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T cell therapy of Epstein–Barr virus and adenovirus infections after hemopoietic stem cell transplant
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Blood Cells, Molecules, and Diseases 40 (2008) 68 – 70 www.elsevier.com/locate/ybcmd
T cell therapy of Epstein–Barr virus and adenovirus infections after hemopoietic stem cell transplant P. Comoli ⁎, S. Basso, M. Labirio, F. Baldanti, R. Maccario, F. Locatelli Pediatric Hematology/Oncology, and Virology Service, Fondazione IRCCS Policlinico S. Matteo, University of Pavia, Viale Golgi 19, 27100 Pavia, Italy Submitted 22 June 2007 Available online 29 September 2007 (Communicated by M. Lichtman, M.D., 30 June 2007)
Abstract Epstein–Barr virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD) and adenovirus (AdV)-related pathologies are lifethreatening complications of immunosuppression in recipients of hematopoietic stem cell transplantation (HSCT). In certain cohorts (unrelated and haploidentical donor HSCT, T-cell-depleted allograft), the risk of developing these complications is higher. Here we describe the impact of T cell therapy, within programs of specific routine surveillance and preemptive treatment, on the course of EBV infection, and development of related disease, in pediatric recipients of T-cell-depleted, HLA-haploidentical HSCT. Future prospectives include the transfer of this technology to treat AdV-related complications following HSCT. © 2007 Published by Elsevier Inc. Keywords: Epstein–Barr virus; Post-transplant lymphoproliferative disease; Rituximab; Adoptive cellular therapy; Adenovirus; Hemopoietic stem cell transplantation
Introduction Viral infections remain a major problem for the recipient of a T-cell-depleted stem cell transplant (HSCT) from a HLAhaploidentical donor [1]. In particular, primary infection or reactivation of EBV in these immunocompromised hosts may progress to onset of a post-transplant lymphoproliferative disorder (PTLD), a complication mostly associated with the proliferation of EBVinfected B cells, whose expansion in immunocompetent individuals is controlled by cytotoxic T lymphocytes (CTLs) [2–4]. The occurrence of PTLD is still associated with a high mortality rate, notwithstanding the use of specific therapeutic approaches [5,6]. Therapy for PTLD is more effective and safer if initiated early [7]; at best, prevention of PTLD, through frequent monitoring of EBV DNA in the peripheral blood by quantitative polymerase chain reaction (PCR) to identify the pre-clinical phase of the disease and application of preemptive treatment, is the option of choice in high-risk cohorts. While waiting for the definition of a
⁎ Corresponding author. Fax: +39 0382 527976. E-mail address: [email protected] (P. Comoli). 1079-9796/$ - see front matter © 2007 Published by Elsevier Inc. doi:10.1016/j.bcmd.2007.06.020
highly specific predictive test, effective treatments with low toxicity are required for a preemptive approach. EBV-related PTLD derive from uncontrolled proliferation of B cells carrying latent EBV. Thus, in the last decade, development of therapeutic strategies that allow selective abrogation of the whole B cell compartment, such as anti-B-cell monoclonal antibodies [8–10] or those B cells bearing EBV, such as DLI [11] or EBVspecific CTL transfer [12,13], has provided relatively safe means to treat PTLD. Infusion of EBV-specific CTLs for prevention of PTLD in recipients of HSCT from a full HLA-haplotype mismatched family donor, who do not usually receive post-transplant immunosuppression, could theoretically increase the risk of acute GVHD. Recently, it has been shown that preemptive therapy with rituximab may improve outcome in recipients of partially T-celldepleted HSCT from an HLA-matched family donor or unrelated volunteer at increased risk for PTLD [14]. Thus, for our program of pediatric HSCT from a HLA-haploidentical donor, we elected to start an EBV surveillance program based on serial blood Q-PCR monitoring of EBV DNA and firstline preemptive treatment with rituximab, followed by adoptive T cell therapy with EBV-specific CTL in patients unresponsive to anti-CD20. Preliminary data [15] show that, with this EBV
P. Comoli et al. / Blood Cells, Molecules, and Diseases 40 (2008) 68–70
