Tanzina Haque

Treatment of Epstein-Barr-virus-positive post-transplantation lymphoproliferative disease with partly HLA-matched allogeneic cytotoxic T cells

BACKGROUND Epstein-Barr virus (EBV)-associated post-transplantation lymphoproliferative disease (PTLD) is a common, often fatal, complication of bone-marrow and solid-organ transplantation. Since tumour growth results from inadequate T-cell control of latent EBV, new immunotherapeutic approaches to treatment are being pioneered. METHODS In a phase 1/2 trial, eight patients with progressive PTLD unresponsive to conventional treatment were given one to six infusions of partly HLA-matched allogeneic EBV-specific cytotoxic T lymphocytes (CTLs) from a frozen bank of CTLs derived from healthy blood donors. FINDINGS Of the five patients who completed treatment, three had complete remission and two had no clinical response. One patient partly responded after two infusions. No graft-versus-host disease or allo-specific antibodies were detected, and graft function improved in three cases. Tumour responses were mainly seen in those with early, localised, polyclonal disease. EBV load in peripheral blood fell to undetectable levels in all patients who responded to treatment, but was more variable in those who did not. INTERPRETATION Treatment of EBV-associated PTLD with partly HLA-matched CTLs grown from unrelated donors is effective. Spontaneous remission is very unlikely to account for tumour regression in our patients; however, a larger, controlled trial is needed to assess this treatment further. The frozen bank of allogeneic CTLs is less prohibitively labour intensive and expensive for wide scale use than treatment with autologous CTLs. Such banks could be established to treat other infectious and neoplastic diseases in many patients.

Complete regression of posttransplant lymphoproliferative disease using partially HLA-matched Epstein Barr virus-specific cytotoxic T cells.

BACKGROUND Adoptive immunotherapy with autologous and donor-derived cytotoxic T lymphocytes (CTL) has recently been used to treat Epstein Barr virus (EBV)-positive posttransplant lymphoproliferative disease (PTLD). METHODS AND RESULTS We report complete regression of EBV-positive PTLD in an 18-month-old small bowel and liver transplant recipient after one infusion of partially human leukocyte antigen (HLA)-matched EBV-specific CTL grown ex vivo from an EBV seropositive unrelated blood donor. No infusion-related toxicity or evidence of graft-versus-host disease was observed. The tumor showed signs of regression within 1 week and EBV load in peripheral blood dropped to undetectable levels. Limiting dilution analyses (LDA) detected no EBV-specific CTL precursor (CTLp) cells before the infusion, and high numbers of CTLp at 4 hr and 24 hr post-CTL infusion. There was a reversal of the CD4/8 ratio in peripheral blood and an increase in HLA-DR positive CD8 cells. The patient has been in complete remission for 24 months. CONCLUSION If this success is repeated in more PTLD patients, then stored CTL could be used for antiviral and antitumor therapies in immunocompromised patients.

Establishment and characterization of a bank of cytotoxic T lymphocytes for immunotherapy of epstein-barr virus-associated diseases.

Adoptive immunotherapy using Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) generated ex vivo can be an effective treatment of EBV-positive posttransplantation lymphoproliferative disease (PTLD). We describe the establishment of a cryopreserved repository of allogeneic virus-specific CTL lines, to our knowledge the first of its kind in the world. CTL lines were grown by weekly stimulation with autologous EBV immortalized lymphoblastoid cell lines (LCLs) from 96 EBV-seropositive blood donors. Analysis of 60 CTL lines grown continuously for 7 to 10 weeks showed an average proportional weekly increase in cell numbers of 1.4, with an overall increase ranging from 1.1 to 83.4. The greatest increase occurred during the early culture period. After four rounds of stimulation, killing of autologous LCLs was generally high (mean 48%); however, most lines required 9 or 10 stimulations to reduce the killing of nonspecific targets. Overall, 79% of CTLs generated showed acceptable levels of specific killing. Phenotypically, the CTL lines consisted of TCR&agr;β+, CD8+ T cells (medians 97% and 90% respectively) with a minority population of CD4+ T cells (median 2%). Most cells expressed the activation and differentiation markers, HLA-DR, CD26, CD45RO, CD69, and CD150. Favorable results have been obtained in an open trial using partially HLA-matched, allogeneic CTLs from this bank to treat PTLD patients. This now represents a single resource that can provide therapeutic CTLs rapidly on a countrywide basis, superseding the time-consuming, expensive practice of generating autologous CTLs from each patient requiring treatment. Additionally, other patient groups, such as those with EBV-positive Hodgkin disease, may benefit from CTL treatment.

