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Summary. Idiotype (Id) vaccination provides an interesting immunotherapeutic strategy against B-cell lymphomas. In multiple myeloma (MM), however, the therapeutic efficacy of Id vaccination has been disappointing. In an attempt to improve the antitumoral potential, we added granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 2 (IL-2) to the protocol. Balb/c mice were inoculated i.p. (d 2) with different doses (1–5 × 105 ) of HOPC myeloma cells secreting the IgHOPC Id protein. Two days later, animals were injected with 10 000 U GM-CSF i.p. for 6 d consecutively (d 0–5). On d 5 and 11, myeloma-specific immunoglobulin (IgHOPC ) was administered i.p. together with incomplete Freund adjuvans followed by IL-2 (2 × 10 000 U/d; i.p) for 10 d (d 5–14). In animals inoculated with 105 myeloma cells, treatment with IL-2 given as a single agent prolonged the median survival time (MST, 67 d) when compared with the tumour control group (MST 48 d), whereas GM-CSF did not elicit any survival benefit (MST 49 d). Complete tumour rejection could be achieved in 27% (4/15) by the combination of Id vaccination and GM-CSF. Additional treatment with IL-2 further increased antimyeloma activity. In this case, 59% of the animals showed no signs of tumour recurrence. In mice with high tumour burden (5 × 105 ), no treatment modality achieved long-term survivors. Both natural killer (NK) cells and CD8+ T cells may be involved in the antitumoural immune response. These data provide evidence for the combined use of GM-CSF and IL-2 to enhance the therapeutic effectiveness of clinical cancer vaccination protocols. [ABSTRACT FROM AUTHOR]

Summary. Idiotype (Id) vaccination provides an innovative treatment modality against B-cell malignancies. In multiple myeloma patients, however, the antitumoral potential of this immunotherapeutic concept is limited. In an attempt to improve the therapeutic effectiveness of Id vaccination, we added Flt3 ligand (Flt3-L) and interleukin 2 (IL-2) to the protocol. Balb/c mice were inoculated i.p. (d -2) with different doses (1–5 × 105 ) of HOPC myeloma cells, secreting the IgHOPC Id-protein. Two days later, animals were treated with Flt3-L (10 µg per mouse/d, given i.p) for 10 consecutive days (d 0–9). On d 5 and d 11, myeloma-specific immunoglobulin (IgHOPC ) was administered s.c., together with incomplete Freund adjuvans (IFA) followed by the administration of IL-2 (2 × 10·000/d given i.p) for 10 d (d 5–14). Whereas IgHOPC , Flt3-L or IL-2, given alone, did not elicit long-term survival, the combination of IL-2 or Flt3-L with Id vaccination achieved a complete tumour rejection in 27% and 41% of mice respectively. However, the most powerful antimyeloma effects were induced by Flt3-L + Id vaccination + IL-2: 81% of the treated animals experienced long-term survival (> 180 d). Both natural killer (NK) cells and CD8+ T cells may be involved in the antitumoral immune response. These data suggest that the combination of Flt3-L and IL-2 can be used to enhance the therapeutic effectiveness of clinical cancer vaccination protocols. [ABSTRACT FROM AUTHOR]

Chronic myeloid leukemia (CML) is a myeloproliferative disorder associated with a characteristic chromosomal translocation called the Philadelphia chromosome. This oncogene is generated by the fusion of breakpoint cluster region (BCR) and Abelson leukemia virus (ABL) genes and encodes a novel fusion gene translating into a protein with constitutive tyrosine kinase activity. The discovery and introduction of tyrosine kinase inhibitors (TKIs) irreversibly changed the landscape of CML treatment, leading to dramatic improvement in long-term survival rates. The majority of patients with CML in the chronic phase have a life expectancy comparable with that of healthy age-matched individuals. Although an enormous therapeutic improvement has been accomplished, there are still some unresolved issues in the treatment of patients with CML. One of the most important problems is based on the fact that TKIs can efficiently target proliferating mature cells but do not eradicate leukemic stem cells, allowing persistence of the malignant clone. Owing to the resistance mechanisms arising during the course of the disease, treatment with most of the approved BCR-ABL1 TKIs may become ineffective in a proportion of patients. This article highlights the different molecular mechanisms of acquired resistance being developed during treatment with TKIs as well as the pharmacological strategies to overcome it. Moreover, it gives an overview of novel drugs and therapies that are aiming in overcoming drug resistance, loss of response, and kinase domain mutations.

