Original Article

Bone Marrow Transplantation (2014) 49, 280–286; doi:10.1038/bmt.2013.170; published online 11 November 2013

Viral Infection

EBV-associated post-transplant lymphoproliferative disorder following in vivo T-cell-depleted allogeneic transplantation: clinical features, viral load correlates and prognostic factors in the rituximab era

C P Fox1,2, D Burns2,3, A N Parker4, K S Peggs5, C M Harvey1, S Natarajan6, D I Marks7, B Jackson8, G Chakupurakal3, M Dennis9, Z Lim10, G Cook11, B Carpenter12, A R Pettitt13, S Mathew14, L Connelly-Smith15, J A L Yin5, M Viskaduraki16, R Chakraverty12, K Orchard17, B E Shaw8, J L Byrne1, C Brookes16, C F Craddock2,3 and S Chaganti2,3

  1. 1Department of Clinical Haematology, Nottingham University Hospitals NHS Trust, Nottingham, UK
  2. 2School of Cancer Sciences, University of Birmingham, Birmingham, UK
  3. 3University Hospitals Birmingham NHS Trust, Birmingham, UK
  4. 4The Beatson, West of Scotland Cancer Centre, Glasgow, UK
  5. 5University College London, Cancer Institute, London, UK
  6. 6Manchester Royal Infirmary, Manchester, UK
  7. 7University Hospitals Bristol NHS Trust, Bristol, UK
  8. 8The Royal Marsden, London, UK
  9. 9The Christie NHS Foundation Trust, Manchester, UK
  10. 10King's College Hospital NHS Trust, London, UK
  11. 11St James's Institute of Oncology, Leeds, UK
  12. 12Royal Free London NHS Trust, London, UK
  13. 13University of Liverpool, Liverpool, UK
  14. 14The Newcastle upon Tyne Hospitals NHS Trust, Newcastle upon Tyne, UK
  15. 15University Hospitals of Leicester NHS Trust, Leicester, UK
  16. 16Cancer Research UK Clinical Trials Unit, University of Birmingham, UK
  17. 17University Hospital Southampton NHS Trust, Southampton, UK

Correspondence: Dr CP Fox, Department of Clinical Haematology, Nottingham University Hospitals (City Campus), Hucknall Road, Nottingham NG5 1PB, UK. E-mail christopher.fox2@nuh.nhs.uk

Received 10 May 2013; Revised 11 August 2013; Accepted 11 September 2013
Advance online publication 11 November 2013

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Abstract

EBV-associated post-transplant lymphoproliferative disease (PTLD) following Alemtuzumab-based allo-SCT is a relatively uncommon and challenging clinical problem but has not received detailed study in a large cohort. Quantitative-PCR (qPCR) monitoring for EBV reactivation post allo-SCT is now commonplace but its diagnostic and predictive value remains unclear. Sixty-nine patients with PTLD following Alemtuzumab-based allo-SCT were studied. Marked clinicopathological heterogeneity was evident; lymphadenopathy was frequently absent, whereas advanced extranodal disease was common. The median viral load at clinical presentation was 49300 copies/mL (50–65200000 copies/mL) and, notably, 23% and 45% of cases, respectively, had less than or equal to10000 and less than or equal to40000 copies/mL. The overall response rate to rituximab as first-line therapy was 70%. For rituximab failures, chemotherapy was ineffectual but DLIs were successful. A four-parameter prognostic index predicted response to therapy (OR 0.30 (0.12–0.74); P=0.009] and PTLD mortality (hazard ratio (HR) 1.81 (1.12–2.93) P=0.02) on multivariate analysis. This is the largest detailed series of EBV-associated PTLD after allo-SCT. At clinical presentation, EBV-qPCR values are frequently below customary thresholds for pre-emptive therapy, challenging current paradigms for monitoring and intervention. A four-point score identifies a proportion of patients at risk of rituximab-refractory disease for whom alternative therapy is needed.

