Transfer of minimally manipulated CMV-specific T cells from stem cell or third-party donors to treat CMV infection after allo-HSCT.

Cytomegalovirus (CMV) infection is a common, potentially life-threatening complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT). We assessed prospectively the safety and efficacy of stem cell-donor- or third-party-donor-derived CMV-specific T cells for the treatment of persistent CMV infections after allo-HSCT in a phase I/IIa trial. Allo-HSCT patients with drug-refractory CMV infection and lacking virus-specific T cells were treated with a single dose of ex vivo major histocompatibility complex-Streptamer-isolated CMV epitope-specific donor T cells. Forty-four allo-HSCT patients receiving a T-cell-replete (D+ repl; n=28) or T-cell-depleted (D+ depl; n=16) graft from a CMV-seropositive donor were screened for CMV-specific T-cell immunity. Eight D+ depl recipients received adoptive T-cell therapy from their stem cell donor. CMV epitope-specific T cells were well supported and became detectable in all treated patients. Complete and partial virological response rates were 62.5% and 25%, respectively. Owing to longsome third-party donor (TPD) identification, only 8 of the 57 CMV patients transplanted from CMV-seronegative donors (D-) received antigen-specific T cells from partially human leukocyte antigen (HLA)-matched TPDs. In all but one, TPD-derived CMV-specific T cells remained undetectable. In summary, adoptive transfer correlated with functional virus-specific T-cell reconstitution in D+ depl patients. Suboptimal HLA match may counteract expansion of TPD-derived virus-specific T cells in D- patients.

Advances in clinical NK cell studies: Donor selection, manufacturing and quality control.

Natural killer (NK) cells are increasingly used in clinical studies in order to treat patients with various malignancies. The following review summarizes platform lectures and 2013-2015 consortium meetings on manufacturing and clinical use of NK cells in Europe and United States. A broad overview of recent pre-clinical and clinical results in NK cell therapies is provided based on unstimulated, cytokine-activated, as well as genetically engineered NK cells using chimeric antigen receptors (CAR). Differences in donor selection, manufacturing and quality control of NK cells for cancer immunotherapies are described and basic recommendations are outlined for harmonization in future NK cell studies.

Long-term follow-up of the AML97 study for patients aged 60 years and above with acute myeloid leukaemia: a study of the East German Haematology and Oncology Study Group (OSHO).

INTRODUCTION: Treatment of patients (pts) with acute myelogenous leukaemia (AML) above 60 years remains a challenge. We report long-term follow-up of the AML97 study, where pts were registered at diagnosis and received treatment dependent on their comorbidities: dose-intense cytarabine (AraC) and anthracycline in the curative arm, and low-dose chemotherapy in the palliative arm or best supportive care. MATERIALS AND METHODS: A total of 618 pts were enrolled in this protocol (curative 471, palliative 115 and supportive 32). In the curative arm, complete remission (CR) was obtained in 66.8 % of pts and the estimated probability of being alive at 2 years was 0.30 (±0.02 SE). In multivariate analysis, gender (p = 0.005), performance status (p = 0.04) and cytogenetics (p = 0.002) were significant factors for CR. With a median follow-up of 10 (range 0.1-11.8) years, the estimated probability of being event-free after 2 and 5 years according to cytogenetics was 0.48 ± 0.11 and 0.48 ± 0.11 for favourable, 0.20 ± 0.03 and 0.09 ± 0.03 for normal, 0.18 ± 0.06 and 0.10 ± 0.05 for other standard risk and 0.10 ± 0.03 and 0.05 ± 0.02 for unfavourable karyotypes, respectively. The median survival time for pts treated with palliative chemotherapy was 54 and 11 days with best supportive care only. CONCLUSION: In conclusion, treatment of older AML pts with dose-intense AraC is feasible in the majority of pts and induces high rates of CR. Nevertheless, except for favourable karyotype, OS and event-free survival remain low. These results need to be viewed in relation to the new modalities including stem cell transplantation following non-myeloablative conditioning, epigenetic and molecular therapies.

Combination of immunoglobulins and natural killer cells in the context of CMV and EBV infection.