Rabbit antithymocyte globulin-Genzyme™ is used to prevent graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Common disadvantages of treatment are infectious complications. The effects of rabbit antithymocyte globulin-Genzyme™ on thymic function have not been well-studied. Multicolor flow cytometry was used to analyze the kinetics of conventional and regulatory T cells in adult patients treated (n=12) or not treated (n=8) with rabbit antithymocyte globulin-Genzyme™ during the first 6 months after allogeneic hematopoietic stem cell transplantation. Patients treated with rabbit antithymocyte globulin-Genzyme™ had almost undetectable levels of recent thymic emigrants (CD45RA(+)CD31(+)) of both conventional and regulatory CD4T cells throughout the 6 months after allogeneic hematopoietic stem cell transplantation whereas CD4(+)CD45RA-memory T cells were less affected, but their levels were also significantly lower than in patients not treated with rabbit antithymocyte globulin-Genzyme™. In vitro, rabbit antithymocyte globulin-Genzyme™ induced apoptosis and cytolysis of human thymocytes, and its cytotoxic effects were greater than those of rabbit antithymocyte globulin-Fresenius™. Rabbit antithymocyte globulin-Genzyme™ in combination with a conditioning regimen strongly impairs thymic recovery of both conventional and regulatory CD4(+) T cells. The sustained depletion of conventional and regulatory CD4(+)T cells carries a high risk of both infections and graft-versus-host disease. Our data indicate that patients treated with rabbit antithymocyte globulin-Genzyme™ could benefit from thymus-protective therapies and that trials comparing this product with other rabbit antithymocyte globulin preparations or lymphocyte-depleting compounds would be informative.

Background: Allogeneic hematopoietic stem cell transplantation (alloHSCT) is often performed in cases of advanced hematological diseases, but because of the associated mortality and a high risk of relapse it is life prolonging only in some patients., Patients and Methods: A retrospective multi-center analysis of 401 patients was conducted to analyze the variables associated with outcome after alloHSCT in advanced hematological diseases. The Cox proportional hazards model was used to assess the independence of overall survival (OS) and disease-free survival (DFS) from prognostic factors in a multivariate model., Results: The 5-year OS and DFS were 27.3 and 21.1% respectively. Multivariate analysis showed that the underlying malignancy had a significant influence on OS and DFS (p < 0.001 and p < 0.011, respectively), whereas development of severe acute graft versus host disease (GvHD) had a negative impact on OS (p < 0.001). Development of chronic GvHD showed a trend to a better OS (p = 0.085) and DFS (p = 0.199). No impact was seen for the intensity of conditioning., Conclusion: Development of chronic GvHD but not the conditioning regimen improved the outcome after alloHSCT for advanced malignancies, underlining the importance of immunological rather than cytotoxic effects., (Copyright © 2012 S. Karger AG, Basel.)

