development of the CAM- PATH antibodies. Cytotherapy 2001; 3: Small TN, Papadopoulos EB, Boulad F et al. Comparison of immune reconstitution after unrelated and related T-celldepleted bone marrow transplantation: effect of patient age and donor leukocyte infusions. Blood 1999; 93: Rebello P, Cwynarsky K, Varughese M et al. Pharmacokinetics of Campath-1H in bone marrow transplant patients. Cytotherapy 2002; 3: Chakrabarti S, Mautner V, Osman H et al. Adenovirus infections following allogeneic stem cell transplantation: the incidence and outcome in relation to graft manipulation, immunosuppression and immune recovery. Blood 2002; 100: Chakrabarti S, Collingham KE, Marshall T et al. Respiratory virus infections in adult T cell-depleted transplant recipients: the role of cellular immunity. Transplantation 2001; 72: Chakrabarti S, Osman H, Collingham K et al. Polyoma viruria in stem cell transplant recipients T cell depleted with Campath- 1H: the incidence and outcome in relation to graft manipulation, donor type and conditioning. Bone Marrow Transplant 2003; 31: Chakraverty R, Robinson S, Peggs K et al. Excessive T cell depletion of peripheral blood stem cells has an adverse effect upon outcome following allogeneic stem cell transplantation. Bone Marrow Transplant 2001; 28: Grob JP, Grundy JE, Prentice HG et al. Immune donors can protect marrow-transplant recipients from severe cytomegalovirus infections. Lancet 1987; 1: Lin TS, Zahrieh D, Weller E et al. Risk factors for cytomegalovirus reactivation after CD6+ T-cell-depleted allogeneic bone marrow transplantation. Transplantation 2002; 74: Einsele H, Ehninger G, Hebart H et al. Polymerase chain reaction monitoring reduces the incidence of cytomegalovirus disease and the duration and side effects of antiviral therapy after bone marrow transplantation. Blood 1995; 86: Reusser P, Einsele H, Lee J et al. The Infectious Diseases Working Party of the European Group for Blood and Marrow Transplantation. Randomized multicenter trial of foscarnet versus ganciclovir for pre-emptive therapy of cytomegalovirus infection after allogeneic stem cell transplantation. Blood 2002; 99: Williamson EC, Millar MR, Steward CG et al. Infections in adults undergoing unrelated donor bone marrow transplantation. Br J Haematol 1999; 104: Marr KA, Carter RA, Boeckh M et al. Invasive aspergillosis in allogeneic stem cell transplant recipients: changes in epidemiology and risk factors. Blood 2002; 100: Grigoleit GU, Riegler S, Hebart H et al. Human cytomegalovirus induces a direct inhibitory effect on antigen presentation by monocytes-derived immature dendritic cells. Bone Marrow Transplant 2002; 29 (Suppl.): S Winston DJ, Ho WG, Bartoni K et al. Ganciclovir prophylaxis of cytomegalovirus infection and disease in allogeneic bone marrow transplant recipients. Results of a placebo-controlled, double-blind trial. Ann Intern Med 1993; 118: Ljungman P, Brand R, Einsele H et al. Donor CMV serological status influences the outcome after unrelated donor stem cell transplantation; an EBMT Megafile analysis. Bone Marrow Transplant 2002; 29 (Suppl.): S32.