Cytomegalovirus (CMV)-specific hyperimmunoglobulin (CMV-HIG) is used to treat and prevent CMV infection in immunocompromised patients, and anti-CD20 monoclonal antibody is successfully used in the treatment for post-transplant lymphoproliferative disease caused by Epstein-Barr virus (EBV). Two immunological approaches have been suggested to further improve the control of viral reproduction in patients with active disease: first, the use of monoclonal antibodies with specificity against viral epitopes and second, coadministration of cells with the capacity to promote antibody-dependent cell-mediated cytotoxicity. Here, we have evaluated the effectiveness of these strategies in vitro (alone and in combination) with neutralization and cytotoxicity assays. Our results indicate that monoclonal antibodies (in particular SM5-1) can be as effective as CMV-HIG in neutralizing-cell-free CMV. Moreover, our data indicate that antibody-mediated elimination (either by moAb or by HIG) of EBV-infected cells can be significantly enhanced by NK cells. Using human NK cells that have been purified, cultured and expanded under GMP conditions, we were able to demonstrate that the combination of NK cells and antibodies could represent a feasible and highly effective clinical approach to achieve control of EBV infections. Especially in leukopenic patients with low numbers of ADCC-promoting cells, the combination of adoptively transferred NK cells and antiviral antibodies offers a promising strategy that should be tested in clinical trials.

Risk-adapted, treosulfan-based therapy with auto- and allo-SCT for relapsed/refractory aggressive NHL: a prospective phase-II trial.

Since the outcome of relapsed/refractory aggressive non-Hodgkin's lymphoma (NHL) is highly variable, a risk-adapted treatment approach was evaluated. After two cycles of DHAP, patients received high-dose treosulfan/etoposide/carboplatinum (TEC) and autologous stem cell rescue. After TEC, low-risk patients with late relapse (>1 year after first CR who achieved CR after DHAP received no further treatment. Patients with late relapse who achieved CR or PR only after TEC underwent a second cycle of TEC. High-risk patients with early relapse/refractory disease received treosulfan/fludarabine followed by allogeneic transplantation. Rituximab was added in patients with B-cell lymphoma (86%). At entry, 36% of all 57 patients had refractory disease, 32% early and 32% late relapse. During DHAP treatment, progression occurred in 32% of patients. Of 33 patients who received TEC, 5 received second TEC and 15 allogeneic transplantation. Main toxicity after TEC was oral mucositis (CTC grades 3 and 4 in 50% and 13%, respectively). In total, 42% patients achieved CR. Median OS was 21.4 months for all patients and 32.6 for those who underwent allogeneic transplantation. International prognostic index (IPI) at study entry was highly discriminative at predicting OS (P<0.0001). Risk-adapted, treosulfan-based therapy with auto- and allo-SCT is feasible. Long-term survival is possible with allogeneic transplantation.

Azacitidine and donor lymphocyte infusions as first salvage therapy for relapse of AML or MDS after allogeneic stem cell transplantation.

The combination of azacitidine and donor lymphocyte infusions (DLI) as first salvage therapy for relapse after allogeneic transplantation (allo-HSCT) was studied in 30 patients with acute myeloid leukemia (AML; n=28) or myelodysplastic syndromes (MDS; n=2) within a prospective single-arm multicenter phase-II trial. Treatment schedule contained up to eight cycles azacitidine (100 mg/m(2)/day, days 1-5, every 28 days) followed by DLI (from 1-5 × 10(6) to 1-5 × 10(8) CD3(+)cells/kg) after every second azacitidine cycle. A median of three courses azacitidine (range 1-8) were administered, and 22 patients (73%) received DLI. Overall response rate was 30%, including seven complete remissions (CRs, 23%) and two partial remissions (7%). Five patients remain in CR for a median of 777 days (range 461-888). Patients with MDS or AML with myelodysplasia-related changes were more likely to respond (P=0.011), and a lower blast count (P=0.039) as well as high-risk cytogenetics (P=0.035) correlated with the likelihood to achieve CR. Incidence of acute and chronic graft-versus-host disease was 37% and 17%, respectively. Neutropenia and thrombocytopenia grade III/IV occurred during 65% and 63% of treatment cycles, while infections were the most common grade III/IV non-hematological toxicity. Azacitidine and DLI as salvage therapy is safe, induces long-term remissions and may become an alternative for patients with AML or MDS relapsing after allo-HSCT.

Detection of a novel HLA-A allele, designated A*02:334.

We report a novel allele HLA-A*02:334 with the transition G→A at nucleotide position 282.

Antiviral function and efficacy of polyvalent immunoglobulin products against CMV isolates in different human cell lines.