infection management program, we can successfully reduce the incidence and mortality of PTLD in pediatric recipients of T-celldepleted, HLA-haploidentical HSCT from family donors. We now present results of a larger cohort and discuss the opportunity for extending this surveillance program to other viruses relevant to HSCT. Patients and methods From August 2001 to date, a total of 46 patients received a Tcell-depleted HSCT from an HLA partially matched family donor at the Pediatric Haematology/Oncology Unit, Fondazione IRCCS Policlinico S. Matteo, Pavia, and were subsequently routinely monitored for EBV infection. Patients' parents gave written informed consent at the time of enrollment and the study was conducted according to Institutional guidelines. Patient's EBV DNA levels in PBMC were monitored by quantitative PCR [16] weekly for the first 3 months, and monthly thereafter until 1 year post-transplant. Upon detection of N1000 copies/105 PBMC (or N1000 copies/10 μl whole blood in patients prior to hematopoietic reconstitution) in two subsequent samples, patients were treated with rituximab (375 mg/m2/dose × 2 doses, 1 week apart). In the presence of EBV disease and/or persistent or increasing EBV DNA levels, patients were infused with EBVspecific CTLs generated from the donor. Following GMP standard procedures, donor EBV-specific CTLs were reactivated and expanded in vitro from fresh or frozen PBMC according to a method previously described [17]. Before cryopreservation, T cells were examined for immunophenotype, sterility, and EBV specificity in a standard 51Cr-release assay against a panel of targets including autologous B-lymphoblastoid cell lines (LCL) and recipient PHA blasts, and HLA-mismatched allogeneic B-LCL. Validated CTL lots were administered to patients not responding to rituximab (persistent or recurrent positivity for EBV DNA in blood), in escalating doses and at weekly intervals, starting from a dose of 0.5 × 106 cells/kg body weight. To evaluate the effects of CTL infusion on EBV-specific T cell immunity, IFN-γ secreting lymphocytes were measured by ELISPOT assay and specific cytotoxicity of reactivated CTL lines evaluated by 51-chromium release assay, on peripheral blood samples collected immediately before rituximab treatment, 2– 4 weeks after anti-CD20 infusion, and 2–8 weeks after CTL infusion, following methods previously described [15,17,18]. Results A total of 46 HSCT recipients have been evaluated so far, with a median follow-up of 28 months. Twelve of the 46 patients developed sustained viremia, requiring treatment. Rituximab was well tolerated and, after a two-dose course, clearance of peripheral blood B lymphocytes and of EBV-load were observed in 10/12 children, while in two patients low levels of CD19+ B cells and EBV DNA persisted. Surprisingly, however, in four of the ten patients who initially responded to anti-CD20 therapy, a new increase of EBV load was observed shortly after the last rituximab administration. In the six HSCT recipients with unsatisfactory response to rituximab, the persistence or new increase of EBV
69
DNA levels was accompanied by fever and fatigue (1 patient) and overt PTLD (5 patients). In four of the five patients who developed PTLD, viremia and appearance of clinical symptoms coincided with the emergence of a subset of CD20-negative, CD19+ B cells with clonal rearrangement in peripheral blood, as evaluated by a semi-nested PCR protocol for amplification of the rearranged heavy chain immunoglobulin genes [19]. The six patients with PTLD and/or relapsed viremia received one or more doses of EBV CTLs as rescue therapy. No adverse events were recorded after EBV CTL infusion, and none of the patients developed acute GVHD as a consequence of HLA-haploidentical donor CTL administration. EBV CTL therapy was able to induce clearance of EBV DNA levels and circulating CD19+ B lymphocytes in all patients, and the five HSCT recipients with PTLD achieved complete remission after a median time of 20 days (range 13–50 days). Analysis of EBV-specific immunity revealed that viral clearance and clinical remission coincided with reconstitution of virus-specific T cell responses. In particular, production of IFNγ, measured in an ELISPOT assay, and specific cytotoxicity of reactivated EBV-CTL after 10 days culture, which were low/ absent at baseline, and after anti-CD20 infusion, reached protective levels after adoptive T cell transfer. Discussion Our updated results indicate that an EBV surveillance program and preemptive/early