The preparation of myeloma-specific T cells activated with dendritic cells loaded with nonapeptides derived from mucin protein MUC1 and catalytic subunit of telomerase hTERT.

BACKGROUND: Multiple myeloma is an incurable hematological disease. High-dose chemotherapy including autologous stem cell transplantation is recently considered a standard therapy for myeloma. Unfortunately, a relapse of the disease is inevitable. Therefore, new approaches such as immunotherapy have been considered recently. A specific activation of cytotoxic T cells can be reached using dendritic cells loaded with tumor-specific antigens. The HLA-A2-specific nonapeptides as hTERT derived from catalytic subunit of telomerase and MUC1 derived from mucin protein can be used. DESIGN AND SUBJECTS: Activation, identification, separation and expansion of myeloma-specific T cells from healthy HLA-A2 blood donors were tested in an in vitro study using hTERT and MUC1 nonapeptides as tumor-specific antigens. METHODS AND RESULTS: T cells and dendritic cells were obtained from peripheral blood. T cells were repeatedly stimulated with hTERT and MUC1 nonapeptide-loaded dendritic cells. Activated myeloma-specific T cells produced interferon gamma and were evaluated by flow cytometry. The activated T cells were immunomagnetically separated and in vitro expanded to the number usable in clinical trials. CONCLUSIONS: This study demonstrates feasibility of a specific activation, identification, separation and expansion of tumor-specific T cells that can be used in myeloma therapy

In vitro activation of cytotoxic T-lymphocytes by hTERT-pulsed dendritic cells.

Multiple myeloma has been considered a weakly immunogenic malignancy that can cause profound defects in the immune system. An important issue for the immunotherapy of myeloma is the identification of appropriate tumor-associated antigens (TAAs). Recently, hTERT (human telomerase reverse transcriptase) was detected on a majority of human malignancies. In the studies reported here, we studied antigen-specific and HLA-A2-restricted cytotoxic activity against an ARH77 myeloma cell line in vitro. An HLA-A2-specific hTERT-derived nonapeptide (540ILAKFLHWL548) was used as a TAA. Myeloma-specific cytotoxic activity of hTERT-reactive cytotoxic lymphocytes (CTLs) was established by repeated stimulation of the CTLs via dendritic cells loaded with hTERT-derived nonapeptide. These studies were able to demonstrate that hTERT-reactive T-lymphocytes can be identified and expanded using relatively simple in vitro techniques consisting of antigen-specific stimulation, immunomagnetic sorting, and then induction of rapid expansion

Generation of dendritic cells using cell culture bags--description of a method and review of literature.

Anticancer immunotherapy using dendritic cell-based vaccines is a strategy aimed at the induction and maintenance of immune responses against cancer cells. Clinical applications of dendritic cells (DCs) require stringent adherence to Good Manufacturing Practice (GMP) methods and rigorous standardization of DC-based vaccine preparation. Recently, closed systems for DC culture have been developed with a goal to minimize the risk of contamination. Here, we compare the yield, immunophenotype, and functional properties of DCs generated in Lifecell X-Fold culture bags and in plastic wells, both from adherence-selected monocytes, and review the current literature on closed systems for DC generation. We found that both the overall yield and the yield of CD83+ cells in cell culture bags was lower than in the standard culture method. No statistically significant differences were observed in the expression of DC immunophenotypic markers. The capability of DCs cultured in bags and in wells to induce the proliferation of allogeneic mononuclear cells were equivalent. The performance of DCs in mixed lymphocyte reaction correlated significantly (p = 0.005) with the CD83 expression but not with the CD80, CD86, HLA-DR, CD1a, and CD1c expression. We conclude that the immunophenotype and stimulatory properties of DCs cultured in closed cell culture bags are similar to those generated by conventional method using cell culture wells

Impact of prior treatment on patients with relapsed multiple myeloma treated with carfilzomib and dexamethasone vs bortezomib and dexamethasone in the phase 3 ENDEAVOR study

The randomized phase 3 ENDEAVOR study (N=929) compared carfilzomib and dexamethasone (Kd) with bortezomib and dexamethasone (Vd) in relapsed multiple myeloma (RMM). We performed a subgroup analysis from ENDEAVOR in patients categorized by number of prior lines of therapy or by prior treatment. Median progression-free survival (PFS) for patients with one prior line was 22.2 months for Kd vs 10.1 months for Vd, and median PFS for patients with ⩾2 prior lines was 14.9 months for Kd vs 8.4 months for Vd. For patients with prior bortezomib exposure, the median PFS was 15.6 months for Kd vs 8.1 months for Vd, and for patients with prior lenalidomide exposure the median PFS was 12.9 months for Kd vs 7.3 months for Vd. Overall response rates (Kd vs Vd) were 81.9 vs 65.5% (one prior line), 72.0 vs 59.7% (⩾2 prior lines), 71.2 vs 60.3% (prior bortezomib) and 70.1 vs 59.3% (prior lenalidomide). The safety profile in the prior lines subgroups was qualitatively similar to that in the broader ENDEAVOR population. In RMM, outcomes are improved when receiving treatment with carfilzomib compared with bortezomib, regardless of the number of prior therapy lines or prior exposure to bortezomib or lenalidomide

The preparation of anticancer vaccine for patients with multiple myeloma on the base of monoclonal immunoglobulin loaded dendritic cells.

