Given that a highly significant reduction in risk of EBV reactivation had been observed
amongst patients with NHL, pre-transplant use of Rituximab was examined as a possible
factor (Table 19). Thus, Rituximab use was documented in 28/29 (97%) patients with NHL,
with the last infusion administered a median 3 months (IQR 2 – 5 months) before transplant.
Prior Rituximab was also recorded in 11/12 (92%) patients with CLL but the last infusion of
Rituximab was delivered substantially longer before transplant, at median 14 months (IQR 8 –
23 months). Importantly, only 1/25 (4%) patients who received Rituximab less than 6 months
Page 139 EBV ≥ 500 copies/ml HR 95% CI P Age > 50 Years 1.46 0.85 – 2.50 0.168 Diagnosis AML/MDS 1.00 - Ref NHL 0.18 0.06 – 0.58 0.004 ALL 0.90 0.46 – 1.77 0.762 HL 1.77 0.69 – 4.53 0.236 CLL 0.98 0.48 – 2.00 0.955 MPD 0.97 0.44 – 2.15 0.940 Other 2.50 0.94 - 6.64 0.067 Conditioning
Flu Mel 1.00 - Ref
Cy TBI 0.77 0.39 – 1.52 0.452
BEAM+/- Flu No Events - -
Other 0.35 0.08 – 1.51 0.159
T Cell Depletion
ATG vs Alemtuzumab 7.36 1.75 – 30.9 0.006
Table 18. Multivariate Analysis of Risk Factors for EBV Reactivation after Allo-HSCT In a multivariate Cox proportional hazards model including the age, diagnosis, conditioning and T-cell depletion, NHL was a highly significant predictor for lack of EBV reactivation. Prior Rituximab therapy was also a highly significant predictor for lack of EBV reactivation in a separate model that excluded diagnosis. Significance for prior Rituximab was eliminated when diagnosis was included in this model. However, severe confounding between prior Rituximab therapy and diagnostic category was apparent. Hazard ratios (HR) with 95% confidence intervals (95% CI) were determined for categories within each variable, relative to a reference category (Ref).
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Table 19. Prior Rituximab Therapy and EBV Reactivation after Allo-HSCT
The interval (months) between the last infusion of pre-transplant Rituximab and day 0 of the transplant is given for all study patients with NHL and CLL. The corresponding day of EBV reactivation or high-level EBV reactivation post-transplant is shown. EBV reactivation events occurring within 1 year after transplant appear in red, whilst those occurring after 1 year appear in blue. Only 1 patient who received Rituximab within 6 months prior to transplant reactivated EBV in the first year after transplant.
EBV DNAemia after 1 year EBV DNAemia within 1 year
Diagnosis Rituximab: Months
prior to transplant Day EBV qPCR positive Day EBV >20,000 copies/ml NHL 1 . . NHL 2 . . NHL 2 . . NHL 2 . . NHL 2 . . NHL 2 . . NHL 2 . . NHL 2 . . NHL 2 565 . NHL 2 . . NHL 2 . . NHL 2 . . NHL 2 . . NHL 2 . . CLL 2 372 . NHL 3 . . NHL 3 380 . NHL 4 . . NHL 4 . . NHL 4 . . NHL 4 . . NHL 5 . . CLL 5 78 78 NHL 6 . . NHL 6 . . NHL 7 . . CLL 7 . . CLL 9 29 43 CLL 10 104 . CLL 14 . . CLL 15 273 . CLL 15 217 . NHL 17 . . NHL 24 57 . NHL 29 . . CLL 30 41 . NHL 31 . . CLL 37 218 337 CLL 47 85 99 NHL None . . CLL None . .
Page 141 Rituximab 5 months prior to transplant and who developed high-level EBV reactivation on
day 78. In contrast, 8/14 (57%) patients who received Rituximab more than 6 months before
transplant developed EBV reactivation, including 3 patients with high-level EBV reactivation.
