CD40L-transfected myeloma cells transfer prolonged immunity in vivo.

BACKGROUND: In a large number of patients with multiple myeloma, chemotherapy is the only therapeutic option. During recent years, major effort has been put into immunotherapeutic approaches for this malignancy. MATERIALS AND METHODS: In this study, wild-type (wt) myeloma cells (5x10(5)) were injected subcutaneously into Balb/c mice. CD40L-transfected myeloma cells (5x10(5)) were subsequently injected intratumorally into the established (>100 mm(3)) wt tumor nodules. Overall survival and tumor growth were measured. RESULTS: Out of eight animals receiving wt tumor cells, one died prior to the formation of a solid tumor nodule. Following the CD40L-transfected myeloma cell injection, stable complete remission at day 60 with all the animals surviving resulted. On day 60, a re-challenge was performed with wt myeloma cells. No tumor growth was observed after 120 days out of seven remaining animals, one died. CONCLUSION: Intratumoral injection of CD40L-transfected myeloma cells induces complete tumor remission and long lasting immunity against tumor recurrence.

Long-term survival of a patient with small cell lung cancer after nine lines of chemotherapy and radiation.

BACKGROUND: Small cell lung cancer (SCLC) results in death within 1-2 months if left untreated. Although therapeutic standards only comprise first- and second-line chemotherapy due to poor prognosis, a subset of patients may warrant a trial of further chemotherapy, as demonstrated in the following case. CASE REPORT: We present a 52-year-old man with confirmed anaplastic SCLC who survived 10 years while receiving 9 lines of chemotherapy including high-dose chemotherapy. Thoracic radiation, afterloading and radionuclide therapy supplemented the therapeutic management. Remarkably, he had two very long remission periods following topotecan. CONCLUSION: This case indicates that frequently repeated chemotherapy beyond second-line treatment in a subset of patients with limited-disease SCLC may result in long-term survival, even without ever achieving a complete remission. Close surveillance of responsiveness and appropriate in-time cytostatic treatment is proposed in the management of patients with SCLC who remain in good performance.

Prevention of lethal tumor growth and generation of long-lasting immunity in vivo using CD137L and interleukin-12 gene transfer.

In a wide range of solid tumors, overexpression of CD137L has been shown to induce tumor immunity partly due to the stimulation of CD8+ CTL, which was even increased when immunotherapy with interleukin-12 (IL12) was additionally employed. However, little in known regarding hematologic neoplasias in this respect. Of the 8 animals receiving IL12-secreting tumor cells, 2 died. Animals treated with CD137L-expressing tumor cells and the combination group, all animals survived. Interestingly, re-challenge with wild-type tumor cells was rejected by all animals in the CD137L group and all remaining animals in the IL12 group, while these in the control group died. IL12- and CD137L-transfected plasmocytoma cells prevented tumor growth and induced long-lasting immunity. Our results warrant follow-up for future clinical use in patients with myeloma.

Minimal size MIDGE vectors improve transgene expression in vivo.

Viral and plasmid vectors may cause immunological side-effects resulting from the expression of therapeutically unwanted genes and from CpG motifs contained in their sequence. A new vector type for minimalistic, immunological-defined gene expression (MIDGE) may overcome these problems. MIDGE is a minimal size gene transfer unit consisting of the expression cassette, including promotor, gene and RNA-stabilizing sequences, flanked by two short hairpin oligonucleotide sequences. DNA not encoding the desired gene is reduced to a minimum. To compare transfection efficiencies in vivo hydrodynamics-based, systemic transfection was performed in BALB/c mice with MIDGE vectors and corresponding plasmids. The transfection efficiencies of the MIDGE vectors as measured by luciferase expression were significantly higher in liver (2.5-fold), lung (3.5-fold), kidneys (3.9-fold) and heart (17-fold) as compared to plasmids. The mean numbers of MIDGE vector molecules per cell as measured by quantitative PCR were also significantly higher. These advantages suggest the preferential use of this new vector type for clinical gene therapy studies.

Cholecystitis in neutropenic patients: retrospective study and systematic review.