Keywords:

alemtuzumab; DLI; EBV; immunotherapy; PTLD; SCT

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Introduction

Post-transplant lymphoproliferative disease (PTLD), consequent upon opportunistic expansion of EBV-transformed B lymphocytes in the T-cell-compromised host, remains an important cause of morbidity and mortality following allo-SCT. The reported incidence ranges from <1 to 11%,1, 2 influenced by a number of risk factors principally related to T-cell depletion of the graft. Selective T-cell depletion in vivo using anti-thymocyte globulin confers a high risk, whereas combined depletion of both T and B cells, with the anti-CD52 MoAb Campath, has been reported to confer a much smaller PTLD risk (0.4–1.3%).2, 3 However, these reports date from the era of Campath 1M and 1G (rat MoAbs), superceded by the humanised Campath 1H Ab (Alemtuzumab) over a decade ago.4 Alemtuzumab has a considerably longer in vivo half-life (15–21 days) compared with Campath 1G (<1 day), resulting in marked delays in T-cell reconstitution.5, 6 Notably, EBV-specific T-cell responses are rarely detected within 6 months of Alemtuzumab-based allo-SCTa7 and, based on small numbers of cases, the PTLD risk appears higher in this context.8 EBV-associated disease remains a significant clinical problem following Alemtuzumab-based allo-SCT, although there is a scarcity of published data in this setting.

Indeed, since the first descriptions of PTLD following allo-SCT (SCT-PTLD) in the 1980s9, 10 there has been a paucity of the literature available on the detailed clinical, radiological and laboratory features of this EBV-driven disease. Before the development of rituximab, treatment was limited to de-escalation of immunosuppression, chemotherapy or unselected DLIs,11, 12 and mortality rates exceeded 80%.13 Current treatment strategies, namely rituximab and EBV-specific CTL therapy have significantly reduced the risk of death in established SCT-PTLD,14, 15 although there are no randomised studies evaluating the efficacy of these interventions.

The widespread availability of quantitative PCR assays (qPCR) for EBV DNA in peripheral blood has allowed targeted or routine monitoring for EBV reactivation post transplant.16, 17, 18 Furthermore, ‘pre-emptive’ treatment strategies in response to EBV DNAemia, with or without accompanying clinical evidence of EBV-associated disease, have been widely incorporated into clinical practice.14, 16, 18, 19, 20, 21 However, this has been predominantly based on uncontrolled, single-centre studies encompassing heterogeneous methods of T-cell depletion and small numbers of PTLD events.

We studied the clinical, laboratory and radiological characteristics of a large series of PTLD occurring following Alemtuzumab-based allo-SCT and analysed the relationship with the viral load data. We also sought to identify factors predictive of outcome in the Rituximab era.

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Materials and methods

Patients and inclusion criteria

This retrospective multicentre study invited the UK SCT centres to contribute data on consecutive patients diagnosed with probable or proven EBV-associated disease following Alemtuzumab-based allo-SCT from 2001 to 2010. Following review of local SCT databases, 15 centres submitted detailed clinical and laboratory data for a total of 74 cases. Limited detail on six cases has been previously reported.8, 22 Original histopathology and radiology reports were requested for central review. Patients undergoing an allo-SCT were prospectively consented for anonymised data collection and analysis.

All submitted data were critically reviewed by CPF and DB and recently published definitions23 were applied: proven EBV disease (PTLD or other end-organ disease)=biopsy-confirmed with corresponding clinical manifestations; probable EBV disease=significant lymphadenopathy (or other end-organ disease) with high EBV DNAemia in the absence of another cause. Importantly, EBV DNAemia per se, irrespective of the magnitude of EBV genome load, was deemed insufficient for study inclusion unless accompanied by robust evidence of probable or proven disease.

Transplant conditioning protocols

All patients received Alemtuzumab as in vivo T-cell depletion with myeloablative or reduced-intensity preparative conditioning according to the institutional protocols. Alemtuzumab was typically administered i.v. from day 5 or day 3 (before stem cell infusion) for a total of 5 or 3 doses, respectively. GVHD prophylaxis comprised CsA with or without MTX or mycophenolate mofetil.

EBV-PCR data

EBV-qPCR values at all time points post allo-SCT were submitted for each patient. For patients with more than one EBV-qPCR result around the time of PTLD diagnosis, the value most proximate to the clinical presentation of PTLD (and always pre-treatment) was selected for analysis.

