Impaired circulating myeloid DCs from myeloma patients

BACKGROUND: In clinical trials, cancer patients have received immunotherapy based on DCs generated from leukapheresed blood. It would therefore be an advantage to be able to measure blood levels and estimate the phenotype of DC before leukapheresis, to estimate the yield required for preparation of vaccines, or ex vivo stimulation of T cells for adoptive immunotherapy. METHODS: Recently, circulating lineage negative (Lin-) myeloid DC cells and their precursors have been identified by flow cytometry. We apply this strategy to the screening of blood samples from patients with multiple myeloma, in an attempt to characterize and quantitate the subset. By a direct flow cytometry approach, the blood levels of circulating lineage (CD3, CD19, CD14) negative, CD33++, HLA-DR+ cells were estimated before and following ex vivo cell differentiation, and phenotyped by MAbs with specificity against HLA-DR, HLA-ABC, CD1a, CD11c, CD33, CD40, CD49d, CD49e, CD54, CD80, CD83, and CD86. RESULTS: This study demonstrated that multiple myeloma patients have a 50% reduced blood level of Lin-, CD33++, HLA-DR+ myeloid DC, but a DC-precursor level within normal range. Furthermore, GM-CSF and IL-4 ex vivo stimulated DCs demonstrated an impaired up-regulation of the co-stimulatory molecule CD80 and the adhesion molecule CD54. DISCUSSION: These results may have clinical implications as a predictor for yield and functionality of the harvested DCs to be used in vaccination of myeloma patients.     

Impaired NHEJ function in multiple myeloma

Multiple myeloma (MM) is characterized by multiple chromosomal aberrations. To assess the contribution of DNA repair to this phenotype, ionizing radiation was used to induce DNA double strand breaks in three MM cell lines. Clonogenic survival assays showed U266 (SF4 = 15.3 + 6.4%) and RPMI 8226 (SF4 = 12.6.0 + 1.7%) were radiation sensitive while OPM2 was resistant (SF4 = 78.9 + 4.1%). Addition of the DNA-PK inhibitor NU7026 showed the expected suppression in radiation survival in OPM2 but increased survival in both radiation sensitive cell lines. To examine non-homologous end joining (NHEJ) repair in these lines, the ability of protein extracts to support in vitro DNA repair was measured. Among the three MM cell lines analyzed, RPMI 8226 demonstrated impaired blunt ended DNA ligation using a ligation-mediated PCR technique. In a bacterial based functional assay to rejoin a DNA break within the β-galactosidase gene, RPMI 8226 demonstrated a 4-fold reduction in rejoining fidelity compared to U266, with OPM2 showing an intermediate capacity. Ionizing radiation induced a robust γ-H2AX response in OPM2 but only a modest increase in each radiation sensitive cell line perhaps related to the high level of γ-H2AX in freshly plated cells. Examination of γ-H2AX foci in RPMI 8226 cells confirmed data from Western blots where a significant number of foci were present in freshly plated untreated cells which diminished over 24 h of culture. Based on the clonogenic survival and functional repair assays, all three cell lines exhibited corrupt NHEJ repair. We conclude that suppression of aberrant NHEJ function using the DNA-PK inhibitor NU7026 may facilitate access of DNA ends to an intact homologous recombination repair pathway, paradoxically increasing survival after irradiation. These data provide insight into the deregulation of DNA repair at the site of DNA breaks in MM that may underpin the characteristic genomic instability of this disease.     

Reduced bone marrow stem cell pool and progenitor mobilisation in multiple myeloma after melphalan treatment

The content of stem cells was analysed in bone marrow samples from 75 multiple myeloma patients. In unstimulated bone marrow the percentage of CD34+cells was significantly reduced in 11 patients previously treated with melphalan-prednisolone (MP)(median= 0.15%) compared to median 0.87% in 31 untreated patients (P=0.0001). The bone marrow cellularity in the two groups did not differ. There was no correlation between the number of courses or total dose of melphalan and content of CD34+cells in the bone marrow. The clonogenicity as, well as the ability to expand the marrow stem cell pool during growth factor treatment were also reduced in MP treated patients compared to untreated patients. Analysis of different subsets of CD34+ cells revealed no influence on the pre B cell compartment in the bone marrow by MP treatment, but the committed stem cells (CD34+CD38+) were reduced more than the uncommitted stem cells (CD34+CD38—) in the MP treated group compared to the untreated patients. Mobilisation to and harvest of total number of CD34+ cells from peripheral blood was also reduced in the MP treated group. There was, however, no difference in the distribution between CD34+CD38+and CD34+CD38—populations in the leukapheresis products in the untreated and the melphalan-treated group, suggesting selective mobilisation of CD34+CD38+ cells and/or differentiation of CD34+ CD38-cells during growth factor stimulation. We conclude that melphalan decreased the number of stem cells in the bone marrow, the ability to expand the stem cell pool and mobilise stem cells to the pheripheral blood.     