treatment of PTLD, including the use of virus-specific cytotoxic T cells, may help reduce the incidence of PTLD and abrogates mortality from the disease in pediatric recipients of T-cell-depleted HSCT from HLA-haploidentical family donors. Using this strategy, we were able to identify 12 patients with EBV DNA levels N 1000 copies/105 peripheral blood mononuclear cells, eligible for preemptive treatment. Of these 12 patients, only six showed stable viral clearance after rituximab therapy. Thus, the 50% rate of stable response to rituximab, and ≈40% incidence of PTLD after anti-CD20 preemptive therapy observed in our preliminary analysis [15] is confirmed by this follow-up study conducted on this larger cohort of pediatric T-cell-depleted HSCT recipients. PTLD development coincided with the appearance/ expansion in the peripheral blood of CD19+, CD20-escape mutants with characteristics of tumor cells rather than resting B cells. In patients unresponsive to rituximab treatment, transfer of donor EBV-specific CTL at the doses commonly employed for preemptive therapy [7,12,17] was able to induce viral DNA clearance, and remission of established PTLD in all patients. The success of T cell transfer in all patients treated for EBV-related PTLD may be related to the fact that (i) EBV CTLs are prevalently of CD8 phenotype, the subset that mediates clearance of virally infected cells, (ii) are polyclonal and polyspecific, thus reducing the risk of selecting escape mutants, and (iii) the number of T cells infused is relatively high. This study indicates that rituximab may represent a useful tool, devoid of major complications, to control viral replication; however, even in a preemptive treatment setting, its efficacy is suboptimal. Thus, in high-risk cohorts, such as recipients of extensively
70
P. Comoli et al. / Blood Cells, Molecules, and Diseases 40 (2008) 68–70
T-cell-depleted haplo-HSCT, the availability of additional, lowtoxicity therapeutic tools, such as EBV-specific CTLs, is paramount for a significant improvement in outcome. In order to implement our strategy for management of viral infection in recipients of extensively T-cell-depleted haplo-HSCT, we elected to start a surveillance program for adenovirus (AdV) infection. AdV infections in immunocompromised hosts are usually not recognized until a late stage of disease is reached, because clinical symptoms of infection, such as diarrhea and fever, are non-specific. This has prompted an ongoing collaborative effort (in conjunction with the Virology Service, IRCCS Policlinico San Matteo) to develop a real-time quantitative PCR, which should aid the early detection of circulating virus and guide clinical treatment decisions. In conjunction with the optimization of the PCR technology to detect AdV DNA, we are also conducting scale-up experiments to validate a method of in vitro culture to expand T cells specific for AdV. These AdV-specific T cells can be prepared, under GMP conditions, within a time period of 26 days from the majority of donors using a set of five AdV– hexon protein-derived peptides [20]. We plan to proceed to preemptive treatment of HSCT recipients with positive blood viral load, by administration of antiviral chemotherapy in association with AdV-specific T cells. Acknowledgments The authors wish to thank Laurene Kelly for editing the manuscript. This work was supported in part by grants from the Associazione Italiana Ricerca sul Cancro (AIRC) to P.C. and F.L.; grants RFM/03, RFM/04, RFM/05 to F.L., R.M. and F.B.; grant FP6-Allostem to F.L.; grant AIFA05 to F.L. This paper is based upon a presentation at a Focused Workshop on Haploidentical Stem Cell Transplantation sponsored by The Leukemia & Lymphoma Society held in Catania, Italy from 4th to 6th October 2007. References [1] F. Aversa, A. Tabilio, A. Velardi, et al., Treatment of high risk acute leukemia with T-cell depleted stem cells from related donors with one fully mismatched HLA haplotype, N. Engl. J. Med. 339 (1998) 1186–1193. [2] R.S. Shapiro, K. McClain, G. Frizzera, et al., Epstein–Barr virus associated B cell lymphoproliferative disorders following bone marrow transplantation, Blood 71 (1988) 1234–1243. [3] R. O'Reilly, T.N. Small, E. Papadopulos, et al., Biology and adoptive cell therapy of Epstein–Barr-virus associated lymphoproliferative disorders in recipients of marrow allografts, Immunol. Rev. 157 (1997) 195–216.