Response and survival

Response to therapy was evaluated on the basis of submitted clinical and radiological data and categorised by consensus of CPF and DB into CR, PR or progressive disease, in line with the Cheson criteria.24 Owing to the nature of the study, the timing and modality of response evaluation were clinically determined and included computed tomography, positron-emission tomography and ultrasound, according to disease sites and the clinical course. Some patients experienced rapid disease progression and died before, or soon after, initiation of treatment. To be designated response-evaluable, the administration of at least two rituximab doses, at least 1week apart was stipulated. OS was calculated from the date of PTLD diagnosis to the date of death or last follow-up.

Statistical analysis

Response to treatment was analysed using logistic regression methods. Survival estimates were calculated by the Kaplan–Meier methodology and Cox proportional hazard modelling. For PTLD-specific mortality, censorship at the time of non-PTLD death or at the date of last contact was performed. Continuous variables were investigated for non-linearity and the best fitting transformation assessed by comparing AIC between models, with and without the transformations applied. Factors with a demonstrable prognostic trend in univariate analyses were analysed multivariately and a backward-elimination selection procedure applied. Differences in patient characteristics were assessed using Fisher’s exact test and the Wilcoxon rank-sum test. All analyses were carried out using SAS/STAT statistical software version 9.1 (SAS Institute, Cary, NC, USA).

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Results

Among the 74 submitted cases, five did not meet the inclusion criteria. The mean EBV-qPCR value in these five cases was 385689 copies/mL (range 6309–1513561) and, although EBV-associated disease could not be discounted in these patients, insufficient evidence of PTLD was available for study inclusion. Of the 69 patients included as cases of probable or proven PTLD, 62 cases (89.9%) presented with features of lymphoma/lymphoproliferative disease, whereas seven patients (10.1%) had clinicopathological evidence of: EBV encephalitis (n=2, note the qPCR value in Table 1 is from peripheral blood but EBV DNA was also detected in cerebrospinal fluid), EBV-haemophagocytic lymphohistiocytosis (n=3) or died of multiorgan failure in association with an extremely high EBV genome load (n=2). Given the clinical importance and high mortality of these seven distinct cases, a description is provided in Table 1, but these patients are not included in the response and outcome analyses. Of the 62 patients with EBV-associated lymphoproliferative disease, 46 patients (74%) had biopsy-proven lymphoma, whereas 16 cases (26%) were designated probable PTLD on the basis of the published criteria;23 these 62 cases constitute the study population.


Patient and transplant characteristics

Characteristics of the 62 patients are shown in Table 2. A majority of patients received PBSCs (87%), from matched unrelated (44%), mis-matched unrelated (greater than or equal to1 HLA loci) (27%) and sibling adult donors (29%). Seventeen patients (27%) underwent myeloablative conditioning, whereas 45 (73%) received reduced-intensity conditioning. The median dose of i.v. Alemtuzumab was 90mg (range 30–100mg) with the exception of one patient who received 10mg Alemtuzumab ‘in the bag’, added ex vivo to the stem cells before infusion. GVHD (acute and/or chronic) was clinically evident before PTLD diagnosis in 28 of 53 (52%) evaluable patients.


In 44 cases, data on previous therapies were available. Of note, eight patients had previously undergone an auto-SCT and three patients (7%) had received Alemtuzumab as a disease-specific therapy before and distinct from the allo-SCT conditioning. Further, eight more patients (18%) received anti-thymocyte globulin either before (aplastic anaemia) or following (graft failure or severe GVHD) an allo-SCT.

PTLD characteristics

Table 3 details the clinical characteristics of the PTLD cases. The median age at PTLD diagnosis was 49 years, in 48 male patients (77%) and 14 female patients (23%). PTLD manifested clinically at a median of 120 days (range 39–1056, interquartile range 82–188 days) following allo-SCT, with no significant difference in timing of PTLD onset between myeloablative and reduced-intensity allo-SCT. Notably, six patients (10%) presented over a year following an allo-SCT, of whom five were receiving immunosuppressive agents. Thirty-nine patients (63%) had a poor performance status (Eastern Co-operative Oncology Group (ECOG) 2–4) at PTLD diagnosis. B symptoms were common and, notably, 52 of 62 patients (82%) had a fever at clinical presentation. The majority of patients (75%) presented with advanced stage (III/IV) disease. Extranodal involvement was frequent (62%), with one extranodal site evident in 31% and greater than or equal to2 extranodal sites in a further 31% of patients (detailed in Table 3). Six patients (10%) had evidence of central nervous system involvement at presentation. BM involvement with PTLD was rare; documented in two of 26 evaluable cases (7.7%).