Establishment and operation of a Good Manufacturing Practice-compliant allogeneic Epstein-Barr virus (EBV)-specific cytotoxic cell bank for the treatment of EBV-associated lymphoproliferative disease

Epstein‐Barr virus (EBV) is associated with several malignancies, including post‐transplant lymphoproliferative disorder (PTLD). Conventional treatments for PTLD are often successful, but risk organ rejection and cause significant side effects. EBV‐specific cytotoxic T lymphocytes (CTLs) generated in vitro from peripheral blood lymphocytes provide an alternative treatment modality with few side effects, but autologous CTLs are difficult to use in clinical practice. Here we report the establishment and operation of a bank of EBV‐specific CTLs derived from 25 blood donors with human leucocyte antigen (HLA) types found at high frequency in European populations. Since licensure, there have been enquiries about 37 patients, who shared a median of three class I and two class II HLA types with these donors. Cells have been infused into ten patients with lymphoproliferative disease, eight of whom achieved complete remission. Neither patient with refractory disease was matched for HLA class II. Both cases of EBV‐associated non‐haematopoietic sarcoma receiving cells failed to achieve complete remission. Thirteen patients died before any cells could be issued, emphasizing that the bank should be contacted before patients become pre‐terminal. Thus, this third party donor‐derived EBV‐specific CTL cell bank can supply most patients with appropriately matched cells and most recipients have good outcomes.

Treatment of Epstein-Barr-virus-associated primary CNS B cell lymphoma with allogeneic T-cell immunotherapy and stem-cell transplantation

344 http://oncology.thelancet.com Vol 6 May 2005 An 8-year-old girl presented in October, 2003, with a 3-week history of headaches, vomiting, diplopia secondary to palsies in the IV and VI cranial nerves, dysarthria, and unsteady gait. Over the following 2 weeks, bulbar palsy progressed, and she developed quadraparesis and obtunded consciousness that needed intensive care with assisted ventilation. The patient did not have lymphadenopathy or hepatosplenomegaly. Gadolinium-enhanced cranial MRI showed moderate hydrocephalus and several cerebral and cerebellar ring-like lesions with no central liquefaction (figure 1A,B). Urgent right frontal external ventricular drainage was done. Cerebrospinal fluid taken during the operation showed no white cells, presence of normal proteins, and normal glucose concentrations; the fluid was negative on PCR, microscopy, and culture for bacteria and fungi. 40 380 copies/mL of Epstein-Barr virus (EBV) DNA were detected by PCR of cerebrospinal fluid, and 6 316 copies/mL were found in peripheral blood. Histological analysis of a biopsy sample of the right parietal brain obtained by image guidance showed an infiltrate of CD79a-positive CD30-positive ALK-1-negative, activated B lymphocytes with pleomorphic nuclei and prominent nucleoli. Cells expressed EBV latent membrane protein 1 (LMP1), EBV encoded small RNA (EBER, shown by in-situ hybridisation) and EBV DNA (shown by PCR). Chest and abdominal CT, and bonemarrow biopsy sample were unremarkable, confirming primary CNS EBV-associated, B-cell lymphoma. The patient had had recurrent bacterial respiratory-tract infections during childhood. Furthermore, at age 5 years she was admitted to hospital for 3 weeks because of severe chickenpox, and was absent from school for Lancet Oncol 2005; 6: 344-46