Abdominal infections are life-threatening complications in neutropenic patients. Among these, neutropenic cholecystitis is relatively rare. Nevertheless, its actual relevance is only investigated by anecdotal reports. We present a consecutive retrospective series of nine patients over a 12-year period. We calculated a frequency of 0.4% among all neutropenic episodes in patients with acute leukemia or aggressive lymphoma undergoing myelosuppressive chemotherapy. Only three of these patients had gallstones. Four patients died during the course of cholecystitis but in none of them cholecystitis was the primary cause of death. Systematic review of the literature revealed 45 patients with neutropenic cholecystitis of whom 26.7% died.

DHAP in combination with rituximab vs DHAP alone as salvage treatment for patients with relapsed or refractory diffuse large B-cell lymphoma: a matched-pair analysis.

The addition of rituximab to chemotherapy in patients with diffuse large B-cell lymphoma (DLBCL) has been shown to improve outcome in first-line therapy. However, in patients with relapsed or refractory disease, the value of adding rituximab to salvage chemotherapy is less clearly defined. This study performed a matched-pair analysis of patients with relapsed or refractory DLBCL by comparing the combination of dexamethasone, high-dose cytarabine and cisplatin (DHAP) with rituximab to DHAP alone. Sixty-seven patients with relapsed or refractory DLBCL were collected from two prospective phase II trials from Germany and Italy. Twenty-three patient pairs treated with either DHAP in combination with rituximab or DHAP alone could be analysed after matching for important prognostic factors. The addition of rituximab to the DHAP regimen led to higher complete and similar overall remission rates. However, differences with regard to complete remission rates failed to reach statistical significance, thereby necessitating further evaluation of the role of combined immunochemotherapy in this patient population.

Anti-tumoral capabilities of effector cells after IFN-alpha or CpG-motif treatment of cocultured dendritic cells.

INTRODUCTION: Ex vivo expansion of monocyte-derived dendritic cells (mDCs) and subsequent coculture with autologous cytokine-induced killer (CIK) cells is an established system to create specific and non-specific anti-tumoral immunity. mDCs constitute the most frequently applied DC subset in clinical studies. One recently published approach to optimize the immunological functions of the DC/CIK cell system is the replacement of interleukin (IL)-4 by interferon (IFN)-alpha in the maturation process of the DCs. MATERIALS AND METHODS: The expressions of relevant surface antigens of IL-4-DCs and IFNalpha-DCs by flow cytometry and the anti-tumoral activation of effector cells cocultured with both types of DCs using cytotoxicity assays were compared. In addition, short-term coculture experiments with both types of DCs and IFNgamma-LAK effector cells were performed and compared with standard CIK cell coculture experiments. RESULTS: Regarding the expressions of functionally relevant surface markers, no differences could be detected for CD80, CD83, and HLA-DR between IFNalpha-DCs and IL-4-DCs, whereas the mean fluorescence intensities of CD40, CD86, CD54, and HLA-ABC were decreased and the expression of CD14 was increased for IFNgamma-DCs. Moreover, no enhancement of cytotoxicity of cocultured CIK cells against tumor cell lines (A498 and SW480) was detected by the use of IFNalpha-DCs. Additionally, coculture experiments with IFNgamma-LAK cells were performed and unexpectedly higher lysis rates in comparison with the established IL-4-DC/CIK coculture model was observed. Early incubation of the mDCs with several CpG-ODNs failed to increase the anti-tumoral cytotoxicity of the cocultured IFNgamma-LAK cells. CONCLUSIONS: These results demonstrate that in the mDC/CIK cell system, IFNalpha-DCs are not superior in inducing anti-tumoral cytotoxicity and even moderately inferior regarding the expression of functionally relevant surface markers compared with IL-4-DCs.

Invasive fungal infections in neutropenic enterocolitis: a systematic analysis of pathogens, incidence, treatment and mortality in adult patients.

