What is the outcome in patients with acute leukaemia who survive severe acute graft-versus-host disease?

BACKGROUND: Acute graft-versus-host disease (aGVHD) is a major complication of allogeneic haematopoietic stem cell transplantation (HSCT). With new promising therapies, survival may improve for severe aGVHD. OBJECTIVES: We wanted to analyze the long-term outcome in patients who survive severe aGVHD. METHODS: This study was a landmark analysis of 23 567 patients with acute Leukaemia who survived for more than 6 months after HSCT, 2002-2014. Patients alive after severe aGVHD (n = 1738) were compared to controls. RESULTS: Patients with severe aGVHD had higher non-relapse mortality (NRM) and higher rate of extensive chronic GVHD (cGVHD) than the controls (P < 10-5 ). The probability of relapse was significantly lower in the severe aGVHD group, but Leukaemia-free survival (LFS) and overall survival were significantly lower than for the controls (P < 10-5 ). Five-year LFS in patients with severe aGVHD was 49%, as opposed to 61% in controls with no or mild GVHD and 59% in patients with moderate GVHD. CONCLUSIONS: HSCT patients who survive severe aGVHD have higher risk of developing extensive cGVHD, a higher NRM, a lower relapse probability, and lower LFS than other HSCT patients. This study is a platform for outcome analysis in patients treated with novel therapies for acute GVHD.

Endophthalmitis as primary clinical manifestation of fatal fusariosis in an allogeneic stem cell recipient.

The occurrence of infections due to previously rare opportunistic pathogens is increasing despite the use of novel treatment strategies for immunocompromised patients. Here, we report the case of a patient presenting with fever, muscle pain, and bilateral endophthalmitis after allogeneic hematopoietic stem cell transplantation. Fusarium solani was isolated from peripheral blood samples and identified as the cause of gradual bilateral vision loss, despite appropriate antifungal prophylaxis, and therapy including vitrectomy and intraocular instillation of antifungal agents. The patient became comatose; basal meningitis involving both optic nerves was suspected based on magnetic resonance tomography. The patient died 8 days later due to septic multi-organ failure. Autopsy revealed that both kidneys, but no other organs, were infiltrated by Fusarium. No fungus was found in cerebral tissues or cerebrospinal fluid. Our case demonstrates some of the typical clinical features of systemic fusariosis and its potentially fatal outcome. The clinical observations reported here may help clinicians caring for immunocompromised patients to accelerate diagnosis and initiate treatment early at the onset of this fatal complication, and highlight the urgent need for interdisciplinary management of invasive fusariosis.

Regression of human metastatic renal cell carcinoma after vaccination with tumor cell-dendritic cell hybrids.

Reports of spontaneous regressions of metastases and the demonstration of tumor-reactive cytotoxic T lymphocytes indicate the importance of the host's immune system in controlling the devastating course of metastatic renal cell carcinoma. Recent research indicates that immunization with hybrids of tumor and antigen presenting cells results in protective immunity and rejection of established tumors in various rodent models. Here, we present a hybrid cell vaccination study of 17 patients. Using electrofusion techniques, we generated hybrids of autologous tumor and allogeneic dendritic cells that presented antigens expressed by the tumor in concert with the co-stimulating capabilities of dendritic cells. After vaccination, and with a mean follow-up time of 13 months, four patients completely rejected all metastatic tumor lesions, one presented a 'mixed response', and two had a tumor mass reduction of greater 50%. We also demonstrate induction of HLA-A2-restricted cytotoxic T cells reactive with the Muc1 tumor-associated antigen and recruitment of CD8+ lymphocytes into tumor challenge sites. Our data indicate that hybrid cell vaccination is a safe and effective therapy for renal cell carcinoma and may provide a broadly applicable strategy for other malignancies with unknown antigens.

Reduced-toxicity conditioning with fludarabine and treosulfan prior to allogeneic stem cell transplantation in multiple myeloma.