Plasma cell myeloma: response of melphalan-resistant patients to high-dose intermittent cyclophosphamide

High-dose intermittent cyclophosphamide was used in the treatment of 19 patients with plasma cell myeloma who had demonstrated resistance to melphalan. Good objective improvement was found in six patients and partial responses in five. Patients have survived from three to more than 36 months (estimated median 21 months) after starting cyclophosphamide, and 11 are still alive. The important implications of the lack of cross-resistance between these two alkylating agents are discussed in terms of the mechanisms of cellular resistance to alkylating agents.     

Impaired production of interleukin 6 and tumour necrosis factor alpha in whole blood cell cultures of patients with multiple myeloma

Cytokine production in whole blood cell cultures can be an indicator of immune cellular status in neoplastic patients. Production of interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-alpha) after stimulation with lipopolysaccharide was measured in 1/10 diluted whole blood culture of patients with monoclonal gammopathies [monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM)]. With this system, lower production of IL-6 and TNF-alpha at 4, 24 and 48 h in total blood cultures in patients with symptomatic MM was observed. Thus, the capacity of cytokine production was greater in control subjects and in patients with MGUS and MM in response than in symptomatic MM (P=0.005 at 4 h and P=0. 006 at 24 and 48 h for IL-6 and P<0.0001 at 24 h and P<0.001 at 48 h for TNF-alpha). These differences remained significant after adjustment on the basis of the lymphocyte count (P<0.001 for IL-6 and TNF-alpha at 24 and 48 h). The impaired IL-6 and TNF-alpha production in patients with symptomatic MM is probably due to a tumour-related immunosuppressive status.     

Steroid-carrier proteins in patients with multiple myeloma

Patients with multiple myeloma have transcortin levels lower than normal. This is due in essence to a subgroup of patients producing IGG heavy chains with lambda light chains. Patients producing IGG with predominantly kappa light chains have almost normal transcortin levels. On the other hand, the binding activity of the steroid binding beta globulin (SB beta G) of the kappa type of multiple myeloma is significantly higher than the steroid binding of the lambda type of multiple myeloma. The serum levels of vitamin D binding protein (DBP) fall in the normal range.     

Impaired Cellular Immunity in Kwashiorkor with Improvement after Therapy

Children with kwashiorkor showed a high incidence of deranged cellular immunity as evidenced by impairment of delayed cutaneous hypersensitivity reactions to candida and diphtheria toxoid antigens and of lymphocyte transformation after phytohaemagglutinin stimulation. This may contribute to their susceptibility to infection. A correlation was shown between the degree of impairment of tests of cellular immunity and the severity of the kwashiorkor. Once recovery was initiated the skin tests gave the expected positive results and the lymphocyte transformation index improved. Protein deprivation may result in impaired deoxyribonucleic acid (DNA) synthesis and in atrophy of both the thymus and the lymphoid tissue.     

Prophylaxis of fungal infection in patients with hematologic neoplasms and severe neutropenia after high-dose chemotherapy]

Infection is one of the main causes of death in patients with hemoblastoses. Within the last years there was observed an increase in the ratio of fungal infections in the structure of mortality among hematologic patients with neutropenia. The present study was aimed at comparative estimation of the efficacy of the prophylactic use of various azole antifungal agents in patients with hematologic neoplasms and severe neutropenia. The trial enrolled 88 patients comparable by the diagnosis and chemotherapy characteristics, in whom severe neutropenia developed after intensive therapy. Antifungal drugs were used prophylactically when the neutrophil count lowered below 1.0 x 10(9)/l until its increasing above 1.0 x 10(9)/l or when the signs of fungal infection were evident. Itraconazole was used in cyclodextrin solution in 30 patients in a dose of 0.2 g orally twice a day and fluconazole was used in capsules in 24 patients in a dose of 0.2 g orally once a day. The results were compared with those of the ketoconazole use in a dose of 0.2 g orally twice a day (n = 34). The frequency of fungal infection proved by the clinical documentation was 20.5% in the ketoconazole group (k) (7 out of 34 patients), 8.3% in the fluconazole group (f) (2 out of 24 patients) and 6.6% in the itraconazole group (i) (2 out of 30 patients), p (k-f) = 0.21, p (k-i) = 0.11 and p (f-i) = 0.74. The frequency of fungal infection proved by the microbiological documentation was statistically much higher in the ketoconazole group (38.2%) vs. the fluconazole group (8.3%) (p = 0.013) and the itraconazole group (6.6%) (p = 0.004). The prophylactic use of itraconazole and fluconazole was efficient in preventing development of invasive mycoses in the patients with hemoblastoses and severe neutropenia. Their efficacy was much higher than that of ketoconazole.     