Recombination of Globally Circulating Varicella Zoster Virus

ABSTRACT Varicella-zoster virus (VZV) is a human herpesvirus, which during primary infection typically causes varicella (chicken pox) and establishes lifelong latency in sensory and autonomic ganglia. Later in life, the virus may reactivate to cause herpes zoster (HZ; also known as shingles). To prevent these diseases, a live-attenuated heterogeneous vaccine preparation, vOka, is used routinely in many countries worldwide. Recent studies of another alphaherpesvirus, infectious laryngotracheitis virus, demonstrate that live-attenuated vaccine strains can recombine in vivo, creating virulent progeny. These findings raised concerns about using attenuated herpesvirus vaccines under conditions that favor recombination. To investigate whether VZV may undergo recombination, which is a prerequisite for VZV vaccination to create such conditions, we here analyzed 115 complete VZV genomes. Our results demonstrate that recombination occurs frequently for VZV. It thus seems that VZV is fully capable of recombination if given the opportunity, which may have important implications for continued VZV vaccination. Although no interclade vaccine-wild-type recombinant strains were found, intraclade recombinants were frequently detected in clade 2, which harbors the vaccine strains, suggesting that the vaccine strains have already been involved in recombination events, either in vivo or in vitro during passages in cell culture. Finally, previous partial and complete genomic studies have described strains that do not cluster phylogenetically to any of the five established clades. The additional VZV strains sequenced here, in combination with those previously published, have enabled us to formally define a novel sixth VZV clade. IMPORTANCE Although genetic recombination has been demonstrated to frequently occur for other human alphaherpesviruses, herpes simplex viruses 1 and 2, only a few ancient and isolated recent recombination events have hitherto been demonstrated for VZV. In the present study, we demonstrate that VZV also frequently undergoes genetic recombination, including strains belonging to the clade containing the vOKA strain.

Incidence and Dynamics of Epstein-Barr Virus Reactivation After Alemtuzumab-Based Conditioning for Allogeneic Hematopoietic Stem-Cell Transplantation

Background. Reactivation of Epstein-Barr virus (EBV) infection and posttransplant lymphoproliferative disorder (PTLD) pose a significant risk after T-cell-depleted (TCD) allogeneic hematopoietic stem-cell transplantation (HSCT). The pattern of EBV reactivation in patients receiving allogeneic HSCT, incorporating in vivo or in vitro alemtuzumab as the method of TCD, is not known. Methods. Monitoring for EBV DNA was performed by quantitative polymerase chain reaction on whole blood in 111 consecutive adults undergoing HSCT using alemtuzumab-based TCD. Patients with more than 40,000 copies/mL were screened for PTLD, followed by the withdrawal of immunosuppression and a single infusion of rituximab. Results. The 2-year cumulative incidence of EBV DNAemia was 40.3%. In vivo alemtuzumab was associated with earlier EBV reactivation than in vitro alemtuzumab (100-day incidence 22.7% vs. 2.8%, P=0.006). Eighteen patients (16%) had EBV DNAemia of more than 40,000 copies/mL. In evaluable patients, the initial rate of increase in EBV DNA levels was significantly faster in those who went on to treatment with rituximab than in patients who were left untreated (mean doubling time 3.5 days vs. 4.2 days, P=0.003). Rituximab treatment induced rapid declines in EBV DNA with an average half-life of 1.2±0.7 days. Only one patient (0.9%) had histologic confirmation of PTLD and subsequently attained a complete remission with rituximab that persists at 18 months. Conclusions. Alemtuzumab-based TCD is associated with a high frequency of EBV reactivation but a low (<1%) risk of PTLD using a strategy of preemptive rituximab therapy.

Soluble CD30: A serum marker for Epstein-Barr virus-associated lymphoproliferative diseases

The soluble form of CD30 (sCD30), a member of tumor necrosis factor receptor superfamily, has been used as a marker of disease activity in various lymphomas. Epstein–Barr virus (EBV) is a potent stimulator of CD30 expression. The study aims to evaluate whether sCD30 can be used as a diagnostic marker for EBV‐associated infectious mononucleosis (IM) and post‐transplant lymphoproliferative disease (PTLD). Plasma from EBV seropositive healthy controls (N = 90), acute IM patients (n = 90), non‐PTLD heart/lung transplant recipients (N = 30) and EBV‐positive PTLD patients (N = 23) was tested for sCD30 using a commercially available ELISA kit. EBV DNA was tested by real time quantitative polymerase chain reaction assay. Significantly higher sCD30 levels were observed in acute IM patients (median 242.9 ng/ml) compared to EBV seropositive controls (median 15.7 ng/ml; P < 0.0001). These levels were highest in IM patients within 14 days of onset of illness. PTLD patients had significantly higher sCD30 levels (median 94 ng/ml) than healthy controls (P < 0.0001) and transplant patients (median 27 ng/ml; P = 0.0007). EBV DNA was detected mostly in acute IM and PTLD patients. In both cases there was a significant correlation between sCD30 and EBV DNA levels in plasma (P < 0.0001). This study demonstrates that sCD30 and EBV DNA levels can be used as potential markers for diagnosis of IM and PTLD. J. Med. Virol. 83:311–316, 2011.