Background: An alternative reduced-toxicity conditioning regimen for allogeneic transplantation, based on treosulfan and fludarabine, has recently been identified. The rationale for this study was to investigate the efficacy and safety of this regimen prospectively in patients with a primary myelodysplastic syndrome., Design and Methods: A total of 45 patients with primary myelodysplastic syndromes were conditioned with 3×14 g/m(2) treosulfan and 5×30 mg/m(2) fludarabine followed by allogeneic hematopoietic stem cell transplantation. Subtypes of myelodysplastic syndromes were refractory anemia with excess blasts-2 (44%), refractory cytopenia with multilineage dysplasia (27%), refractory anemia (9%), refractory anemia with ringed sideroblasts (4%), refractory cytopenia with multilineage dysplasia and ringed sideroblasts (4%), refractory anemia with excess blasts-1 (2%), and myelodysplastic syndrome with isolated del (5q) (2%). The myelodysplastic syndrome was unclassified in 7% of the patients. Forty-seven percent of the patients had a favorable karyotype, 29% an unfavorable one, and 18% an intermediate karyotype. Patients were evaluated for engraftment, adverse events, graft-versus-host disease, non-relapse mortality, relapse incidence, overall survival and disease-free survival., Results: All but one patient showed primary engraftment of neutrophils after a median of 17 days. Non-hematologic adverse events of grade III-IV in severity included mainly infections and gastrointestinal symptoms (80% and 22% of the patients, respectively). Acute graft-versus-host disease grade II-IV developed in 24%, and extensive chronic graft-versus-host disease in 28% of the patients. After a median follow-up of 780 days, the 2-year overall and disease-free survival estimates were 71% and 67%, respectively. The 2-year cumulative incidences of non-relapse mortality and relapse were 17% and 16%, respectively., Conclusions: Our safety and efficacy data suggest that treosulfan-based conditioning therapy is a promising treatment option for patients with myelodysplastic syndromes. clinicaltrials.gov identifier: NCT01062490.

The purpose of this prospective multicenter phase 2 trial was to investigate the long-term outcome of reduced-intensity conditioning allogeneic stem cell transplantation (alloSCT) in patients with poor-risk chronic lymphocytic leukemia. Conditioning was fludarabine/ cyclophosphamide-based. Longitudinal quantitative monitoring of minimal residual disease (MRD) was performed centrally by MRD-flow or real-time quantitative polymerase chain reaction. One hundred eligible patients were enrolled, and 90 patients proceeded to alloSCT. With a median follow-up of 46 months (7-102 months), 4-year nonrelapse mortality, event-free survival (EFS) and overall survival (OS) were 23%, 42%, and 65%, respectively. Of 52 patients with MRD monitoring available, 27 (52%) were alive and MRD negative at 12 months after transplant. Four-year EFS of this subset was 89% with all event-free patients except for 2 being MRD negative at the most recent assessment. EFS was similar for all genetic subsets, including 17p deletion (17p-). In multivariate analyses, uncontrolled disease at alloSCT and in vivo T-cell depletion with alemtuzumab, but not 17p-, previous purine analogue refractoriness, or donor source (human leukocyte antigen-identical siblings or unrelated donors) had an adverse impact on EFS and OS. In conclusion, alloSCT for poor-risk chronic lymphocytic leukemia can result in long-term MRD-negative survival in up to one-half of the patients independent of the underlying genomic risk profile. This trial is registered at http://clinicaltrials.gov as NCT00281983.

Forty patients were enrolled in this phase 2 study combining radioimmunotherapy (RIT) using yttrium-90-ibritumomab-tiuxetan (15 MBq [0.4 mCi]/kg) with reduced-intensity conditioning (RIC) using fludarabine (90 mg/m(2)) and 2 Gy total body irradiation followed by allogeneic hematopoietic cell transplantation (HCT) from related (n = 13) or unrelated (n = 27) donors for the treatment of advanced non-Hodgkin lymphoma. Diagnoses were follicular lymphoma (n = 17), chronic lymphocytic leukemia (n = 13), mantle cell lymphoma (n = 8), marginal zone lymphoma (n = 1), and lymphoplasmacytic lymphoma (n = 1). Median age was 55 years (range, 34-68 years). All patients were high risk with refractory disease or relapse after preceding autologous HCT. No additional toxicities attributable to RIT were observed. Engraftment was rapid and sustained. Incidences of acute graft-versus-host disease 2-4 and chronic graft-versus-host disease were 43% and 53%, respectively. Kaplan-Meier-estimated nonrelapse mortality was 45% at 2 years. Twenty-two of 40 patients (55%) are alive, resulting in a Kaplan-Meier-estimated 2-year survival of 51% for all, 67% for follicular lymphoma, 49% for chronic lymphocytic leukemia, and 37% for mantle cell lymphoma patients. The combined use of RIT with RIC is feasible with acceptable toxicity, even in elderly and heavily pretreated patients. This study is registered at www.clinicaltrials.gov as #NCT00302757.

