Survey of CMV Management in Pediatric Allogeneic HSCT Programs, on Behalf of the Inborn Errors, Infectious Diseases and Pediatric Diseases Working Parties of EBMT

ORIGINAL ARTICLE Survey of CMV management in pediatric allogeneic HSCT programs, on behalf of the Inborn Errors, Infectious Diseases and Pediatric Diseases Working Parties of EBMT

T Bontant1, P Sedlac¸ek2, A Balduzzi3, B Gaspar4, S Cesaro5, H Einsele6, C Peters7 and J-H Dalle1

Human CMV infection is a frequent complication after HSC in children with remarkable morbidity and mortality. Antiviral drugs are relatively efﬁcient but have numerous side effects. They are used as prophylactic, pre-emptive or therapeutic medicines. It is still a matter of debate which option is the best strategy. No uniform procedure has emerged regarding these three options, and new immunologic tools have raised more questions for physicians. To assess the current practice in the management of CMV infection, we sent a questionnaire to the EBMT centers performing hematopoietic SCT (HSCT) in children. Fifty-six out of 196 responded to the questionnaire (28.5%). Quantitative PCR was the most common monitoring tool (44/56). Only 4/56 centers use the pp65 antigenemia alone. All centers used pre-emptive strategy (56/56). 21/56 centers also used prophylactic measures, 13/21 after analysis of donor/receptor serologic status. Ganciclovir was the most common ﬁrst-line agent for CMV disease (55/56). The most common dose and duration for induction treatment were 5 mg/kg bid (47/55) for 14 days (20/55). There is no uniform procedure for researching resistance strain, antiviral second-line therapy or cell therapy. A harmonization process should enable sound prospective trials to improve prevention, control and cure of CMV disease in children and adolescents.

Bone Marrow Transplantation (2014) 49, 276–279; doi:10.1038/bmt.2013.164; published online 28 October 2013 Keywords: pediatric; HSCT; CMV; prophylaxis; curative therapy; pre-emptive therapy

INTRODUCTION children. These drugs are active on CMV replication but not on Despite improvements achieved in the allogeneic hematopoietic latency, and they expose patients to toxicities and to emergence of 18–22 SCT(HSCT setting, human CMV infection still remains a relevant resistant strains. The lack of pharmacological studies, complication both in adults and children.1–4 CMV infection in particularly in children, and the lack of clinical trials testing these transplant recipients can cause direct organ lesion and indirect antiviral drugs and the probable benefit/risk ratio make clinician’s 23 effects with a potential role of CMV immunomodulatory abilities, choice difficult. It is well known that beside anti-CMV, particularly in GVHD occurrence or re-flare.2,5–8 To prevent and pharmaceutical cells are necessary to prevent or control CMV control CMV infection, clinicians have to define procedures for infection and/or reactivation. Hence, new immunological tools and 10,24–27 monitoring. Specific and sensitive tests are available—that is, adoptive cell therapy have raised new options to improve pp65 antigenemia and real-time PCR. Both methods are estab- CMV infection detection and outcomes. lished tools but there is no consensus on biological monitoring To our knowledge, no survey has focused on CMV infection schedules.9–12 management in pediatric transplant centers so far. To assess the Different biological and clinical situations have to be distin- current situation, we sent a questionnaire on CMV infection guished: serological status giving evidence of previous CMV management to all EBMT centers performing allogeneic HSCT in contact; CMV reactivation corresponding to detection of either children and adolescents. viral DNA or antigen in blood sample; CMV infection in which DNA or antigen is detectable in association with fever; and CMV disease when clinical symptoms, organ involvement and virus detection MATERIALS AND METHODS are associated.4,5 A 30-item questionnaire regarding CMV infection management was sent to Two strategies are applied to prevent CMV infection: prophylaxis EBMT centers performing pediatric HSCT. The EBMT Pediatric Diseases, Infection Diseases and Inborn Errors Working Parties have previously strategy that exposes all patients to antiviral drugs, regardless of validated this extensive questionnaire. First part was dedicated to screening tests for CMV replication or pre-emptive therapy, which 13–16 virological monitoring: nature of samples, type of test performed, initiates antiviral treatment based on screening test results. monitoring schedule, thresholds, frequency and duration of testing. 17 There is no evidence that one strategy is superior to the other. Questions of the second part were asked about prophylactic or pre- Different antiviral drugs are used; however, none is licensed for emptive strategies and about antiviral treatment modalities. Last part