[4] A.B. Rickinson, D.J. Moss, Human cytotoxic T lymphocyte responses to Epstein–Barr virus infection, Annu. Rev. Immunol. 15 (1997) 405–431. [5] R.E. Curtis, L.B. Travis, P.A. Rowlings, et al., Risk of lymphoproliferative disorders after bone marrow transplantation: a multi-institutional study, Blood 94 (1999) 2208–2216. [6] T.G. Gross, M. Steinbuch, T. DeFor, et al., B cell lymphoproliferative disorders following hematopoietic stem cell transplantation: risk factors, treatment and outcome, Bone Marrow Transplant 23 (1999) 251–258. [7] P. Comoli, C.M. Rooney, Treatment of Epstein–Barr virus infections, in: A. Tselis, H.B. Jenson (Eds.), Epstein–Barr virus, Taylor & Francis, New York, 2006, pp. 353–374. [8] A. Fischer, S. Blanche, J. LeBidois, et al., Anti-B-cell monoclonal antibodies in the treatment of severe B-cell lymphoproliferative syndrome following bone marrow and organ transplantation, N. Engl. J. Med. 324 (1991) 1451–1456. [9] I. Kuehnle, M.H. Huls, Z. Liu, et al., CD20 monoclonal antibody (rituximab) for therapy of Epstein–Barr virus lymphoma after hemopoietic stem-cell transplantation, Blood 95 (2000) 1502–1505. [10] S. Choquet, V. Leblond, R. Herbrecht, et al., Efficacy and safety of rituximab in B-cell post-transplantation lymphoproliferative disorders: results of a prospective multicenter phase 2 study, Blood 107 (2006) 3053–3057. [11] E.B. Papadopoulos, M. Ladanyi, D. Emanuel, et al., Infusions of donor leukocytes as treatment of Epstein–Barr virus associated lymphoproliferative disorders complicating allogeneic marrow transplantation, N. Engl. J. Med. 330 (1994) 1185–1191. [12] C.M. Rooney, C.A. Smith, C. Ng, et al., Use of gene-modified virus-specific T lymphocytes to control Epstein–Barr virus-related lymphoproliferation, Lancet 345 (1995) 9–13. [13] H.E. Heslop, C.Y.C. Ng, C. Li, et al., Long-term restoration of immunity against Epstein–Barr virus infection by adoptive transfer of gene-modified virus-specific T lymphocytes, Nat. Med. 2 (1996) 551–555. [14] J.W.J. van Esser, H.G.M. Niesters, B. van der Holt, et al., Prevention of Epstein–Barr virus-lymphoproliferative disease by molecular monitoring and preemptive rituximab in high risk patients after allogeneic stem cell transplantation, Blood 99 (2002) 4364–4369. [15] P. Comoli, S. Basso, M. Zecca, et al., Preemptive therapy of EBV-related lymphoproliferative disease after pediatric haploidentical stem cell transplantation, Am. J. Transplant 7 (2007) 1648–1655. [16] F. Baldanti, P. Grossi, M. Furione, et al., High levels of Epstein–Barr virus DNA in blood of solid-organ transplant recipients and their value in predicting posttransplant lymphoproliferative disorders, J. Clin. Microbiol. 38 (2000) 613–619. [17] P. Comoli, M. Labirio, S. Basso, et al., Infusion of autologous Epstein– Barr virus (EBV)-specific cytotoxic T cells for prevention of EBV-related lymphoproliferative disorder in solid organ transplant recipients with evidence of active virus replication, Blood 99 (2002) 2592–2598. [18] P. Comoli, P. Pedrazzoli, R. Maccario, et al., Cell therapy of stage IV nasopharyngeal carcinoma with autologous EBV-targeted cytotoxic T-lymphocytes, J. Clin. Oncol. 23 (2005) 8942–8949. [19] A. Achille, A. Scarpa, M. Montresor, et al., Routine application for polymerase chain reaction in the diagnosis of monoclonality of B-cell lymphoid proliferations, Diagn. Mol. Pathol. 4 (1995) 14–24. [20] L.A. Veltrop-Duits, B. Heemskerk, C.C. Sombroek, et al., Human CD4+ Tcells stimulated by conserved adenovirus 5 hexon peptides recognize cells infected with different species of human adenovirus, Eur. J. Immunol. 36 (2006) 2410–2423.