Primary infection and reactivation of human cytomegalovirus (CMV) remain a major problem in immunocompromised patients, frequently resulting in a life threatening CMV disease. Intravenous polyvalent (hyper)-immunoglobulins (IVIG) can be administered for therapy and prophylaxis of CMV infections. However, only limited data about the efficacy and mechanism of action of IVIG products against viral infections in vitro are available so far. In this study, the effect of IVIG on CMV infection in vitro was investigated using isolates from CMV-infected patients as well as the laboratory strains AD169 and TB40. A qualitative and quantitative comparison of five different commercially available IVIG products in different human cell lines was performed concerning their ability (1) to neutralize cell-free virus, (2) to inhibit cell-to-cell spread and cell-associated transmission and (3) to influence CMV mRNA levels. All IVIG tested exhibited a high neutralization activity in epithelial and endothelial cell cultures (50% inhibition dose <0.1 mg/ml). However, qualitative differences between the products could be demonstrated in neutralization tests using human embryonal lung fibroblasts (HELF). The IVIG products also significantly differed in their ability to inhibit cell-to-cell spread within an CMV-infected HELF monolayer displaying inhibition rates that varied between 61 and 100%. No correlation between the ability to neutralize cell-free virus and to inhibit cell-to-cell spread could be observed. The incubation with IVIG influenced the amount of CMV immediate early and late mRNA, as indicated by a significant reduction in CMV mRNA in infected epithelial cells after incubation with IVIG in a dose-dependent manner. This study suggests different antiviral functions of polyvalent IVIG and confirms their potential to inhibit a CMV infection in vitro, with profound differences between the hereby used IVIG products.

Allogeneic hematopoietic SCT in patients with AML following treosulfan/fludarabine conditioning.

An alternative reduced-toxicity conditioning regimen for allogeneic transplantation, based on treosulfan and fludarabine, has recently been identified. The safety and efficacy of this new conditioning regimen has been investigated prospectively in patients with AML. A total number of 75 patients with AML in CR were treated with 3 × 14 g/m(2) treosulfan and 5 × 30 mg/m(2) fludarabine, followed by matched sibling or unrelated SCT. Patients were evaluated for engraftment, adverse events, GVHD, and for non-relapse mortality, relapse incidence, overall and disease-free survival (DFS). All patients showed primary engraftment of neutrophils after a median of 20 days. Non-hematological adverse events grade III-IV in severity included mainly infections (59%) and gastrointestinal symptoms (7%). Acute GVHD grade II-IV occurred in 21% and extensive chronic GVHD occurred in 16% of the patients. After a median follow-up of 715 days, the 2-year overall and DFS estimates were 61% and 55%, respectively. The 2-year incidences of relapse and non-relapse mortality reached 34% and 11%, respectively. In summary, our data confirm promising safety and efficacy of the treosulfan-based conditioning therapy in AML patients, ClinicalTrials.gov Identifier: NCT01063660.

A new HLA-B*52 allele, B*52:23, detected in a patient before bone marrow transplantation.

The new allele HLA-B*52:23 differs from B*52:01:01 at position 208 in exon 2.

Immune reconstitution and cytomegalovirus infection after allogeneic stem cell transplantation: the important impact of in vivo T cell depletion.

We analyzed cytomegalovirus (CMV) infection risk factors and immune reconstitution kinetics in 89 patients after allogeneic stem cell transplantation (allo-SCT). The use of alemtuzumab for in vivo T cell depletion (TCD) had, besides the donor/recipient CMV serostatus, the strongest influence on the CMV infection risk in univariate and multivariate analyses. In comparison to without use of in vivo TCD, the CMV infection risk [hazard ratio (HR)] was 4.82-fold after TCD with alemtuzumab, but only 1.40-fold after TCD with antithymocyte globulin (ATG). Alemtuzumab strongly depressed CD4(+) and CD8(+) T cell reconstitution, whereas ATG only delayed CD4(+) T cell reconstitution. Considering the reconstitution kinetics of CD4(+) and CD8(+) T cells, CMV-specific CD8(+) T cells, NK cells and the IgG concentration, only a low day +60 NK cell count (< or =161 versus >161/microl) was significantly associated with CMV infection development (HR 2.92, p = 0.034). CMV-specific CD8(+) T cells were detected in 57% of patients with a CMV-seropositive donor, but in none of the patients with a CMV-seronegative donor on day +30 (p = 0.01). Our data indicate that the type of in vivo TCD (alemtuzumab or ATG) differentially influences both the CMV infection risk and CD4(+)/CD8(+) T cell reconstitution kinetics in patients after allo-SCT.