On the basis of the well-validated international prognostic index (IPI) applied in de novo diffuse large B cell lymphoma,25 we calculated a prognostic score for each patient (Table 3). However, serum lactate dehydrogenase was excluded as a parameter because of missing data in 20 patients, resulting in a maximum score of 4. Eighteen patients (29%) had a score of greater than or equal to3.

EBV-qPCR data

EBV-qPCR evaluation post allo-SCT varied, both between the UK centres and chronologically over the study period, as the clinical importance of this parameter was increasingly recognised. Thus, for some patients, a series of viral load values were available, whereas others had a single result around the time of PTLD diagnosis. Viral load data were available for 56 patients (90%); this was a whole-blood assay in 43 patients (77%) and a plasma assay in 13 patients (23%). To evaluate the diagnostic utility of EBV qPCR, we selected the viral load value closest to the onset of PTLD symptomatology for each patient. In 88% of cases, this value was within 7 days of clinical presentation (median day 0, range −10 to +22 days) and, importantly, all values were before PTLD treatment.

There was a marked variation in EBV qPCR at clinical presentation of PTLD (Figure 1a); the median value was 49300 copies/mL (range 50–65200000 copies/mL, interquartile range 11000–525000 copies/mL). There was no statistically significant difference between the median viral load in the plasma assay group as compared with the whole-blood assay group. The observed heterogeneity in EBV-qPCR values was not simply explained by inter-laboratory variation, as pronounced differences were seen both within and between laboratories (Figure 1b). Notably, at clinical presentation of PTLD, a substantial proportion of patients had viral loads below commonly adopted thresholds for pre-emptive intervention;18, 19, 20, 21 EBV-qPCR values at symptom onset were less than or equal to40000 copies/mL and less than or equal to10000 copies/mL in 45% and 23% of cases, respectively.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

The distribution of EBV-qPCR values at the time of clinical presentation of PTLD is shown. Each dot represents an individual PTLD case. Panel a shows the inter-patient variation over a log10 scale. Panel b demonstrates the distribution of EBV-qPCR values within and between participating transplant centres.

Full figure and legend (18K)

Histology

The current WHO classification system of PTLD26 is predominantly based on a consensus opinion relating to PTLD in the context of solid-organ transplantation. The clinical utility of this classification in the SCT setting remains unclear. Nonetheless, on the basis of the available histopathology reports (n=41), we classified the biopsy-proven SCT-PTLD cases as polymorphic (n=13), monomorphic (n=21) and unclassifiable (n=7). There was a single case of T-cell lymphoma. Interestingly, six cases of B-cell lymphoma were CD20 negative, although five of these patients were treated with rituximab before biopsy, confounding interpretation.

Details of therapy

Eleven cases (17.7%) were post-mortem diagnoses patients who or died rapidly after initiation of therapy, leaving 51 patients evaluable for treatment response. Notably, 36% of non-evaluable patients had undergone a prior auto-SCT compared with 8% in the evaluable group (P=0.03), and the median absolute lymphocyte count was 0.4 × 109/L in non-evaluable patients compared with 0.9 × 109/L in evaluable patients (P= 0.04).

All 51 evaluable patients had reduction/cessation in immunosuppression if relevant, and the vast majority (47/51, 92%) simultaneously received rituximab as a first-line treatment of PTLD (median total rituximab dose 1500mg/m2 (range 375–4500mg/m2) typically 375mg/m2 weekly). This was Rituximab monotherapy in 46/47 patients; one patient received a single cycle of CVP chemotherapy concurrent with rituximab. Three patients underwent de-escalation of immunosuppression without further specific therapy, and one patient received DLI as a first-line therapy.