Blood Cells, Molecules, and Diseases 40 (2008) 68 – 70 www.elsevier.com/locate/ybcmd
T cell therapy of Epstein–Barr virus and adenovirus infections after hemopoietic stem cell transplant P. Comoli ⁎, S. Basso, M. Labirio, F. Baldanti, R. Maccario, F. Locatelli Pediatric Hematology/Oncology, and Virology Service, Fondazione IRCCS Policlinico S. Matteo, University of Pavia, Viale Golgi 19, 27100 Pavia, Italy Submitted 22 June 2007 Available online 29 September 2007 (Communicated by M. Lichtman, M.D., 30 June 2007)
Abstract Epstein–Barr virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD) and adenovirus (AdV)-related pathologies are lifethreatening complications of immunosuppression in recipients of hematopoietic stem cell transplantation (HSCT). In certain cohorts (unrelated and haploidentical donor HSCT, T-cell-depleted allograft), the risk of developing these complications is higher. Here we describe the impact of T cell therapy, within programs of specific routine surveillance and preemptive treatment, on the course of EBV infection, and development of related disease, in pediatric recipients of T-cell-depleted, HLA-haploidentical HSCT. Future prospectives include the transfer of this technology to treat AdV-related complications following HSCT. © 2007 Published by Elsevier Inc. Keywords: Epstein–Barr virus; Post-transplant lymphoproliferative disease; Rituximab; Adoptive cellular therapy; Adenovirus; Hemopoietic stem cell transplantation
Introduction Viral infections remain a major problem for the recipient of a T-cell-depleted stem cell transplant (HSCT) from a HLAhaploidentical donor [1]. In particular, primary infection or reactivation of EBV in these immunocompromised hosts may progress to onset of a post-transplant lymphoproliferative disorder (PTLD), a complication mostly associated with the proliferation of EBVinfected B cells, whose expansion in immunocompetent individuals is controlled by cytotoxic T lymphocytes (CTLs) [2–4]. The occurrence of PTLD is still associated with a high mortality rate, notwithstanding the use of specific therapeutic approaches [5,6]. Therapy for PTLD is more effective and safer if initiated early [7]; at best, prevention of PTLD, through frequent monitoring of EBV DNA in the peripheral blood by quantitative polymerase chain reaction (PCR) to identify the pre-clinical phase of the disease and application of preemptive treatment, is the option of choice in high-risk cohorts. While waiting for the definition of a
⁎ Corresponding author. Fax: +39 0382 527976. E-mail address: [email protected] (P. Comoli). 1079-9796/$ - see front matter © 2007 Published by Elsevier Inc. doi:10.1016/j.bcmd.2007.06.020
highly specific predictive test, effective treatments with low toxicity are required for a preemptive approach. EBV-related PTLD derive from uncontrolled proliferation of B cells carrying latent EBV. Thus, in the last decade, development of therapeutic strategies that allow selective abrogation of the whole B cell compartment, such as anti-B-cell monoclonal antibodies [8–10] or those B cells bearing EBV, such as DLI [11] or EBVspecific CTL transfer [12,13], has provided relatively safe means to treat PTLD. Infusion of EBV-specific CTLs for prevention of PTLD in recipients of HSCT from a full HLA-haplotype mismatched family donor, who do not usually receive post-transplant immunosuppression, could theoretically increase the risk of acute GVHD. Recently, it has been shown that preemptive therapy with rituximab may improve outcome in recipients of partially T-celldepleted HSCT from an HLA-matched family donor or unrelated volunteer at increased risk for PTLD [14]. Thus, for our program of pediatric HSCT from a HLA-haploidentical donor, we elected to start an EBV surveillance program based on serial blood Q-PCR monitoring of EBV DNA and firstline preemptive treatment with rituximab, followed by adoptive T cell therapy with EBV-specific CTL in patients unresponsive to anti-CD20. Preliminary data [15] show that, with this EBV
P. Comoli et al. / Blood Cells, Molecules, and Diseases 40 (2008) 68–70