From 2002 to 2007, 103 patients with primary myelofibrosis or postessential thrombocythemia and polycythemia vera myelofibrosis and a median age of 55 years (range, 32-68 years) were included in a prospective multicenter phase 2 trial to determine efficacy of a busulfan (10 mg/kg)/fludarabine (180 mg/m(2))-based reduced-intensity conditioning regimen followed by allogeneic stem cell transplantation from related (n = 33) or unrelated donors (n = 70). All but 2 patients (2%) showed leukocyte and platelet engraftment after a median of 18 and 22 days, respectively. Acute graft-versus-host disease grade 2 to 4 occurred in 27% and chronic graft-versus-host disease in 43% of the patients. Cumulative incidence of nonrelapse mortality at 1 year was 16% (95% confidence interval, 9%-23%) and significantly lower for patients with a completely matched donor (12% vs 38%; P = .003). The cumulative incidence of relapse at 3 years was 22% (95% confidence interval, 13%-31%) and was influenced by Lille risk profile (low, 14%; intermediate, 22%; and high, 34%; P = .02). The estimated 5-year event-free and overall survival was 51% and 67%, respectively. In a multivariate analysis, age older than 55 years (hazard ratio = 2.70; P = .02) and human leukocyte antigen-mismatched donor (hazard ratio = 3.04; P = .006) remained significant factors for survival. The study was registered at www.clinicaltrials.gov as #NCT 00599547.

This study investigated the immunogenicity of Wilms tumor gene product 1 (WT1)-peptide vaccination in WT1-expressing acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients without curative treatment option. Vaccination consisted of granulocyte-macrophage colony-stimulating factor subcutaneously days 1 to 4, and WT1.126-134 peptide and 1 mg keyhole limpet hemocyanin on day 3. The initial 9 patients received 4 vaccinations biweekly, then monthly, and the subsequent 10 patients received continual biweekly vaccination. Seventeen AML patients and 2 refractory anemia with excess blasts patients received a median of 11 vaccinations. Treatment was well tolerated. Objective responses in AML patients were 10 stable diseases (SDs) including 4 SDs with more than 50% blast reduction and 2 with hematologic improvement. An additional 4 patients had clinical benefit after initial progression, including 1 complete remission and 3 SDs. WT1 mRNA levels decreased at least 3-fold from baseline in 35% of patients. In 8 of 18 patients, WT1-tetramer(+) T cells increased in blood and in 8 of 17 patients in bone marrow, with a median frequency in bone marrow of 0.18% at baseline and 0.41% in week 18. This WT1 vaccination study provides immunologic, molecular, and preliminary evidence of potential clinical efficacy in AML patients, warranting further investigations.

Effects of angiotensin (Ang)-(1-7), an AngII metabolite, on bone marrow-derived hematopoietic cells were studied. We identified Ang-(1-7) to stimulate proliferation of human CD34(+) and mononuclear cells in vitro. Under in vivo conditions, we monitored proliferation and differentiation of human cord blood mononuclear cells in NOD/SCID mice. Ang-(1-7) stimulated differentially human cells in bone marrow and accumulated them in the spleen. The number of HLA-I(+) and CD34(+) cells in the bone marrow was increased 42-fold and 600-fold, respectively. These results indicate a decisive impact of Ang-(1-7) on hematopoiesis and its promising therapeutic potential in diseases requiring progenitor stimulation.