Five patients received chemotherapy following rituximab failure: CHOP (n=1); rituximab-CHOP (n=2); rituximab-CY (n=1); and rituximab and high-dose Cytarabine (n=1). Five patients received DLIs as salvage therapy and, with the exception of one DLI product that was CD8-depleted, all administered DLIs were unstimulated, unmanipulated T cells from the original stem cell donor. DLIs were typically administered as one infusion with a median administered dose of 1 × 106 CD3+ cells/kg (range 1 × 105–1 × 1.5 × 107). No EBV-specific T-cell products were used.1

Response to therapy

Thirty-three of 47 rituximab-treated patients (70%) achieved CR/CRu. Three patients (all with biopsy-proven PTLD presenting at 78, 225 and 1056 days post transplant) had de-escalation of immunosuppression without further specific therapy: two patients achieved CR, whereas one died of progressive PTLD. One patient received DLI as a first-line therapy at 55 days post transplant for biopsy-proven EBV+DLBCL and achieved CR. Fourteen patients (30%) progressed despite rituximab monotherapy (median total dose 1500mg/m2 (range 750–4500mg/m2)), of whom three died rapidly from progressive PTLD. Ten of the 14 rituximab failures received further treatment: five underwent chemotherapy, three received DLIs and two underwent chemotherapy followed by DLI. No patient achieved PR or CR with chemotherapy after failing rituximab, whereas three of five patients who received DLIs as salvage therapy achieved CR. Notably, one patient (20%) experienced grade III skin- and gut-GVHD following DLI. Strikingly, there were no documented relapses amongst patients who achieved a PR or CR; however, this was achieved.

Factors predicting response to therapy

An univariate analysis identified two factors associated with response to treatment: stem cell source (OR 6.7 (1.2–36.6) in favour of PBSC vs BM, P=0.03) and greater than or equal to2 extranodal sites (OR 0.08 (0.013–0.50), P=0.01). Incorporating the 4-point IPI within a univariate analysis also predicted response to therapy (linear OR 0.39 (0.18–0.85), P=0.02). In multivariate analyses, cell source (OR 19.5 (1.92–196.9) in favour of PBSCs; P=0.01) and the 4-point IPI (OR 0.30 (0.12–0.74); P=0.009) were both statistically significant as independent prognostic factors for response in a backward-elimination selection model. Of note, there was no association between response to therapy and magnitude of viral load (presentation or peak value), or absolute lymphocyte count.

Survival

With a median follow-up of 1.7 years (20.5 months, interquartile range 13.0–42.7) for living patients, the 1- and 2-year survival rates were 46.3% (95% confidence interval (CI) 38.8–58.8) and 39.7% (95% CI 26.9–52.5), respectively (Figure 2a). Nineteen deaths (31% of total cohort) were attributable to PTLD (Figure 2b), seven from other non-relapse mortality and seven from relapse of primary disease. In two cases, the cause of death was uncertain, although PTLD could not be excluded as a contributory factor. Patients with rituximab-refractory PTLD died rapidly at a median of 33 days (13–257 days) from diagnosis.

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Kaplan–Meier plots showing all-cause mortality measured from date of PTLD diagnosis (a) and PTLD-specific mortality (b).

Full figure and legend (49K)

Factors predicting death from PTLD

An univariate analysis identified three factors predictive of PTLD-specific mortality: ECOG performance score (PS) greater than or equal to2 (HR 3.8, 95% CI 1.1–12.5, P=0.03); IPI (HR 9.94, 95% CI 1.16–85.21, P=0.048); and, somewhat unexpectedly, an absence of peripheral lymphadenopathy (HR 6.21 95% CI 2.20–17.55, P=0.0006). Both the 4-point IPI (HR 1.81 (1.12–2.93) P=0.02) and (absence of) lymphadenopathy (HR 4.29 (1.45–12.56), P=0.008) retained significance in a multivariate model (Figure 3). Further evaluation of those patients without clinically evident lymphadenopathy revealed that such patients were either diagnosed post mortem or died rapidly (median 16 days) after initiation of treatment. There was no apparent association between death from PTLD and donor type, conditioning intensity, Alemtuzumab dose, absolute lymphocyte count, presentation or the peak viral load. However, this analysis is based upon a small number of events and therefore lacks statistical power.

Figure 3.
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Kaplan–Meier plots showing the impact of palpable lymphadenopathy (a) and a 4-point IPI score (b) on PTLD mortality.