1Department of Hematology and Immunology, Hospital Robert Debre, Paris 7—Paris Diderot University, Paris, France; 2Department of Pediatric Hematology and Oncology, University Hospital Motol, 2nd Medical School, Charles University, Prague, Czech Republic; 3Clinica Pediatrica Universita` degli Studi di Milano Bicocca, Ospedale San Gerardo, Monza (Milano), Italy; 4Chair of EBMT Inborn Error Working Party, Molecular Immunology Unit, UCL Institute of Child Health, London, UK; 5Chair of EBMT Infectious Disease Working Party, Paediatric Haematology Oncology Policlinico G.B. Rossi, Verona, Italy; 6Division of Hematology, Department of Internal Medicine II, University Hospital of Wu¨rzburg, Wu¨rzburg, Germany and 7Chair of EBMT Paediatric Diseases Working Party, St Anna Children’s Hospital, Vienna, Austria. Correspondence: Professor J-H Dalle, Department of Hematology and Immunology, Hospital Robert Debre, Paris 7—Paris Diderot University, 48 Boulevard Serurier, Paris Cedex 19, Paris 75935, France. [email protected] Received 15 February 2013; revised 7 August 2013; accepted 8 August 2013; published online 28 October 2013 CMV in pediatric transplantation: results of an EBMT survey T Bontant et al 277 asked the centers about definition of treatment failure, second-line therapy Pre-emptive strategy and cell therapy. This questionnaire was sent in December 2009. Pre-emptive therapy was used by all the responding centers. Responders had the possibility to choose more than one answer in most Treatment of CMV infection started after either one positive or two cases. Questionnaire is available in the appendix. Responses were collected until April 2010. Except for some questions (indicated in text), all results are positive results in 26 and 27 centers, respectively; three centers given out of 56 responding centers. However, as multiple responses have used both signals. The most common first-line pre-emptive been allowed, the total may be 456 for some results. therapy was Ganciclovir (55 programs, including before engraftment in 22). Foscarnet was used in 19 centers. Valganciclovir was used in 18 centers, including 3/18 before the occurrence of RESULTS engraftment. Finally, four centers used Cidofovir. From 12 December 2009 to 01 April 2010, 196 centers received the questionnaire and 56 EBMT centers (28. 5%) from 26 countries Schedule and drug doses completed the questionnaire and sent it back. All responding Ganciclovir. The dose of induction therapy is 5 mg/kg per 12 h centers performed pediatric transplantation either exclusively or (47/55, one center did not specify) during 14 days in 20 out of associated with adult HSCT. Most of these centers performed both 47 responding centers to this question. Twenty centers shortened related and unrelated HSCT, including for some of them this treatment when virus became undetectable. Thirty-nine (71%) alternative HSCT from partially matched unrelated cord blood or centers used a maintenance therapy. Among them, 32 gave the haplo-identical HSCT. According to EBMT 2010 annual activity dose of 5 mg/kg once a day either for 14 days (20 centers) or 5 report, all together these 56 centers performed about 2150 days a week for 2 weeks (10 centers). Two centers performed allogeneic HSCT a year—that is, 17.5% of all Promise-database Ganciclovir plasma-level monitoring in order to adapt dosage. registered allogeneic HSCT and around 45% of pediatric activity.28 The median annual activity for responding centers is 30 allogeneic Foscarnet. This antiviral drug is used in 45 centers. The dose of HSCT a year (range: 1–142). induction treatment is 90 mg/kg twice daily in 24 centers (53%), As indicated above, except for some questions (number 60 mg/kg twice daily in 15 centers (33%) and 60 mg/kg per 8 h in specified in text), all results are given out of the 56 responding 