In recent years, reduced-intensity conditioning (RIC) regimens before allogeneic stem cell transplantation (SCT) are increasingly used in patients not eligible for conventional conditioning. We did a retrospective, multicenter analysis to assess the feasibility of conditioning with fludarabine and treosulfan before allogeneic SCT in multiple myeloma patients. Thirty-four patients with a median age of 51.5 years were included in the analysis. All patients underwent myeloablation after conditioning followed by stable engraftment, and 29 of 31 evaluable patients (94%) showed early complete hematopoietic chimerism. Non-hematological toxicities were limited and encompassed mainly fever in neutropenia and infections. Grade II-IV acute and chronic graft-versus-host disease was observed in 33 and 39%, respectively. With a median follow-up of 708 days (range 60-1729 days), the median progression-free survival was 180 days. The treatment-related mortality was 10% on day 100 and 25% after 1 year. The median overall survival has not yet been reached. Our data indicate that conditioning with fludarabine and treosulfan before allogeneic SCT is feasible in intensively pretreated multiple myeloma patients and leads to stable engraftment and complete hematopoietic chimerism. Randomized trials are warranted to determine if this approach might be incorporated in an algorithm of multiple myeloma treatment.

Tumor necrosis factor alpha and CD40 ligand antagonize the inhibitory effects of interleukin 10 on T-cell stimulatory capacity of dendritic cells.

Interleukin (IL)-10 secretion by tumor cells was demonstrated to be one of the mechanisms by which tumor cells can escape immunological recognition and destruction. In dendritic cells (DCs), which are currently used for vaccination therapies for malignant diseases, IL-10 inhibits IL-12 production and induces a state of antigen-specific anergy in CD4- and CD8-positive T cells. We therefore analyzed the effects of different activation stimuli including lipopolysaccharide (LPS), tumor necrosis factor (TNF)-alpha, and CD40 ligation on IL-10 mediated inhibition of DC development and stimulatory capacity. In our study, the addition of IL-10 to the cultures containing granulocyte/macrophage-colony stimulating factor and IL-4 with or without LPS completely inhibited the generation of DCs from peripheral blood monocytes. These cells remained CD14 positive and expressed high levels of IL-10 receptor (IL-10R), suggesting that IL-10 mediates its effects by up-regulating the IL-10R. In contrast, the simultaneous incubation of monocytes with IL-10 and TNF-alpha or soluble CD40 ligand (sCD40L) resulted in the generation of CD83-positive DCs, induction of nuclear localized RelB, and inhibition of IL-10R up-regulation. DCs grown in the presence of IL-10 and TNF-alpha or sCD40L elicited efficient CTL responses against viral and tumor-associated peptide antigens, which, however, were reduced as compared with DC cultures generated without IL-10. IL-10 decreased the production of IL-6 and the expression of IL-12 in the presence of TNF-alpha or sCD40L, but it had no effect on IL-15, IL-18, and TNF-alpha secretion. Our results show that TNF-alpha or CD40 ligation can antagonize the IL-10-mediated inhibition on DC function, suggesting that depending on activation stimuli, the presence of IL-10 does not necessarily result in T-cell anergy.

Her-2/neu-derived peptides are tumor-associated antigens expressed by human renal cell and colon carcinoma lines and are recognized by in vitro induced specific cytotoxic T lymphocytes.

The Her-2/neu oncogene encodes a Mr 185,000 transmembrane protein with homology to the epidermal growth factor receptor. It is overexpressed in 30-40% of breast and ovarian cancers, and this overexpression was shown to correlate with aggressiveness of malignancy and poor prognosis. Using tumor-associated lymphocytes isolated from patients with ovarian or breast cancer, several HLA-A2-restricted, Her-2/neu-derived peptides were identified. Further studies revealed that these tumor-associated CTLs can also lyse other tumors, including non-small cell lung and pancreatic cancer cells, suggesting that Her-2/neu epitopes are shared between several distinct types of epithelial tumors. To analyze whether Her-2/neu epitopes are tumor-associated antigens for renal cell carcinoma (RCC) and colon carcinoma, we induced Her-2/neu peptide-specific CTL responses by primary in vitro immunization and used these CTLs to determine the presentation of Her-2/neu epitopes on human tumor lines. Autologous dendritic cells (DCs) generated from peripheral blood monocytes were pulsed with Her-2/neu-derived peptides E75 and GP2 and used as antigen-presenting cells for CTL priming. High CTL activity toward peptide-pulsed targets was obtained after two weekly restimulations. CTLs induced with DCs generated in the presence of TNF-alpha elicited a higher cytotoxic activity when they were stimulated with the cognate peptide than did CTLs induced with DCs grown in granulocyte macrophage colony-stimulating factor and interleukin 4 alone. The cytotoxicity of induced CTLs was antigen specific and HLA-A2 restricted. Furthermore, these CTLs lysed, in a MHC- and antigen-restricted fashion, not only breast cancer cells but also colon carcinoma and RCC cell lines expressing Her-2/neu. The cytotoxic activity against tumor cells was blocked by cold HLA-A2-positive targets pulsed with the cognate peptide in cold target inhibition assay and by anti-HLA-A2 monoclonal Ab. These results suggest that epitopes derived from Her-2/neu protein might be attractive candidates for broadly applicable vaccines and may prove useful for adoptive immunotherapies designed for colon carcinoma or RCC.