In univariate Cox analysis, there was a highly significant association between pre-transplant
Rituximab therapy and lack of EBV reactivation, using definitions of pre-transplant
Rituximab of either ‘prior Rituximab ever’ (HR 0.35, CI 0.19 – 0.64; P = 0.0007) or a cut-off of 6 months prior to transplant (HR 0.17, CI 0.06-0.46; P = 0.0005). These differences did not
persist in multivariate testing but this analysis was complicated for several reasons. Firstly,
there was strong confounding between diagnostic category and prior use of Rituximab, such
that almost all patients with NHL and CLL received Rituximab at some time before
transplant. Secondly, there was uncertainty regarding what constitutes an appropriate cut-off
(months before transplant) to use to define pre-transplant Rituximab. Finally, few patients
with NHL reactivated EBV, even if they received Rituximab more than 6 months before
transplant, unlike patients with CLL (Table 19).
Regarding the safety of pre-transplant Rituximab, testing revealed no significant difference in
overall survival for patients treated with Rituximab at any time before transplant (HR 0.72, CI
0.41 – 1.28; P = 0.262) or within 6 months before transplant (HR 0.75, CI 0.38 – 1.5; P =
0.420). Similarly no significant differences were seen for non-relapse mortality for
Rituximab given at any time before transplant (HR 0.93 CI 0.43 – 2.02; P = 0.856) or using a
6 month cut-off (HR 0.91, CI 0.36 – 2.33; P = 0.845). An association between pre-transplant
Rituximab (defined as any time before transplant or using a 6 month cut-off) and risk of either
Page 142 4.5 Discussion
This study reports incidence and risk factors for EBV reactivation and PTLD amongst
patients undergoing TCD allo-HSCT at a major UK transplant centre, and includes the largest
number of transplants using Alemtuzumab reported thus far. A relatively high overall
incidence of EBV DNAemia was observed amongst Alemtuzumab-treated patients, with 48%
of individuals exhibiting EBV qPCR positivity by 12 months post-transplant and 18%
developing high-level EBV reactivation. This incidence was significantly higher amongst
ATG-treated patients, with 80% exhibiting EBV qPCR positivity and 30% developing high-
level reactivation. Importantly, PTLD was diagnosed, coincident with high-level EBV
reactivation, in 11 patients. Three of these had received ATG, whilst Alemtuzumab had been
used in 8, giving a crude incidence of PTLD amongst Alemtuzumab-treated patients of 4.3%.
This is greater than that reported in the study by Carpenter et al., in which only 0.9% of
Alemtuzumab-treated patients developed PTLD409, although it is within the range report by
other studies412,414,415,500. However, it is notable that around a third of patients in the Carpenter
study received Alemtuzumab ‘in the bag’ (in which the drug is used to treat the stem cell graft in vitro, before it is infused into the recipient) rather than in vivo, whereas the latter was used for all patients in the present study. Higher peak concentrations and persistence of
Alemtuzumab have been reported when it is used in vivo577, and this is also associated with
delayed reconstitution of EBV-specific immunity383. These alternative approaches to TCD
may account for at least some of the variation observed in PTLD incidence between these
studies.
Regarding the kinetics of EBV DNAemia, most cases of EBV reactivation occurred between
Page 143 earlier than in those treated with Alemtuzumab. However, some instances were documented
up to and beyond 12 months. Given this, it is notable that recent guidelines have advised that
high risk patients should be monitored for 3 months after allo-HSCT496. The current study
supports extending this recommendation to 6 months, at least for Alemtuzumab-treated
patients. Furthermore, given that over 80% of high-level reactivations were seen to occur
within 2 weeks of initial EBV qPCR positivity, particular scrutiny should be exercised in the
first weeks following initial EBV qPCR positivity.