6 centers (14%). Induction treatment is systematically admini- centers. However, as multiple responses have been allowed, the stered during 14 days in 34 centers, whereas 11 centers shorten this total may be 456 for some results. treatment if virus is no longer detectable. Thirty centers out of 45 use maintenance therapy. Dose of maintenance therapy is either 60 Biological monitoring or 90 mg/kg once a day in 8 and 22 centers, respectively. It is Quantitative PCR was performed in 44 centers, qualitative (that is, pursued every day for 14 days in 12/30 centers and 5 days a week no quantitative PCR) PCR in 17 and pp65 antigenemia alone in 4. during 2 weeks in 10/30 centers; eight centers did not specify. Seventeen out of 56 programs used both PCR and pp65 antigenemia, and 15 programs used both quantitative and Cidofovir. This component (Cidofovir, Gilead, Forster City, CA, qualitative PCR. Biological material used for viral replication tests USA) is used in 26 centers, at the dose of 5 mg/kg once a week in 20/ was whole blood in 38 centers, plasma in 22 centers or separated 26 centers and of 1 mg/kg three times a week in six centers. Duration cells in 7 centers. In 51 centers, 91% results were expressed as of treatment is 2 weeks in 21 centers; five centers did not specify. CMV DNA copies/mL and in 12 as qualitative response—that is, positive or negative. Twelve out of 56 centers validated their Valganciclovir. Valganciclovir (Glaxo-SmithKline, London, UK) is results in copies of DNA of CMV reported to be X cells. Nine used in 27 centers; 4/27 prescribe it even before engraftment. centers had precise X values: 10 000 (1 center), 20 000 (1 center), Dose widely differs between centers: 15 mg/kg per 12 h, 18 mg/kg 2 100 000 (5 centers), 200 000 (1 center), 250 000 (1 center). bid, 20 mg/kg bid, 520 mg/m bid, 900 mg total dose bid and Biological monitoring of viral replication starts after day þ 7 450 mg/day. Treatment duration is 14 days in 18 centers. It is following the graft in 36 programs, in which 13 centers start shortened in seven centers if virus replication becomes undetect- monitoring after engraftment and 5 when leukocyte count is able. A maintenance treatment is used in 16 centers and 3 monitor above 500/mL. This monitoring was performed once a week in 34 the Valganciclovir plasma levels. centers and twice a week in 18 centers. Two out of 56 centers monitor CMV three times per week. Treatment failure Monitoring was carried out until discontinuation of immuno- Treatment failure was defined by persistence of positive suppression in 30 centers, up to immune reconstitution in antigenemia in 12/21 centers or decrease in viral load of less 29 centers (without given precisions about definition of immune than 1 log in 27/44 centers, after 1 week of first-line therapy for reconstitution) and up to day þ 100 after HSCT in 20 centers. 11/56 centers and 2 weeks for 29/56 centers. Fourteen centers modulated their monitoring schedule regarding stem cell source and donor type. Positive threshold for real-time PCR was 1000 copies/mL in 33/ Second-line therapy 44 centers and 5000 copies/mL in 4/44 centers. Positive threshold In the case of treatment failure, change of antivirals was the was 1 cell/200 000 in 8/21 centers and 2 cells/200 000 in 6/21 option in 51 centers. If Ganciclovir was the first-line drug, it could centers using pp65 antigenemia (threshold has been not indicated be switched for Forscarnet in 49 centers or for Cidofovir in 5. If by the seven remaining centers). Foscarnet was the first-line drug, it could be switched for Ganciclovir in 13 centers or for Cidofovir in 9. Prophylactic strategy Association of two antiviral drugs is an option in 21 centers: Ganciclovir–Foscarnet association is used in 18 centers, A prophylaxis approach was used in 21 programs. Among these Ganciclovir–Cidofovir in 5 centers and Foscarnet–Cidofovir in programs, 13/21 choose this option after the analysis of the 1 center. serological donor/recipient CMV status. Antivirals for this indica- tion were aciclovir (Glaxo-SmithKline, London, UK) for 9/21, ganciclovir (Roche, Basel, Switzerland) for 8/21 and foscarnet Resistant strains (Clinigen Healthcare, Burton-on-Trent, UK) for 1/21; three centers In 23 centers, the presence of a drug-resistant CMV mutant was did not specify the drug used. checked in case of treatment failure: in 9 centers by restriction