Clofarabine salvage therapy before allogeneic hematopoietic stem cell transplantation in patients with relapsed or refractory AML: results of the BRIDGE trial.

In patients with relapsed or refractory (r/r) acute myeloid leukemia (AML), long-term disease control can only be achieved by allogeneic hematopoietic stem cell transplantation (HSCT). We studied the safety and efficacy of clofarabine-based salvage therapy. The study was designed as phase II, multicenter, intent-to-transplant (ITT) study. A total of 84 patients with r/r AML were enrolled. All patients received at least one cycle of CLARA (clofarabine 30 mg/m(2) and cytarabine 1 g/m(2), days 1-5). Chemo-responsive patients with a donor received HSCT in aplasia after first CLARA. Generally, HSCT was performed as soon as possible. The conditioning regimen consisted of clofarabine (4 × 30 mg/m(2)) and melphalan (140 mg/m(2)). The median patient age was 61 years (range 40-75). On day 15 after start of CLARA, 26% of patients were in a morphologically leukemia-free state and 79% exposed a reduction in bone marrow blasts. Overall, 67% of the patients received HSCT within the trial. The primary end point, defined as complete remission after HSCT, was achieved by 60% of the patients. According to the ITT, overall survival at 2 years was 43% (95% confidence interval (CI), 32-54%). The 2-year disease-free survival for transplanted patients was 52% (95% CI, 40-69%). Clofarabine-based salvage therapy combined with allogeneic HSCT in aplasia shows promising results in patients with r/r AML.

Human tumour and dendritic cell hybrids generated by electrofusion: potential for cancer vaccines.

Hybrid cells created by fusion of antigen presenting and tumour cells have been shown to induce potent protective and curative anti-tumour immunity in rodent cancer models. The application of hybrid cell vaccines for human tumour therapy and the timely intervention in disease control are limited by the requirement to derive sufficient autologous cells to preserve homologous tumour antigen presentation. In this study, the efficiency of various methods of electrofusion in generating hybrid human cells have been investigated with a variety of human haemopoietic, breast and prostate cell lines. Cell fusion using an electrical pulse is enhanced by a variety of stimuli to align cells electrically or bring cells into contact. Centrifugation of cells after an exponential pulse from a Gene Pulser electroporation apparatus provided the highest yield of mixed cell hybrids by FACS analysis. An extensive fusogenic condition generated in human cells after an electrical pulse contradicts the presumption that prior cell contact is necessary for cell fusion. Alignment of cells in a concurrent direct current charge and osmotic expansion of cells in polyethylene glycol also generated high levels of cell fusion. Waxing of one electrode of the electroporation cuvette served to polarize the fusion chamber and increase cell fusion 5-fold. Optimisation of a direct current charge in combination with a fusogenic pulse in which fusion of a range of human cells approached or exceeded 30% of the total pulsed cells. The yield of hybrid prostate and breast cancer cells with dendritic cells was similar to the homologous cell fusion efficiencies indicating that dendritic cells were highly amenable to fusion with human tumour cells under similar electrical parameters. Elimination of unfused cells by density gradient and culture is possible to further increase the quantity of hybrid cells. The generation and purification of quantities of hybrid cells sufficient for human vaccination raises the possibility of rapid, autologous tumour antigen presenting vaccines for trial with common human tumours.

Successful transplantation of highly selected CD34+ peripheral blood stem cells in a HLA-sensitized patient treated with immunoadsorption onto protein A.