In vivo diagnosis of acute intestinal graft-versus-host disease by confocal endomicroscopy.

BACKGROUND AND STUDY AIMS: Conventional histology with hematoxylin and eosin (H&E) staining is the accepted standard for diagnosing acute intestinal graft-versus-host disease (GvHD). Confocal endomicroscopy (CEM) is a noninvasive method that allows in vivo histology to be performed during endoscopy. The aim of this study was to evaluate CEM for the diagnosis of acute intestinal GvHD. PATIENTS AND METHODS: This observational pilot study conducted between September 2006 and August 2008 included patients with acute diarrhea after stem cell transplantation, infectious diarrhea, or active ulcerative colitis. CEM (EC-3870CIFK, Pentax, Tokyo, Japan) was performed after intravenous injection of fluorescein 10% and topical application of acriflavine 0.05%. RESULTS: A total of 35 patients with acute diarrhea after stem cell transplantation were examined. In 16 patients, CEM and histology showed no evidence of GvHD. In 14/19 patients with histologically confirmed GvHD, the diagnosis could already be established by CEM during ongoing endoscopy. In GvHD grade IV, near complete destruction of the colonic crypts ("flat mucosa") was visible. Control patients with infectious colitis (N = 15) or ulcerative colitis (N = 15) displayed inflammatory changes but no evidence of GvHD. Altogether, sensitivity of CEM was 74% and specificity was 100 %. CONCLUSIONS: CEM improves rapid diagnosis of acute intestinal GvHD with high accuracy while performing endoscopy. Platelet transfusions and unnecessary biopsy acquisition can be avoided once acute intestinal GvHD has been diagnosed in vivo.

Adoptive T-cell therapy of a lung transplanted patient with severe CMV disease and resistance to antiviral therapy.

Infections with cytomegalovirus (CMV) can induce severe complications after transplantation, particularly in patients resistant to virostatic therapy. Adoptive transfer of CMV-specific T-cell lines has demonstrated promising results in patients after hematopoietic stem cell transplantation. However, the generation of specific T-cell lines ex vivo and their function in vivo is complicated in solid organ transplant (SOT) recipients. Here, we present the successful adoptive transfer of autologous CMV-specific T cells to a lung transplant recipient with ganciclovir-resistant CMV-pneumonia requiring mechanical ventilation. Infused T cells rapidly expanded in vivo and efficiently inhibited viral replication as confirmed by extensive longitudinal immunological monitoring. After full recovery, the patient was released from the clinic. After 4 weeks, the infection reappeared and persisted at a low level even after a second T-cell infusion. Our experimental data indicate that this could be the consequence of the late differentiated phenotype of the infused T cells and therefore their insufficient longevity in vivo. In summary, our report signifies the high therapeutic potential of adoptive immunotherapy in the treatment of SOT recipients when all other measures show no effect. Further studies have to elucidate the most potent strategies to generate antigen-specific T cells with high functional capacity and robust long-term persistence.

Prophylactic i.v. Igs in patients with a high risk for CMV after allo-SCT.

We developed a novel algorithm to define the need for high-dose prophylactic i.v. Igs (IVIG) in periods of high risk for CMV to patients after allo-SCT. IVIG were administered only if at least one of the following, monthly-assessed, criteria was fulfilled: (1) IgG concentration <4 g/l, (2) NK (natural killer) cell count <100/microl, (3) CD4(+) cell count <100/microl, (4) acute or chronic GVHD. The primary endpoint was to determine the cumulative incidence of CMV infection in patients who received prophylactic IVIG according to the algorithm (intervention group) and compare it with that of a sequentially assessed control group, to which prophylactic IVIG were not administered. The study included 79 patients (44 in the intervention and 35 in the control group). The estimated cumulative incidence of CMV infection in the intervention and control group did not differ significantly (44 and 36%; P=0.31). Additionally, prophylactic IVIG did not reduce the frequency of CMV infection episodes. CMV disease was rare in both cohorts (5 and 9%; P=0.65). We conclude that prophylactic IVIG should not be administered after allo-SCT, even if administered selectively in a high dose to patients with delayed immune reconstitution or GVHD.