& 2014 Macmillan Publishers Limited Bone Marrow Transplantation (2014) 276 – 279 CMV in pediatric transplantation: results of an EBMT survey T Bontant et al 278 analysis and in 14 centers by sequencing. Delays of result indicated that both strategies still remained useful.24 However, validation fluctuate from 3 days to 6 weeks. there was no available study, either in adult or pediatric population, allowing clear recommendations for choosing either prophylactic or pre-emptive strategy. By definition, prophylactic Cell therapy strategy exposes all patients to antiviral drugs and to side This treatment technique was performed in 11 centers. Indications effects.13–16 It also could be possible—but not demonstrated— were as follows: CMV disease and treatment failure in 10, delay of that this strategy may facilitate the emergence of resistant viral immune reconstitution in 4, development or co-existence of active strains. On the other hand, some studies report an excess of late GVHD in three programs and antiviral drug toxicity in one center. CMV reactivation as compared with prophylactic strategy and may be more late CMV diseases.14 For therapy, Ganciclovir is the first-line therapy chosen by DISCUSSION almost all the centers (55/56). The dose of induction therapy is This EBMT survey demonstrates that each responding center has 5 mg/kg per 12 h (47/55) during 14 days in (20/47) centers. Other its own management protocol for CMV infection. In this survey, we medicines are less often prescribed and there is much more did not distinguish management between reactivation/infection variation in the dosage, especially for Valganciclovir. and disease. This situation is probably because of the absence of For second-line therapy, analysis of results show that definition sufficient and comprehensive data leading to stringent treatment of treatment failure is not so clear and that 23/56 centers seek for protocols and underlines the need for both, academic and resistant strain in this situation. There is no common approach industry studies. Many centers have developed protocols, about antiviral switch or association of two molecules.10 This last generally on previous experience or local conditions (laboratory option is chosen in 22/56 programs. equipment, drug availability and cost). Differences regarding toxic profile—for example, renal toxicity For example, and as mentioned below, biological monitoring of for foscarnet and bone marrow toxicity for ganciclovir—limited viral replication, biological tests and their schedules are probably antiviral activity (primary or secondary viral strain resistance), chosen in collaboration with laboratories. The most frequently added to the absolute lack of trials studying pharmacokinetics of used technique is real-time PCR (41/56). This technique is easy to these different compounds or comparing the results obtained perform for laboratories, as it can be performed in batches on with different drugs and different schedules could explain the stored samples and can be automated. While pp65 antigenemia wide heterogeneity we report here. needs a 6-hours manual technique to be performed on fresh We did not ask any question regarding the use of either blood sample specimen, requires subjective interpretation and is polyvalent or CMV-specific IgIV in the center strategies. However, not reliable in neutropenic patients.29,30 However, Marchetti none of the centers specified to use it in free comment space et al.31 demonstrated that RT–PCR is a reliable diagnostic tool included in the questionnaire. and that it can be more effective than pp65-based assays in Cell therapy—a very recent treatment modality—seems to be monitoring CMV infection progression and in guiding therapy in used in 11/56 of the responding centers. To date, cell therapy immunocompromised patients.31 Procedure differences between techniques are difficult to perform, in terms of logistics for the centers exist also in the performance of real-time PCR: in the production of virus-specific T cells. A new technique, described by sample type (whole-blood sample (38/56), plasma (22/56) or Feuchtinger et al.25 has been described and used in a clinical trial. separated cells (7/56)), in the description of the results (CMV DNA The development of multi-virus-specific T cells associated to the copies/mL (12/56), copies of CMV