infection management program, we can successfully reduce the incidence and mortality of PTLD in pediatric recipients of T-celldepleted, HLA-haploidentical HSCT from family donors. We now present results of a larger cohort and discuss the opportunity for extending this surveillance program to other viruses relevant to HSCT. Patients and methods From August 2001 to date, a total of 46 patients received a Tcell-depleted HSCT from an HLA partially matched family donor at the Pediatric Haematology/Oncology Unit, Fondazione IRCCS Policlinico S. Matteo, Pavia, and were subsequently routinely monitored for EBV infection. Patients' parents gave written informed consent at the time of enrollment and the study was conducted according to Institutional guidelines. Patient's EBV DNA levels in PBMC were monitored by quantitative PCR [16] weekly for the first 3 months, and monthly thereafter until 1 year post-transplant. Upon detection of N1000 copies/105 PBMC (or N1000 copies/10 μl whole blood in patients prior to hematopoietic reconstitution) in two subsequent samples, patients were treated with rituximab (375 mg/m2/dose × 2 doses, 1 week apart). In the presence of EBV disease and/or persistent or increasing EBV DNA levels, patients were infused with EBVspecific CTLs generated from the donor. Following GMP standard procedures, donor EBV-specific CTLs were reactivated and expanded in vitro from fresh or frozen PBMC according to a method previously described [17]. Before cryopreservation, T cells were examined for immunophenotype, sterility, and EBV specificity in a standard 51Cr-release assay against a panel of targets including autologous B-lymphoblastoid cell lines (LCL) and recipient PHA blasts, and HLA-mismatched allogeneic B-LCL. Validated CTL lots were administered to patients not responding to rituximab (persistent or recurrent positivity for EBV DNA in blood), in escalating doses and at weekly intervals, starting from a dose of 0.5 × 106 cells/kg body weight. To evaluate the effects of CTL infusion on EBV-specific T cell immunity, IFN-γ secreting lymphocytes were measured by ELISPOT assay and specific cytotoxicity of reactivated CTL lines evaluated by 51-chromium release assay, on peripheral blood samples collected immediately before rituximab treatment, 2– 4 weeks after anti-CD20 infusion, and 2–8 weeks after CTL infusion, following methods previously described [15,17,18]. Results A total of 46 HSCT recipients have been evaluated so far, with a median follow-up of 28 months. Twelve of the 46 patients developed sustained viremia, requiring treatment. Rituximab was well tolerated and, after a two-dose course, clearance of peripheral blood B lymphocytes and of EBV-load were observed in 10/12 children, while in two patients low levels of CD19+ B cells and EBV DNA persisted. Surprisingly, however, in four of the ten patients who initially responded to anti-CD20 therapy, a new increase of EBV load was observed shortly after the last rituximab administration. In the six HSCT recipients with unsatisfactory response to rituximab, the persistence or new increase of EBV
69
DNA levels was accompanied by fever and fatigue (1 patient) and overt PTLD (5 patients). In four of the five patients who developed PTLD, viremia and appearance of clinical symptoms coincided with the emergence of a subset of CD20-negative, CD19+ B cells with clonal rearrangement in peripheral blood, as evaluated by a semi-nested PCR protocol for amplification of the rearranged heavy chain immunoglobulin genes [19]. The six patients with PTLD and/or relapsed viremia received one or more doses of EBV CTLs as rescue therapy. No adverse events were recorded after EBV CTL infusion, and none of the patients developed acute GVHD as a consequence of HLA-haploidentical donor CTL administration. EBV CTL therapy was able to induce clearance of EBV DNA levels and circulating CD19+ B lymphocytes in all patients, and the five HSCT recipients with PTLD achieved complete remission after a median time of 20 days (range 13–50 days). Analysis of EBV-specific immunity revealed that viral clearance and clinical remission coincided with reconstitution of virus-specific T cell responses. In particular, production of IFNγ, measured in an ELISPOT assay, and specific cytotoxicity of reactivated EBV-CTL after 10 days culture, which were low/ absent at baseline, and after anti-CD20 infusion, reached protective levels after adoptive T cell transfer. Discussion Our updated results indicate that an EBV surveillance program and preemptive/early treatment of PTLD, including the use of virus-specific cytotoxic T