BACKGROUND: Haploidentical bone marrow transplantation with preexisting anti-HLA antibodies is associated with a high risk of graft failure. METHODS: A 27-year-old female patient with chronic myeloid leukemia and evidence of several osseous chloromas had no suitable matched bone marrow donor, and fluorescence cytometric cross-match (FCXM) revealed antibodies against donor-specific HLA-molecules. Immunoadsorption onto staphylococcal protein A was applied to remove these antibodies, and peripheral stem cell transplantation was performed from her haploidentical sister after a negative FCXM was documented after immunoadsorption and conditioning treatment. RESULTS: FCXM for donor lymphocytes and stem cells remained negative throughout the posttransplant period, and engraftment of donor cells was documented on day +69. CONCLUSION: Immunoadsorption onto protein A should be considered in stem cell transplantation even from an haploidentical donor where anti-HLA antibodies and a positive FCXM are documented.

Approaches to dendritic cell-based immunotherapy after peripheral blood stem cell transplantation.

High-dose chemotherapy with peripheral blood progenitor cell transplantation (PBPCT) is a potentially curative treatment option for patients with both hematological malignancies and solid tumors, including breast cancer. However, based on a number of clinical studies, there is strong evidence that minimal residual disease (MRD) persists after high-dose chemotherapy in a number of patients, which eventually results in disease recurrence. Therefore, several approaches to the treatment of MRD are currently being evaluated, including treatment with dendritic cell (DC)-based cancer vaccines. DCs, which play a crucial role with regard to the initiation of T-lymphocyte responses, can be generated ex vivo either from CD34+ hematopoietic progenitor cells or from blood monocytes. They can be pulsed in vitro with tumor-derived peptides or proteins, and then used as a professional antigen-presenting cell (APC) vaccine for the induction of antigen-specific T-lymphocytes in vivo. This paper summarizes our preclinical studies on the induction of primary HER-2/neu specific cytotoxic T-lymphocyte (CTL) responses using peptide-pulsed DC. As HER-2/neu is overexpressed on 30-40% of breast and ovarian cancer cells, this novel vaccination approach might be particularly applicable to advanced breast or ovarian cancer patients after high-dose chemotherapy and autologous PBPCT.

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.

CD8+ T-cell responses to tumor-associated antigens correlate with superior relapse-free survival after allo-SCT.

The GVL effect following allo-SCT is one of the most prominent examples showing the ability of the immune system to eliminate malignant hematological diseases. Tumor-associated Ags (TAA), for instance WT1 and proteinase-3, have been proposed as targets for T cells to establish a GVL effect. Here, we examined an additional TAA (MUC1) as a possible T-cell target of GVL-related immune responses. We have defined new peptide epitopes from the MUC1 Ag to broaden patients' screening and to expand the repertoire of immunologic monitoring as well as for therapeutic approaches in the future. Twenty-eight patients after allo-SCT have been screened for T-cell responses toward TAA (proteinase-3, WT1, MUC1). We could detect a significant relationship between relapse and the absence of a TAA-specific T-cell response, whereby only 2/13 (15%) patients with TAA-specific CTL relapsed, in contrast to 9/15 (60%) patients without TAA-specific CTL responses (P<0.05). In conclusion, CD8(+) T-cell responses directed to TAA might contribute to the GVL effect. These observations highlight both the importance and the potential of immunotherapeutic approaches after allo-SCT.

Collaboration of helper and cytotoxic T lymphocytes.

Cytotoxic T lymphocytes are major effector cells in response to viral infections and in allograft rejection and are implicated in many other immunological reactions. Efficient induction of cytotoxic activity in these cells in many but not all cases depends upon helper T and antigen-presenting cells so that at least three different cell types have to work together. Here we present an in vitro model for the helper T cell-dependent induction of cytotoxic T lymphocytes which allows the investigation of the collaboration of helper and cytotoxic T cells. First results demonstrate that linkage of helper and killer epitopes on the surface of one antigen-presenting cell is a prerequisite for productive interaction between the two T cells that results in induction of cytolytic activity. T helper 1 and T helper 2 cells are equally efficient. The crucial roles of interleukin-2 and interferon-gamma in this process were confirmed. Activated CD4 cells can influence cytotoxic T lymphocytes in such a way that they produce interferon-gamma independent from recognition of cognate peptide. The possibility of direct T-T contacts mediated by adhesion molecules that promote collaboration of the two cells is discussed.

Recruitment of helper T cells for induction of tumour rejection by cytolytic T lymphocytes.