BACKGROUND: Neutropenic enterocolitis is a life-threatening complication most frequently occurring after intensive chemotherapy in acute leukaemias. Gramnegative bacteria constitute the most important group of causative pathogens. Fungi have also been reported, but their practical relevance remains unclear. The guidelines do not address concrete treatment recommendations for fungal neutropenic enterocolitis. METHODS: Here, we conducted a metaanalysis to answer the questions: What are frequency and mortality of fungal neutropenic enterocolitis? Do frequencies and microbiological distribution of causative fungi support empirical antimycotic therapy? Do reported results of antimycotic therapy in documented fungal neutropenic enterocolitis help with the selection of appropriate drugs? Following a systematic search, we extracted and summarised all detail data from the complete literature. RESULTS: Among 186 articles describing patients with neutropenic enterocolitis, we found 29 reports describing 53 patients with causative fungal pathogens. We found no randomised controlled trial, no good quality cohort study and no good quality case control study on the role of antifungal treatment. The pooled frequency of fungal neutropenic enterocolitis was 6.2% calculated from all 860 reported patients and 3.4% calculated from selected representative studies only. In 94% of the patients, Candida spp. were involved. The pooled mortality rate was 81.8%. Most authors did not report or perform antifungal therapy. CONCLUSION: In patients with neutropenic enterocolitis, fungal pathogens play a relevant, but secondary role compared to bacteria. Evidence concerning therapy is very poor, but epidemiological data from this study may provide helpful clues to select empiric antifungal therapy in neutropenic enterocolitis.

Retroviral IFN-alpha gene transfer combined with gemcitabine acts synergistically via cell cycle alteration in human pancreatic carcinoma cells implanted orthotopically in nude mice.

Standard chemotherapy for pancreatic carcinoma is based on the use of gemcitabine. The clinical benefit of interferon-alpha (IFN-alpha) in advanced pancreatic cancer has been shown. However, it has been demonstrated that to be effective, there is a need for a constant amount of IFN-alpha at the site of the tumor. Therefore, we examined transfection of the human pancreatic cancer cell line DAN-G with a retrovirus encoding for IFN-alpha and the effect of IFN-alpha gene expression alone or in combination with gemcitabine on growth inhibition of DAN-G pancreatic cancer cells in vitro and in vivo in orthotopically implanted DAN-G cells in nude mice. DAN-G cells could be efficiently transfected retrovirally by the human IFN-alpha gene and significantly enhanced the levels of IFN-alpha mRNA. In vitro gemcitabine led to an alteration of G1/S phase progression in transduced as well as untransduced cells, whereas IFN-alpha led to a significant decrease in cell viability in the transduced cells via delay in the progression of the S phase but no alteration of G1/S phase progression. In vivo, tumor volume in mice was reduced significantly with gemcitabine combined with IFN-alpha (76% +/- 8.3%) compared with gemcitabine alone (62.9% +/- 7.3%) or IFN-alpha alone (24.4% +/- 5.2%) compared with untreated animals. We conclude that gemcitabine and IFN-alpha concomitantly inhibited tumor cell proliferation significantly.

High-dose chemotherapy followed by autologous stem cell transplantation as first-line therapy in aggressive non-Hodgkin's lymphoma: a meta-analysis.

BACKGROUND AND OBJECTIVES: High-dose chemotherapy followed by autologous stem cell transplantation (HDT/ASCT) has proven to be superior to conventional chemotherapy in patients with chemosensitive relapse of aggressive non-Hodgkin's lymphoma (NHL). Therefore, HDT/ASCT was evaluated as part of first-line therapy. Several trials generated conflicting results. This meta-analysis summarizes the available evidence from all suitable studies. DESIGN AND METHODS: Prospective, randomized trials with HDT/ASCT as first-line therapy of aggressive lymphoma were included in this meta-analysis. The primary outcome was overall mortality. Statistical analysis applied the odds ratio (OR) and a fixed effects model. RESULTS: Eleven trials with 2228 patients were eligible for meta-analysis. Overall mortality was comparable in the HDT/ASCT and in control arms (OR=0.97, 95% CI: 0.69;1.36, p=0.9), with statistically significant heterogeneity between the trials. To resolve this, we tried to identify variables that could explain this heterogeneity. Among a range of methodological, patient- or treatment-related factors, subgroups formed by the proportion of bulky disease in treated patients, the type of therapy prior to HDT/ASCT, the drop-out rate from the HDT/ASCT arm, and the presence of high or high-intermediate risk IPI showed significant benefit for any of the treatment modalities. However, such post-hoc subgroup analysis may be considerably influenced by random or systemic biases. INTERPRETATION AND CONCLUSIONS: Overall, the analysis of published evidence reveals very heterogeneous results and no overall survival benefit. Therefore, HDT/ASCT cannot be recommended as standard first line treatment for patients with aggressive NHL. However, the exploratory analyses presented here may help to design new trials for this treatment modality.