cells, may help reduce the incidence of PTLD and abrogates mortality from the disease in pediatric recipients of T-cell-depleted HSCT from HLA-haploidentical family donors. Using this strategy, we were able to identify 12 patients with EBV DNA levels N 1000 copies/105 peripheral blood mononuclear cells, eligible for preemptive treatment. Of these 12 patients, only six showed stable viral clearance after rituximab therapy. Thus, the 50% rate of stable response to rituximab, and ≈40% incidence of PTLD after anti-CD20 preemptive therapy observed in our preliminary analysis [15] is confirmed by this follow-up study conducted on this larger cohort of pediatric T-cell-depleted HSCT recipients. PTLD development coincided with the appearance/ expansion in the peripheral blood of CD19+, CD20-escape mutants with characteristics of tumor cells rather than resting B cells. In patients unresponsive to rituximab treatment, transfer of donor EBV-specific CTL at the doses commonly employed for preemptive therapy [7,12,17] was able to induce viral DNA clearance, and remission of established PTLD in all patients. The success of T cell transfer in all patients treated for EBV-related PTLD may be related to the fact that (i) EBV CTLs are prevalently of CD8 phenotype, the subset that mediates clearance of virally infected cells, (ii) are polyclonal and polyspecific, thus reducing the risk of selecting escape mutants, and (iii) the number of T cells infused is relatively high. This study indicates that rituximab may represent a useful tool, devoid of major complications, to control viral replication; however, even in a preemptive treatment setting, its efficacy is suboptimal. Thus, in high-risk cohorts, such as recipients of extensively
70
P. Comoli et al. / Blood Cells, Molecules, and Diseases 40 (2008) 68–70
T-cell-depleted haplo-HSCT, the availability of additional, lowtoxicity therapeutic tools, such as EBV-specific CTLs, is paramount for a significant improvement in outcome. In order to implement our strategy for management of viral infection in recipients of extensively T-cell-depleted haplo-HSCT, we elected to start a surveillance program for adenovirus (AdV) infection. AdV infections in immunocompromised hosts are usually not recognized until a late stage of disease is reached, because clinical symptoms of infection, such as diarrhea and fever, are non-specific. This has prompted an ongoing collaborative effort (in conjunction with the Virology Service, IRCCS Policlinico San Matteo) to develop a real-time quantitative PCR, which should aid the early detection of circulating virus and guide clinical treatment decisions. In conjunction with the optimization of the PCR technology to detect AdV DNA, we are also conducting scale-up experiments to validate a method of in vitro culture to expand T cells specific for AdV. These AdV-specific T cells can be prepared, under GMP conditions, within a time period of 26 days from the majority of donors using a set of five AdV– hexon protein-derived peptides [20]. We plan to proceed to preemptive treatment of HSCT recipients with positive blood viral load, by administration of antiviral chemotherapy in association with AdV-specific T cells. Acknowledgments The authors wish to thank Laurene Kelly for editing the manuscript. This work was supported in part by grants from the Associazione Italiana Ricerca sul Cancro (AIRC) to P.C. and F.L.; grants RFM/03, RFM/04, RFM/05 to F.L., R.M. and F.B.; grant FP6-Allostem to F.L.; grant AIFA05 to F.L. This paper is based upon a presentation at a Focused Workshop on Haploidentical Stem Cell Transplantation sponsored by The Leukemia & Lymphoma Society held in Catania, Italy from 4th to 6th October 2007. References [1] F. Aversa, A. Tabilio, A. Velardi, et al., Treatment of high risk acute leukemia with T-cell depleted stem cells from related donors with one fully mismatched HLA haplotype, N. Engl. J. Med. 339 (1998) 1186–1193. [2] R.S. Shapiro, K. McClain, G. Frizzera, et al., Epstein–Barr virus associated B cell lymphoproliferative disorders following bone marrow transplantation, Blood 71 (1988) 1234–1243. [3] R. O'Reilly, T.N. Small, E. Papadopulos, et al., Biology and adoptive cell therapy of Epstein–Barr-virus associated lymphoproliferative disorders in recipients of marrow allografts, Immunol. Rev. 157 (1997) 195–216.