On June 2006, phase II clinical trial focused on anticancer vaccination of multiple myeloma patients, was started. On September 2007, the immune and clinical response evaluation of first four patients was finished.The anticancer vaccine contained dendritic cells loaded with monoclonal immunoglobulin produced by myeloma cells. METHODS AND PATIENTS: Within the frame of phase II clinical trial were vaccinated four myeloma patients with stable disease. It was administered six vaccines for each patient, monthly. The dendritic cells were cultured from the patient's peripheral blood mononuclear cells and loaded with autologous monoclonal immunoglobulin under the good manufacturing practice conditions. After the safety and quality control, the satisfactory vaccine was administered to the patient. The functional characteristic of dendritic cells was evaluated using flow cytometry, the immune response was evaluated using ELISpot. The clinical response was monitored using monoclonal immunoglobulin concentration in patient's sera. RESULTS AND CONCLUSION: The immune response detected using ELISpot was observed in 3/4 patients. The monoclonal immunoglobulin concentration was changeable for all twelve months, but never exceeded the range of 25% for minimal clinical response achievement. During the vaccination, no significant toxicities or negative side-effects were observed. The clinical trial is going on with vaccination other patients with multiple myeloma

Critical assessment of automated flow cytometry analysis techniques

Traditional methods for flow cytometry (FCM) data processing rely on subjective manual gating. Recently, several groups have developed computational methods for identifying cell populations in multidimensional FCM data. The Flow Cytometry: Critical Assessment of Population Identification Methods (FlowCAP) challenges were established to compare the performance of these methods on two tasks: (i) mammalian cell population identification, to determine whether automated algorithms can reproduce expert manual gating and (ii) sample classification, to determine whether analysis pipelines can identify characteristics that correlate with external variables (such as clinical outcome). This analysis presents the results of the first FlowCAP challenges. Several methods performed well as compared to manual gating or external variables using statistical performance measures, which suggests that automated methods have reached a sufficient level of maturity and accuracy for reliable use in FCM data analysis

Candida bloodstream infections in intensive care units: analysis of the extended prevalence of infection in intensive care unit study

To provide a global, up-to-date picture of the prevalence, treatment, and outcomes of Candida bloodstream infections in intensive care unit patients and compare Candida with bacterial bloodstream infection. DESIGN: A retrospective analysis of the Extended Prevalence of Infection in the ICU Study (EPIC II). Demographic, physiological, infection-related and therapeutic data were collected. Patients were grouped as having Candida, Gram-positive, Gram-negative, and combined Candida/bacterial bloodstream infection. Outcome data were assessed at intensive care unit and hospital discharge. SETTING: EPIC II included 1265 intensive care units in 76 countries. PATIENTS: Patients in participating intensive care units on study day. INTERVENTIONS: None. MEASUREMENT AND MAIN RESULTS: Of the 14,414 patients in EPIC II, 99 patients had Candida bloodstream infections for a prevalence of 6.9 per 1000 patients. Sixty-one patients had candidemia alone and 38 patients had combined bloodstream infections. Candida albicans (n = 70) was the predominant species. Primary therapy included monotherapy with fluconazole (n = 39), caspofungin (n = 16), and a polyene-based product (n = 12). Combination therapy was infrequently used (n = 10). Compared with patients with Gram-positive (n = 420) and Gram-negative (n = 264) bloodstream infections, patients with candidemia were more likely to have solid tumors (p < .05) and appeared to have been in an intensive care unit longer (14 days [range, 5-25 days], 8 days [range, 3-20 days], and 10 days [range, 2-23 days], respectively), but this difference was not statistically significant. Severity of illness and organ dysfunction scores were similar between groups. Patients with Candida bloodstream infections, compared with patients with Gram-positive and Gram-negative bloodstream infections, had the greatest crude intensive care unit mortality rates (42.6%, 25.3%, and 29.1%, respectively) and longer intensive care unit lengths of stay (median [interquartile range]) (33 days [18-44], 20 days [9-43], and 21 days [8-46], respectively); however, these differences were not statistically significant. CONCLUSION: Candidemia remains a significant problem in intensive care units patients. In the EPIC II population, Candida albicans was the most common organism and fluconazole remained the predominant antifungal agent used. Candida bloodstream infections are associated with high intensive care unit and hospital mortality rates and resource use