Immunotherapy of cancer could be possible in cases in which competent effector T cells can be induced. Such an approach depends on expression of tumour-specific antigens by the tumour cells and on the availability of sufficient costimulatory support for activation of cytotoxic T lymphocytes. Here, a strategy for helper T cell recruitment for induction of tumour-specific cytotoxic immune responses is presented. Allogenic MHC class II molecules were introduced into tumour cells by cell fusion. These hybrid cells, when injected into mice, induced rejection of an established tumour. The contribution of CD4-expressing helper T cells in the induction phase and of CD8-expressing T cells in the effector phase of the immune response was demonstrated. The approach described could be applicable to cases in which a suitable tumour antigen is present but not identified; it employs regulatory interactions that govern physiological immune responses and is designed to be minimally invasive.

Antigen organization regulates cluster formation and induction of cytotoxic T lymphocytes by helper T cell subsets.

Generation of effector cytotoxic T lymphocytes (CTLs) is a process tightly governed by regulatory helper T (Th) cells. The nature of cellular interactions as well as the precise role of distinct Th cell subsets involved in efficient CTL activation remains elusive. Employing in vitro cultures for primary induction of human, peptide-specific CTL, a strict requirement for Th cells and linkage of epitopes for helper and CTLs on the surface of antigen presenting cells was found, suggesting a three cell type cluster as minimal immune regulatory entity. Cognate and antigen-driven interactions of T cells were neither essential nor sufficient to override the need for linked epitopes. Within the three cell type cluster complex, keyhole limpit hemocyanin or tetanus toxoid-reactive Th cells promoted generation of MAGE-3- or HIV-gag-specific CTL. Both type 1 and type 2 Th cells were recruited and induced by CTL. Interleukin 2 and interferon gamma were essential in early stages, and interleukin 4 was utilized in later stages, of CTL maturation. Synergistic effects of CD45RA+ and CD45RO+ Th cells were found. The data reported here suggest a critical link between the innate and adaptive immune system in the initiation process of cytolytic immune responses and offers the basis for efficient vaccine strategies.

Induction of cytotoxic T-lymphocyte responses in vivo after vaccinations with peptide-pulsed dendritic cells.

Vaccination of patients with cancer using dendritic cells (DCs) was shown to be effective for B-cell lymphoma and malignant melanoma. Here we provide evidence that patients with advanced breast and ovarian cancer can be efficiently vaccinated with autologous DCs pulsed with HER-2/neu- or MUC1-derived peptides. Ten patients were included in this pilot study. The DC vaccinations were well tolerated with no side effects. In 5 of 10 patients, peptide-specific cytotoxic T lymphocytes (CTLs) could be detected in the peripheral blood using both intracellular IFN-gamma staining and (51)Cr-release assays. The major CTL response in vivo was induced with the HER-2/neu-derived E75 and the MUC1-derived M1.2 peptide, which lasted for more than 6 months, suggesting that these peptides might be immunodominant. In addition, in one patient vaccinated with the MUC1-derived peptides, CEA- and MAGE-3 peptide-specific T-cell responses were detected after several vaccinations. In a second patient immunized with the HER-2/neu peptides, MUC1-specific T lymphocytes were induced after 7 immunizations, suggesting that antigen spreading in vivo might occur after successful immunization with a single tumor antigen. Our results show that vaccination of DCs pulsed with a single tumor antigen may induce immunologic responses in patients with breast and ovarian cancer. This study may be relevant to the design of future clinical trials of other peptide-based vaccines.

Autologous and allogenic hybrid cell vaccine in patients with metastatic renal cell carcinoma.

OBJECTIVE: To evaluate the safety, acute and long-term toxicity and therapeutic activity of an allogenic and an autologous hybrid cell vaccine in patients with progressive metastatic renal cell carcinoma (RCC). PATIENTS AND METHODS: Eleven patients were vaccinated with a lethally irradiated hybrid cell vaccine of allogenic RCC tumour cells fused with major histocompatibility complex class I-matched and class II-unmatched activated allogenic lymphocytes. These patients were then followed for a mean of 11 months. Another 13 patients were vaccinated with a hybrid cell vaccine of autologous tumour cells fused with allogenic activated lymphocytes and followed for a mean of 6 months. RESULTS: Six of the 11 patients receiving the allogenic vaccination showed an initial response, with two complete and two partial responses to date. Only three patients who received autologous vaccination responded to treatment. CONCLUSIONS: Hybrid cell vaccination is a promising new approach in the treatment of patients with advanced RCC.