[4] A.B. Rickinson, D.J. Moss, Human cytotoxic T lymphocyte responses to Epstein–Barr virus infection, Annu. Rev. Immunol. 15 (1997) 405–431. [5] R.E. Curtis, L.B. Travis, P.A. Rowlings, et al., Risk of lymphoproliferative disorders after bone marrow transplantation: a multi-institutional study, Blood 94 (1999) 2208–2216. [6] T.G. Gross, M. Steinbuch, T. DeFor, et al., B cell lymphoproliferative disorders following hematopoietic stem cell transplantation: risk factors, treatment and outcome, Bone Marrow Transplant 23 (1999) 251–258. [7] P. Comoli, C.M. Rooney, Treatment of Epstein–Barr virus infections, in: A. Tselis, H.B. Jenson (Eds.), Epstein–Barr virus, Taylor & Francis, New York, 2006, pp. 353–374. [8] A. Fischer, S. Blanche, J. LeBidois, et al., Anti-B-cell monoclonal antibodies in the treatment of severe B-cell lymphoproliferative syndrome following bone marrow and organ transplantation, N. Engl. J. Med. 324 (1991) 1451–1456. [9] I. Kuehnle, M.H. Huls, Z. Liu, et al., CD20 monoclonal antibody (rituximab) for therapy of Epstein–Barr virus lymphoma after hemopoietic stem-cell transplantation, Blood 95 (2000) 1502–1505. [10] S. Choquet, V. Leblond, R. Herbrecht, et al., Efficacy and safety of rituximab in B-cell post-transplantation lymphoproliferative disorders: results of a prospective multicenter phase 2 study, Blood 107 (2006) 3053–3057. [11] E.B. Papadopoulos, M. Ladanyi, D. Emanuel, et al., Infusions of donor leukocytes as treatment of Epstein–Barr virus associated lymphoproliferative disorders complicating allogeneic marrow transplantation, N. Engl. J. Med. 330 (1994) 1185–1191. [12] C.M. Rooney, C.A. Smith, C. Ng, et al., Use of gene-modified virus-specific T lymphocytes to control Epstein–Barr virus-related lymphoproliferation, Lancet 345 (1995) 9–13. [13] H.E. Heslop, C.Y.C. Ng, C. Li, et al., Long-term restoration of immunity against Epstein–Barr virus infection by adoptive transfer of gene-modified virus-specific T lymphocytes, Nat. Med. 2 (1996) 551–555. [14] J.W.J. van Esser, H.G.M. Niesters, B. van der Holt, et al., Prevention of Epstein–Barr virus-lymphoproliferative disease by molecular monitoring and preemptive rituximab in high risk patients after allogeneic stem cell transplantation, Blood 99 (2002) 4364–4369. [15] P. Comoli, S. Basso, M. Zecca, et al., Preemptive therapy of EBV-related lymphoproliferative disease after pediatric haploidentical stem cell transplantation, Am. J. Transplant 7 (2007) 1648–1655. [16] F. Baldanti, P. Grossi, M. Furione, et al., High levels of Epstein–Barr virus DNA in blood of solid-organ transplant recipients and their value in predicting posttransplant lymphoproliferative disorders, J. Clin. Microbiol. 38 (2000) 613–619. [17] P. Comoli, M. Labirio, S. Basso, et al., Infusion of autologous Epstein– Barr virus (EBV)-specific cytotoxic T cells for prevention of EBV-related lymphoproliferative disorder in solid organ transplant recipients with evidence of active virus replication, Blood 99 (2002) 2592–2598. [18] P. Comoli, P. Pedrazzoli, R. Maccario, et al., Cell therapy of stage IV nasopharyngeal carcinoma with autologous EBV-targeted cytotoxic T-lymphocytes, J. Clin. Oncol. 23 (2005) 8942–8949. [19] A. Achille, A. Scarpa, M. Montresor, et al., Routine application for polymerase chain reaction in the diagnosis of monoclonality of B-cell lymphoid proliferations, Diagn. Mol. Pathol. 4 (1995) 14–24. [20] L.A. Veltrop-Duits, B. Heemskerk, C.C. Sombroek, et al., Human CD4+ Tcells stimulated by conserved adenovirus 5 hexon peptides recognize cells infected with different species of human adenovirus, Eur. J. Immunol. 36 (2006) 2410–2423.