Amongst patients who developed high-level EBV reactivation, with or without PTLD,
excellent response rates to pre-emptive Rituximab therapy were observed, with 38/41 (93%)
patients exhibiting complete resolution of EBV DNAemia. However, 3 patients developed
Rituximab-refractory PTLD and died from progressive disease, despite the use of cytotoxic
chemotherapy in 2 of them. Unfortunately, although chemotherapy such as CHOP may
achieve good outcomes for Rituximab-refractory disease in the setting of solid organ
transplantation, this has not been the case for PTLD arising after allo-HSCT, where very poor
outcomes are likely to be a consequence of increased toxicity in this patient group520,572. In
light of these experiences we now recommend that all patients developing Rituximab-
refractory PTLD are treated with EBV CTLs. Notably, some studies have recently raised
concerns about the safety of Rituximab in recipients of allo-HSCT, demonstrating a possible
excess of infectious complications, although an effect on survival has not been
established507,575,576. Given this, it is notable that the use of pre-emptive Rituximab was not
associated with an increase in mortality in the present study, although this analysis was based
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Of possible predictors for EBV reactivation we found that ATG treatment, older age at
transplant and the occurrence of acute GvHD were all significant risk factors. In this respect,
our data agree with previous studies on risks associated with EBV reactivation and PTLD
after allo-HSCT392. However, the most striking finding was a dramatically reduced incidence
of EBV reactivation and PTLD amongst patients with a diagnosis of NHL. Therefore,
individuals with NHL exhibited a highly significant reduction in the risk of EBV reactivation
in both univariate and multivariate analyses, and no patients with NHL exhibited high-level
reactivation. Whilst our data should be treated with some caution due to the retrospective
nature of the study, our observation is intriguing because it raises the possibility that
Rituximab therapy delivered prior to transplant might be responsible for this effect. In support
of this we found that individuals with HL did frequently experience EBV reactivation -
patients with HL do not routinely receive treatment with Rituximab. Furthermore, our
observations are consistent with the anecdotal description by Savani et al. of an absence of
EBV reactivation after allo-HSCT in 38 patients who received Rituximab prior to, or
concurrent with, transplantation574. Although our findings have not been observed in other
studies of EBV reactivation and PTLD after allo-HSCT, this may be because these studies
pre-date the routine use of Rituximab for patients with NHL392,407, or because they have not
specifically analysed the risks associated with NHL408,410,411,416-419. Notably, in the study by
Carpenter et al., NHL showed a trend towards lower incidence of EBV reactivation (HR 0.68;
CI 0.25–1.85) but this was not statistically significant (P = 0.45); however it is unknown what
type of NHL these patients had, or indeed whether they had ever received Rituximab501.
It light of the above, it is possible that Rituximab delivered shortly before transplant might be
Page 145 Rituximab might prevent EBV reactivation through 1 of 2 alternative mechanisms. Firstly, it
has a half-life of up to 18 days, with levels remaining detectable in the serum for up to 3
months after administration, and it is known to deplete circulating B-cells for around 6
months497. As such, prior Rituximab might contribute to a delay in the recovery of B-cells
post-transplant. Indeed, van Dorp et al. demonstrated a significantly reduced rate of B-cell
reconstitution in patients who had received Rituximab within 6 months prior to transplant578.
Alternatively, Rituximab may act by reducing recipient pre-transplant virus burden.
Interestingly, the latter supposes that it is predominantly recipient-derived virus that most
commonly leads to EBV reactivation. With regard to this, a small number of (largely
anecdotal) studies have demonstrated that patients with EBV reactivation or PTLD arising
after allo-HSCT acquire a donor or third party EBV strain in approximately 50% of cases579-
582
. However, it is notable that all such studies have been conducted in the setting of
myeloablative transplantation; it is possible that RIC transplantation may increase the
likelihood that recipient-derived virus is important for the pathogenesis of EBV reactivation
and PTLD post allo-HSCT.
To conclude, in this study we have examined EBV reactivation and PTLD amongst a large
cohort of adult patients undergoing TCD allo-HSCT. Importantly, we describe the novel
finding that risk of EBV reactivation appears to be greatly reduced amongst recipients with
NHL. This suggests that Rituximab could be used as effective prophylaxis for PTLD arising
after allo-HSCT. Ultimately, our data make a strong case for prospectively evaluating the role
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