DNA reported to X cells (12/56)), availability of CTL from third-party donors and/or from banks in the time from which monitoring starts (36/56 in the first week could modify the habits of the centers in the future.35 following the graft, 13/56 at engraftment, 5/56 leukocyte count above 500/mL) and the testing frequency (once a week in 34/56 centers but differs in other centers). Another issue is that values CONCLUSION obtained with different real-time PCR protocols are not We conclude in this survey report that CMV monitoring, comparable. An international standard reference for prophylaxis and treatment in children are still an ‘individualized’ amplification of CMV DNA should help to standardize assays of 32 or a ‘center’ process. Our data may provide a basis for the real-time PCR protocol for CMV infection diagnosis. establishment of an algorithm to manage CMV infection in As immunological reconstitution differs between patients different pediatric transplantation scenarios. This survey attests according to stem cell source, donor/recipient HLA match level, that management of this infection varies widely between centers the intensity of conditioning regimen and both prophylaxis and as clinicians have many choices to make with few rational data. treatment of GVHD common standards are not appropriate for the International standards for human CMV for nucleic acid amplifica- different conditions. In the near future, integration of immuno- tion techniques are in development and will probably help to logical parameter monitoring in the algorithm for CMV monitoring reach consensus on biologic monitoring and positive threshold. and therapy should change our approach by detecting patients 26 More studies are needed to help in finding the appropriate who could spontaneously control CMV infection. schedule and dosage in pediatric transplantation, in both first- and All centers that answered to this survey use pre-emptive second-line therapies. The current development of cell therapy strategy, including those using prophylactic strategy also for either has already changed CMV chemorefractory disease management. all or only the high-risk patients. The definition of high-risk New drugs are under development and we have to strongly argue patients differs between the centers, either only seronegative with pharmaceutical companies to convince them of the huge donor/seropositive recipient or all D/R pair with at least one importance of pediatric investigation plans. positive member. Pre-emptive strategy was described for the first time by Boeckh et al. in 1996 and was rapidly and widely used,34 In this paper, Boeckh et al.34 reported a randomized double-blind Propositions study on 226 allogeneic HSCT recipients who received either Response analysis may allow us to propose the basic rules for prophylactic or pre-emptive ganciclovir. They observed more CMV human CMV infection management in pediatric allogeneic HSCT. diseases by day 100 (but not by day 180) in pre-emptive arm than Monitoring should be performed with quantitative PCR, at least in the prophylactic arm and more invasive fungal infection in once a week. We suggest that 14 days of Ganciclovir at 5 mg/kg/bid prophylactic arm than in the preventive one. Finally, OS was or Foscarnet at 90 mg/kg/bid should be the first-line treatment of identical in both arms. In 2011 ASH education program, Boekh CMV infection in an pre-emptive strategy. Cidofovir may be an option

Bone Marrow Transplantation (2014) 276 – 279 & 2014 Macmillan Publishers Limited CMV in pediatric transplantation: results of an EBMT survey T Bontant et al 279 only for second-line treatment. Data are not sufficient to propose the 13 Boeckh M, Nichols WG, Papanicolaou G, Rubin R, Wingard JR, Zaia J. dosage for Cidofovir. Specific immunotherapy using anti-CMV CTL Cytomegalovirus in hematopoietic stem cell transplant recipients: current status, has to be considered as the second- or the third-line therapy. known challenges, and future strategies. Biol Blood Marrow Transplant 2003; 9: A schedule for maintenance therapy cannot be proposed either. 543–558. This survey was not about finding a consensus; however, these 14 Matthes-Martin S, Lion T, Aberle SW, Fritsch G, Lawitschka A, Bittner B et al. propositions are the reflection of the centers’ most shared attitude. 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