Full figure and legend (73K)

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Discussion

With the exception of epidemiological studies evaluating PTLD risk2 (in pre-1994 cohorts), and the literature reviews of treatment outcome,14 we are not aware of a larger published series of SCT-PTLD with detailed clinical features, laboratory data and treatment outcome. This multicentre UK study highlights EBV-associated disease as a significant clinical problem following an Alemtuzumab-based allo-SCT. It remains unclear whether PTLD in this context has genuinely increased in incidence, attributable to the use of the Campath-1H formulation,4, 5, 6, 7, 8 or simply whether more cases are now captured because of the diagnostic advances. Given the diverse and often aggressive clinical presentation, it is conceivable that some cases of SCT-PTLD have been previously misdiagnosed as culture-negative sepsis or organ-failure of non-infective aetiology.

An augmented immunosuppression may have contributed to PTLD susceptibility in a proportion of our cases; over half of patients had GVHD before PTLD onset and 18% had additionally received anti-thymocyte globulin. We were struck by the vast variation in peripheral blood EBV load at the time of PTLD presentation; ranging over a sevenfold log10 scale. However, viral load at symptomatic presentation of SCT-PTLD was frequently below conventionally adopted thresholds;16, 18, 19, 20, 21, 27 less than or equal to40000 copies/mL and less than or equal to10000 copies/mL in 45% and 23% of cases, respectively. It follows that reliance on a viral load threshold per se to predict SCT-PTLD jeopardises timely diagnoses. A high index of suspicion together with clinical, radiological and viral load data is required to improve diagnostic sensitivity and specificity. Parallel quantitation of T-cell reconstitution28 or functional EBV-specific assays17, 29 undoubtedly improves the predictive value of viral load values, but validation across transplant protocols is needed.

Remarkable heterogeneity in the clinical presentation of EBV-driven disease was apparent; a proportion of patients had marked systemic manifestations, some with multiorgan dysfunction and a fulminant clinical course. Interestingly, peripheral lymphadenopathy was frequently absent, potentially resulting in diagnostic delays. An advanced stage and a diverse extranodal disease were common, reflecting either late presentation or a biologically aggressive disease; a significant number of cases were diagnosed at post mortem. Applying a 4-point prognostic score identified patients more likely to have a rituximab-refractory disease and at a high risk of PTLD mortality.

Although rituximab-monotherapy is effective for a majority of patients with established PTLD, response rates remain suboptimal; 30% failed rituximab treatment despite adequate dosing (median 1500mg/m2). Rituximab failure conferred an extremely poor prognosis, with 11 of 14 patients dying rapidly from PTLD at a median of 33 days. Conventional chemotherapy appeared ineffective as salvage treatment for rituximab failures; all five such patients experienced progressive disease and died from PTLD. In contrast, DLI was far more efficacious in both rituximab and rituximab/chemotherapy failures. The efficacy of adoptive T-cell therapies for PTLD post allo-SCT is well established1, 11, 12, 15, 30 and we would support this approach as the optimal salvage for SCT-PTLD refractory to rituximab treatment. Available data suggest that unselected DLIs, donor-derived EBV-specific CTLs and banked third-party EBV-CTLs have equivalent therapeutic efficacy. Unselected DLIs are immediately accessible but confer a risk of GVHD, whereas production of EBV-specific CTLs can be protracted, although do not cause GVHD.1, 15

This detailed analysis of the largest cohort of SCT-PTLD, alongside correlative EBV-qPCR data, highlights the biological heterogeneity of this often clinically aggressive, virus-driven, opportunistic malignancy. Our observation that many patients with biopsy-proven disease have modest viral loads at initial clinical presentation challenges current paradigms for monitoring and intervention. Rituximab has undoubtedly improved the outcome for SCT-PTLD patients but remains ineffective for a significant proportion of patients. Accurate risk stratification may allow the identification of patients requiring adoptive T-cell therapy earlier in the therapeutic algorithm; this urgently requires prospective studies.

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Conflict of interest

CPF has received travel grants and speaker honoraria from Roche UK. ARP has received travel grants, advisory board and speaker honoraria from Roche UK. SC has received travel grants from Roche UK.

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Acknowledgements

CPF received research funding from Leukaemia and Lymphoma Research, UK. DB received research funding from the Wellcome Trust UK.