Treatment options of AIDS-related lymphoma.

Recently, the median survival of patients with AIDS-related lymphoma has changed significantly. This effect is mainly because of changes in the use of antiviral (highly active antiretroviral therapy; HAART) or chemotherapy regimens. Several novel treatment options have been explored in patients with lymphoma. It is hoped that innovative strategies will lead to a survival benefit in these patients. In this review, we present an update of current strategies for the treatment of AIDS-related lymphoma.

Standard of care of patients with Chronic Myeloid Leukemia (CML) treated in community based oncology group practices between 2001-2015 in Rhineland- Palatinate (Germany)

Background: The aim of this study was to assess retrospectively treatment and outcome of CML-patients in community based oncology practices in Germany and whether European LeukemiaNET (ELN) recommendations were followed. Method: All Ph+, BCR-ABL1+ CML-patients who were treated between 11/2001 and 12/2015 in nine oncology group practices were analyzed retrospectively. Results: Two hundred sixty patients with a median age of 60 (18­90) were analyzed. 254 (98%) were in chronic phase, 5 (2%) in accelerated and 1 (0.4%) in blast crisis. 248 patients (95%) received some form of TKI-therapy. 1st line TKI was imatinib in 197 patients (79%), 51 (21%) received a second generation TKI. 75% of TKI-therapies were monitored by PCR. Overall survival after 10 years according to Charlson comorbidity index (CCI) was: CCI 2: 100%; CCI 3­4: 83%; CCI 5­6: 52%; CCI ≥7: 39%. More patients died from comorbidities (8%) than from CML (5%). Whether patients died was strongly correlated to CCI at diagnosis: CCI 2: 3% of patients died, CCI 3­4: 16% of patients died, CCI 5­6: 38% of patients died, CCI ≥ 7: 42% of patients died. Conclusion: CML-patients treated in oncology group practices receive standard of care as recommended by ELN. Overall survival in routine care is comparable to international studies. Molecular monitoring should be improved (AU)

Increase of in vivo antitumoral activity by CD40L (CD154) gene transfer into pancreatic tumor cell-dendritic cell hybrids.

OBJECTIVES: Fusion of dendritic cells (DC) with tumor cells is an approach in immunotherapy combining antigenicity and capacity of antigen presentation to activate T cells for the induction of tumor-specific cytotoxic immunity. Although there have been reports of clinical benefit, response rates have been limited and further improvements are warranted. METHODS: We used murine DC and a novel protocol for an effective fusion of those cells with the murine pancreatic cell line Panc02. RESULTS: We observed 2 events: only moderate in vitro and in vivo cytotoxicity of tumor cell/DC hybrids and a down-regulation of costimulatory molecules on fused cells. Therefore, we transfected tumor cell/DC hybrids with an adenovirus expressing CD154 to improve DC activation and generating antitumor immune response without the need of CD4 T cells. High CD154 expression could be obtained by transfection of DC and Panc02 cells prior fusion. Furthermore, vaccination with CD154-transfected tumor cell/DC hybrid led to a significantly increased induction of cytotoxic T cells in vitro and to an improved antitumoral effect in an orthotopic in vivo mouse model. CONCLUSIONS: CD154-transfected tumor cell/DC hybrids are a promising approach to increase the efficiency of antitumoral response.

Cytokine-induced killer cells are type II natural killer T cells.

BACKGROUND: Until now, cytokine-induced killer (CIK) cells were assumed to be part of the type I natural killer T (NKT) cell population, but it was not yet investigated if this is correct. METHODS: For analysis, CIK cells were generated by various culture conditions. Human type I NKT cells express a T cell receptor (TCR) composed of an invariant V alpha 24-J alpha Q chain combined with one of several V beta chains. The V alpha 24 is a reliable marker for the presence of these TCRs. RESULTS: While comparing cultures stimulated with different substances, we observed the lack of any V alpha 24 on the surface of CIK culture cells. CONCLUSION: We conclude that CIK cells do not belong to the type I NKT cells.

High dose chemotherapy with autologous stem cell transplantation in diffuse large B-cell lymphoma.