Clinical-scale generation of human anti-Aspergillus T cells for adoptive immunotherapy.

Invasive aspergillosis is a major cause of morbidity and mortality in patients undergoing allogeneic hematopoietic SCT. There is a growing body of evidence that T cells are important in the host defense against Aspergillus, and adoptively transferred anti-Aspergillus T-helper 1 (T(H)) 1 cells might reduce infectious mortality in hematopoietic transplant recipients. Here we present for the first time a simple and rapid method for the clinical-scale generation of functionally active anti-Aspergillus T cells according to good manufacturing practice conditions. A total of 1.1 x 10(9) WBCs derived from a leukapheresis product were incubated with Aspergillus antigens. Stimulated cells were selected by means of the IFN-gamma secretion assay and expanded. In three independent experiments, a median number of 2 x 10(7) CD3+CD4+cells (range, 0.9-3.2 x 10(7)) were obtained within 13 days. The cultured CD3+CD4+ cells exhibited almost exclusively a memory activated T-helper cell phenotype. Upon restimulation, the generated T cells produced IFN-gamma, but no IL-4 or IL-10, indicating a T(H)1-cell population. Additionally, the cells proliferated upon restimulation and showed reduced alloreactivity compared to unselected CD4+ cells. This method of generating is suitable for future prospective trials designed to evaluate the effect of adoptive immunotherapy in hematopoietic transplant recipients with invasive aspergillosis.

Serotherapy with thymoglobulin and alemtuzumab differentially influences frequency and function of natural killer cells after allogeneic stem cell transplantation.

Although thymoglobulin and alemtuzumab are frequently used in hematopoietic stem cell transplantation (HSCT), little is known of their effects on NK cells, which mediate important functions in post-transplantation immunology. In the present study, we determined NK cell death in vitro using propidium iodide and Annexin V. The NK cell activity in 34 patients at day +30 after allogeneic HSCT was assessed using the CD107a assay. Alemtuzumab and thymoglobulin were similarly very potent in inducing NK cell death in vitro. Even in low concentrations (<1 microg/ml) the antibodies induced apoptosis and necrosis in a relevant percentage of NK cells (>30%). However, the number of tumor reactive (CD107a+) NK cells was 13.16 per mul and 1.15 per microl (mean) in patients receiving T-cell depletion with 6 mg/kg thymoglobulin and in patients receiving 100 mg alemtuzumab, respectively (P=0.02). Although thymoglobulin and alemtuzumab are equally NK cell toxic in vitro, the recovery of NK cell frequency and anti-tumor reactivity is reduced in recipients of alemtuzumab. Our findings can be explained by a longer half-life of alemtuzumab as compared to active thymoglobulin under therapeutic conditions. Prolonged immunosuppression with increased risk of infections and tumor relapse are a potential threat to patients undergoing HCST and receiving alemtuzumab as T-cell depletion.

Intensified strategies to control vancomycin-resistant enterococci in immunocompromised patients.

Increasing colonization and infection with vancomycin-resistant enterococci (VRE) in immunocompromised patients are associated with increased mortality. Despite contact precautions for VRE control, rapid limitation of its spread is often impossible. We report on a VRE outbreak in a hematologic/oncologic unit including 33 patients. Although 28 of the patients had only VRE colonization, VRE-related infection was probable in 4 patients, and VRE infection of the bloodstream occurred in 1 case. Two patients were identified by VRE screening on admission, 20 were identified by weekly routine VRE screening, and 6 were identified from specimens taken to clarify infections (eg, urine, bronchoalveolar lavage). Five individuals acquired VRE colonization as inpatients (contact patients). Multiple-locus variable-number tandem repeat analysis (MLVA) proved that the outbreak was caused by VanA gene-positive Enterococcus faecium belonging to MLVA genogroup C1(MLVA types 1, 7, 12). The outbreak strains exhibited the potential virulence factor esp(enterococcus surface protein). The outbreak was terminated within 2 months by intensified infection-control measures, including quarantine and the cohorting of patients who tested positive for VRE; however, VRE spread recurred after the measures were discontinued but was again limited by resuming the measures. We conclude that intensive infection-control strategies enable the timely termination of VRE outbreaks, even those involving VRE strains with high epidemic potential on "high-risk wards" (eg, hematologic/oncologic units). Premature discontinuation of infection-control measures may cause recurrence of the VRE spread.