Porous ceramics made of alumina and hydroxyapatite were created using a protein foaming method. Porosity and pore size distribution were successfully varied by means of chemical modification of the foaming protein Bovine serum albumin (BSA). The effectiveness of the BSA and of its chemical modifications as well as the influence of the dispersing agent were investigated using synchrotron tomography. Resulting porous ceramic materials were used as three-dimensional substrates for the cultivation of human peripheral stem cells. The cells proliferated and differentiated in culture. Five cell lines consistent with human blood cell lines were observed.

The list of multisystem diseases involving both kidney and skin is long and includes immunologic disorders like systemic lupus erythematodes, a broad variety of vasculitides, metabolic disorders like diabetes mellitus, and infectious diseases. The present work describes the first case of renal failure due to Schnitzler's syndrome, a rare entity characterized by the association of generalized chronic urticaria, monoclonal IgM gammopathy, and osteosclerotic lesions. The described patient experiences improvement of renal function after treatment with the chimeric anti-CD20 antibody rituximab, indicating that the impairment of renal function might be mediated by B-lymphocytes. Renal insufficiency is known to be a potential complication of hypocomplementemic urticarial vasculitis. The present case reveals that it can occur in normocomplementemic urticarial vasculitides as well. Schnitzler's syndrome is a rare but probably underdiagnosed syndrome. It should be considered in the differential diagnosis of rash and coincidental renal failure of unknown origin. Immunofixation of immunoglobulins constitutes the crucial diagnostic step, since monoclonal gammopathy is a constant clinical sign in this entity.

There is considerable interest in immunotherapeutic approaches for lymphoma. The expression of proteinase inhibitor 9 (PI-9), a molecule that inactivates granzyme B, is considered an immune escape mechanism in lymphoma. Further, lymphomas frequently overexpress the antiapoptotic molecule bcl-2, which is able to inhibit perforin-dependent cytotoxic pathways. In this study, the impact of PI-9 and bcl-2 expression on the sensitivity of lymphomas to T- and natural killer (NK) cell-mediated cytotoxicity was analyzed. We found PI-9 expression in 10 of 18 lymphoma cell lines and in 9 of 14 primary lymphomas. Overexpression of bcl-2 was found in 8 of 18 cell lines and in 12 of 14 primary lymphomas. All lymphoma cells were sensitive to cytolysis by specific T cells and cytokine-activated NK cells, and no difference in sensitivity was observed with respect to PI-9 or bcl-2 expression. Cytolysis was mediated predominantly through perforin-dependent pathways despite expression of PI-9 and bcl-2. Interestingly, the majority of lymphoma cells were resistant to cytolysis by resting allogeneic NK cells. This was due to the failure of lymphomas to induce degranulation of resting NK cells. These results show that resistance to perforin-dependent pathways is not a relevant immune escape mechanism in lymphoma and therefore is unlikely to impair clinical outcome of immunotherapeutic approaches.

Adoptive immunotherapy using natural killer (NK) cells is currently under investigation, especially in situations where anti-neoplastic effect is needed but infusion of T cells is considered hazardous, such as in recipients of haematopoietic stem-cell transplantation (HSCT) from haploidentical donors. NK-cell therapy is mainly but not exclusively investigated in the setting of allogeneic stem-cell transplantation. NK cells may induce potent anti-leukaemic and possibly anti-rejection activity, and may even mitigate graft-versus-host disease (GvHD). It remains to be determined whether such effects are clinically important and whether or not they are mediated mainly or exclusively by KIR-HLA class I interactions. Recent advances in graft engineering has provided methods for isolating large numbers of purified NK cells. Several groups have shown that clinical-grade NK cells at doses up to 10(7)/kg may be collected and purified for the purpose of infusion to patients. Early results in a limited number of patients show that these cell doses may be administered without adverse events and possibly without inducing GvHD. Further study is required to determine whether such infusions will be useful in preventing graft rejection, exerting graft-versus-leukaemia effects, and/or hastening immune recovery.