BACKGROUND: High-dose chemotherapy (HDT) with autologous stem cell transplantation (ASCT) plays an important role in the treatment of aggressive non-Hodgkin's lymphoma (NHL). We report on a retrospective analysis of all patients with diffuse large B-cell lymphoma who were consecutively treated with HDT followed by ASCT at the University Hospital of Bonn, Germany, between 1996 and 2004. METHODS: A total of 25 patients were transplanted for biopsy-proven diffuse large B-cell lymphoma (DLBCL). Eight patients received up-front HDT as first-line therapy, four patients received HDT due to incomplete response to conventional induction chemotherapy, and six patients were treated for primary refractory disease. Seven patients had recurrent lymphoma. RESULTS: A complete remission (CR) was achieved in 14 of 25 patients (56%). Estimated 3-year survival for patients treated with upfront HDT, chemosensitive patients with incomplete response to first line therapy, and patients with chemosensitive relapsed disease was 87.5%, 50.0% and 60.0%, respectively. In contrast, no patient with primary refractory disease or relapsed disease lacking chemosensitivity lived longer than 8 months. Chemosensitivity was the only significant prognostic factor for overall survival (OS) in multivariate analysis. CONCLUSIONS: Our results confirm that HDT and ASCT is a highly effective therapy in patients with DLBCL leading to long-term survival in a substantial proportion of patients. Patients treated upfront for high-risk disease, incomplete response to conventional first-line therapy, or for chemosensitive relapse have a good prognosis. In contrast, patients with primary chemorefractory disease and patients with relapsed disease lacking chemosensitivity do not benefit from HDT with ASCT.

Pegfilgrastim as hematopoietic support for dose-dense chemoimmunotherapy with R-CHOP-14 as first-line therapy in elderly patients with diffuse large B cell lymphoma.

GOALS OF WORK: Recently, 6 cycles of R-CHOP-14 have been recommended as the reference standard regimen for the treatment of elderly patients with diffuse large B-cell lymphoma (DLBCL). Pegfilgrastim has been shown to facilitate dose-dense chemotherapy schedules with a single administration per chemotherapy cycle. The aims of this study were to evaluate the use of pegfilgrastim in combination with the R-CHOP-14 regimen in a homogenous group of previously untreated elderly patients with DLBCL and to assess the pharmacokinetics of pegfilgrastim within this patient population. MATERIALS AND METHODS: Ten patients with DLBCL between 60 and 80 years of age received a single subcutaneous injection of 6 mg pegfilgrastim 24 h after administration of R-CHOP chemoimmunotherapy, which was repeated for 6 to 8 cycles in two-weekly intervals. A total of 348 blood samples were collected. Pegfilgrastim plasma levels and absolute neutrophil counts were measured every other day during the first treatment cycle and twice weekly during all consecutive cycles. MAIN RESULTS: Sixty-three of 72 cycles (87.5%) could be delivered on time and at the planned dose. Median absolute neutrophil nadir was 0.32 g/l on day 9. Grade 3/4 granulocytopenia occurred in all patients. Febrile neutropenia occurred in two patients. Plasma levels of pegfilgrastim remained elevated during the neutropenic phase. At the start of hematologic recovery, plasma concentrations of pegfilgrastim decreased rapidly to baseline levels. Median pegfilgrastim trough plasma level was 0.43 ng/ml on the day preceding the next application. CONCLUSIONS: A single fixed dose of 6 mg of pegfilgrastim given once per cycle of R-CHOP-14 is effective in supporting neutrophil recovery to allow two-weekly drug administration in previously untreated elderly patients with DLBCL. However, close monitoring for infectious complications is mandatory in this patient population.

Chromosomal translocations are associated with poor prognosis in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), chromosomes usually evade detailed cytogenetic analyses because cells poorly respond to the traditionally used set of mitogens. We applied novel technologies, such as stimulation of CLL cells either with CD40 ligand or with a combination of CpG-oligodeoxynucleotides and IL-2, to increase the frequency of metaphase spreads for detailed chromosome analysis in 96 patients with CLL. This approach revealed that translocations occurred in 33 of 96 (34%) of our patients with CLL. The presence of translocations defined a new prognostic subgroup because these patients have significantly shorter median treatment-free survival (24 months vs 106 months; P < .001) and significantly inferior overall survival (OS; median, 94 months) than patients without translocations (346 months; P < .001). In multivariate analysis-including Binet stage, complex karyotype, CD38 expression, and 17p deletions-translocation proved to be the prognostic marker with the highest impact for an unfavorable clinical outcome (P < .001). In summary, we identified a new subgroup of patients with CLL defined by chromosomal trans-locations and poor prognosis. Our data may facilitate the identification of molecular events crucial for transforming activity in this disease and should have implications for risk-adapted clinical management of patients with CLL.