THE ROLE OF ADOPTIVE IMMUNOTHERAPY IN THE PREVENTION AND TREATMENT OF LYMPHOPROLIFERATIVE DISEASE FOLLOWING TRANSPLANTATION

The last two decades have seen an increase in various types of organ transplantation for the treatment of incurable organ diseases. This has been made possible by the development of effective immunosuppressive agents required for survival of the grafted organ. However, iatrogenic immunosuppression renders the recipient susceptible to a wide range of infections and neoplasms. These patients have long been recognized as a risk group for tumour development, the commonest tumours being skin and lip cancers. Lymphoid tumours are the second most common tumours in transplant recipients constituting 24% of all tumours in this patient group (Penn, 1994, 1998). Lymphomas are usually of B-cell origin and characterized by their aggressive behaviour and diverse morphological presentation. These tumours are collectively known as B-cell lymphoproliferative disease (BLPD), and in the transplant setting may also be referred to as post-transplant lymphoproliferative disorder.

Allogeneic T-Cell Therapy for Epstein-Barr Virus-Positive Posttransplant Lymphoproliferative Disease: Long-Term Follow-Up

Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disease (PTLD) is an important cause of morbidity and mortality in transplant patients. EBV-specific cytotoxic T-cell line (CTL) that are in vitro grown and donor derived have been used to prevent and treat EBVpositive PTLD in hematopoietic stem-cell transplant recipients (1). However, donorderived T cells are neither generally available nor appropriate for PTLD arising after solid organ transplantation. Furthermore, although monitoring EBV load in blood may indicate a risk of PTLD after hematopoietic stem-cell transplant, allowing time for CTL preparation, this monitoring gives less convincing results after solid organ transplantation. Additionally, the alternative strategy of generating autologous CTL after PTLD diagnosis often produces unacceptable delays in treatment. To counter these problems, we generated a bank of 100 EBV-specific CTL from healthy blood donors covering more than 95% of common UK human leukocyte antigen (HLA) haplotypes and treated a patient with PTLD with a partially HLA-matched CTL line that resulted in complete tumor regression (2, 3). Subsequently, we used these cell lines in a phase 2 multicenter clinical trial to treat PTLD in a best HLA match basis (4). All 33 trial participants had progressive disease, despite conventional treatments. Our trial recorded a response rate of 52% at 6 months, with 14 patients achieving complete remission (CR), three patients with partial remission (PR), and 16 patients showed no response (NR); five of whom died during the CTL treatment. Significantly better response rates were seen in patients with closer HLA matches and higher numbers of CD4 T cells in the infused CTL. We now report the long-term outcome of trial participants. We obtained follow-up data from 32 of the 33 trial participants, 4 to 9 years after their last CTL infusion. At 6 months, 15 of the 32 patients showed NR and 17 patients were responders (three PR and 14 CR). All those in the PR and NR groups, but none of the CR group, received further PTLD treatment after CTL therapy. Nineteen (59%) of the 32 patients are alive to date and 13 (41%) have died. Of the 14 patients who achieved CR at 6 months, 12 patients (86%) survived and are still in CR after 4 to 9 years. Two patients (14%) from the CR group had died; one at 10 months post-CTL therapy from relapsed PTLD and the other at 5 years from a chest infection while PTLD was in CR. Of the 19 surviving trial participants, 13 participants (68%) were responders (one PR and 12 CR) and six participants (32%) were NR at 6 months. In contrast, of the 13 patients who have died, nine patients (69%) were NR and four patients (31%) were responders (two PR and two CR) at 6 months. There was a significantly increased survival rate among the PR and CR group compared with the NR group (P 0.018; Fig. 1). Our clinical trial showed allogeneic T-cell therapy for PTLD to be safe and effective in the short term, and this 4to 9-year follow-up data are important in showing that, although of indeterminate life-span in vivo, CTL induces long-term remission of PTLD in patients with refractory disease. Therefore, it seems appropriate to use this therapy early in PTLD and for prophylaxis in highrisk cases. We are generating a new bank of clinical-grade EBV-specific CTL under good manufacturing practice conditions to provide partially matched CTL internationally on a not-for-profit basis, funded by The Wellcome Trust, UK.