Dexamethasone, high-dose cytarabine, and cisplatin in combination with rituximab as salvage treatment for patients with relapsed or refractory aggressive non-Hodgkin's lymphoma.

We designed a multicenter Phase II trial to prospectively evaluate the efficacy and safety of the combination of rituximab with the DHAP regimen (dexamethasone, high-dose cytarabine, cisplatin) in patients who relapsed after or were resistant to a CHOP-like regimen. A total of 53 patients with relapsed or resistant aggressive B-cell NHL were analyzed. The overall response rate was 62.3 percent. With a median follow-up of 24.9 months, median overall and progression-free survivals were 8.5 and 6.7 months, respectively. Immunochemotherapy with rituximab and DHAP proved to be feasible and effective in this patient population.

Neutropenic enterocolitis in adults: systematic analysis of evidence quality.

OBJECTIVE: Neutropenic enterocolitis is a life-threatening complication occurring most frequently after intensive chemotherapy in acute leukaemias. The literature is heterogeneous and a systematic review is lacking. METHODS: Following a systematic search we categorised all relevant reports according to their quality and extracted evidence to answer the questions: Which diagnostic criteria are appropriate? What is the incidence of neutropenic enterocolitis? Are there good quality studies supporting specific interventions: Which empiric antimicrobial therapy is recommendable? Is neutropenic enterocolitis without surgical emergency complications an indication for bowel resection? RESULTS: We found and analysed 145 articles of these reports: 64 were reports of single cases, 30 papers reported of two or three cases, 13 were narrative reviews, 34 were retrospective case series of more than three cases and four were prospective diagnostic studies. There were no prospective trials or case control studies on the therapy of neutropenic enterocolitis. There was no consensus on diagnostic criteria. We discuss the difficulty to define diagnostic criteria without having a disease definition. Histology is mostly not available in the living patients. We suggest applying a combination of clinical and radiological criteria: fever, abdominal pain and any bowel wall thickening >4 mm detected by ultrasonography (US) or computed tomography. We calculated a pooled incidence rate from 21 studies of 5.3% (266/5058; 95% CI: 4.7%-5.9%) in patients hospitalised for haematological malignancies, for high-dose chemotherapy in solid tumours or for aplastic anaemia. CONCLUSIONS: This systematic review provides diagnostic criteria for neutropenic enterocolitis, presents a quantitative synthesis on its incidence and discusses its treatment recommendations. Prospective studies are clearly warranted.

Transfection of human monocyte-derived dendritic cells with CpG oligonucleotides.

Monocyte-derived dendritic cells (mDC), the most frequently applied DC subset in clinical studies, which can be obtained easily from peripheral blood monocytes after incubation with GM-CSF and IL-4, have not been clearly demonstrated to be activated by CpG oligodeoxynucleotides (ODN). The development of novel molecular strategies - such as the use of CpG-ODN - to increase immunological functions and thus improve the therapeutic efficiency of mDC vaccines in the treatment of malignant diseases is highly desirable. CpG-ODN need to be internalized into specific intracellular compartments to be active. Therefore, we applied electroporation and lipofection and compared these techniques with incubation to overcome possible defects in localization. Conditions of CpG-ODN transfection of these cells were optimized using fluorescein-marked ODN 2216. We were able to achieve high transfection efficiencies with various methods of delivery. However, we did not observe increased expression of maturation-associated and functionally relevant surface antigens (CD14, HLA-DR, CD40, CD83, CD80 and CD86), significant secretion of IL-12 and IFN-alpha in culture supernatant, or enhanced antitumour activation of cytokine-induced killer cells. In conclusion, our results show that non-viral transfection of CpG-ODN is not sufficient to overcome resistance of mDC to CpG activation.