Characterization of hematopoietic stem cell subsets from patients with multiple myeloma after mobilization with plerixafor

Background aimsPrevious studies have demonstrated that the combination of granulocyte–colony-stimulating factor (G-CSF) + plerixafor is more efficient in mobilizing CD34⁺ hematopoietic stem cells (HSC) into the peripheral blood than G-CSF alone. In this study we analyzed the impact of adding plerixafor to G-CSF upon the mobilization of different HSC subsets.MethodsWe characterized the immunophenotype of HSC subsets isolated from the peripheral blood of eight patients with multiple myeloma (MM) before and after treatment with plerixafor. All patients were supposed to collect stem cells prior to high-dose chemotherapy and consecutive autologous stem cell transplantation, and therefore received front-line mobilization with 4 days of G-CSF followed by a single dose of plerixafor. Samples of peripheral blood were analyzed comparatively by flow cytometry directly before and 12 h after administration of plerixafor.ResultsThe number of aldehyde dehydrogenase (ALDH)^bright and CD34⁺ cells was significantly higher after plerixafor treatment (1.2–5.0 and 1.5–6.0 times; both P < 0.01) and an enrichment of the very primitive CD34⁺ CD38^? and ALDH^bright CD34⁺ CD38^? HSC subsets was detectable. Additionally, two distinct ALDH⁺ subsets could be clearly distinguished. The small ALDH^high subset showed a higher number of CD34⁺ CD38^? cells in contrast to the total ALDH^bright subpopulation and probably represented a very primitive subpopulation of HSC.ConclusionsA combined staining of ALDH, CD34 and CD38 might represent a powerful tool for the identification of a very rare and primitive hematopoietic stem cell subset. The addition of plerixafor mobilized not only more CD34⁺ cells but was also able to increase the proportion of more primitive stem cell subsets.     

CD4(+) CD28(+) lymphocytes on day 5 after high-dose melphalan for multiple myeloma predict a low risk of infections during severe neutropenia and are associated with the number of reinfused T lymphocytes of the autologous stem cell graft

Background aimsNeutropenia following high-dose chemotherapy is associated with a substantial risk of infectious complications. The aim of this study was to identify variables in residual leukocyte subsets during neutropenia that are predictive for neutropenic fever.MethodsResidual leukocytes in the peripheral blood on day 5 after autologous blood stem cell transplantation were analyzed by three-color flow cytometry in 55 consecutive patients with multiple myeloma. Furthermore, the number of T cells transfused with the autografts was determined.ResultsNeutrophil counts at day 5 and neutrophil engraftment were similar in patients with and without neutropenic fever. Low absolute lymphocyte, CD4⁺ CD28⁺ and CD45RO⁺ CD28⁺ counts at day 5 were associated with neutropenic fever. T-cell counts at day 5 correlated with the CD3⁺ cell number in the graft.ConclusionsOur data show that the absolute lymphocyte, CD4⁺ CD28⁺ and CD45RO⁺ CD28⁺ counts play a role in host defense during severe neutropenia. The T-cell number in the graft may help to identify patients at high risk of neutropenic infections.     

Whole body MR in patients with multiple myeloma

BackgroundMultiple myeloma is a cancer of plasma cells which leads to bone marrow infiltration.AimWhole-body MR is the most sensitive imaging method available to detect multiple myeloma lesions.Material and MethodsMR scans were performed in 100 patients with multiple myeloma who were receiving treatment in the Haematology Clinic in Poznań in the years 2005–2006. Whole-body MR scans were performed with general coil 1.0 T in STIR sequences and T1 sequences, in coronal and sagittal planes with scanning area covering the head, neck, trunk and the limbs (FOV for specific regions was 36–48 cm). The bone lesions were classified as focal (monofocal/multifocal lesions), in-filtrative, mixed and “salt and pepper” type. Depending on the size of the lesions the patients were included in one of three groups according to Salmon-Durie Plus classification.ResultsFour main types of multiple myeloma were distinguished based on MR scans: focal (48 patients; monofocal in 10 patients), infiltrative (17 patients), mixed type (19 patients) and “salt and pepper” type (4 patients). The remaining 12 patients had no multiple myeloma lesions in the bone marrow. Additionally, in 18% of patients a soft tissue mass could be observed. According to Salmon-Durie Plus categorisation 27 subjects were classified as having stage I, 16 patients stage and 57 patients stage III disease. In 12% of patients MR data changed the disease staging.ConclusionsWB MR is a sensitive and effective diagnostic method with an important impact on staging and further treatment of multiple myeloma.     

In vitro dendritic cell generation and lymphocyte subsets in myeloma patients: influence of thalidomide and high-dose chemotherapy treatment

While vaccination with antigen-pulsed dendritic cells (DCs) represents a promising therapeutic strategy in multiple myeloma (MM), clinical benefit, so far, has been limited to individual patients. To identify potential problems with this approach, we have analyzed the influence of treatment parameters, in particular high-dose chemotherapy (HD-CTX) and thalidomide, on in vitro DC generation and peripheral blood lymphocyte subsets in MM patients. From a total of 25 MM patients, including 14 patients on thalidomide treatment and 11 after HD-CTX, in vitro DC generation from peripheral blood monocytes under serum-free condition was investigated. In addition, peripheral blood lymphocyte subsets were assessed in 17 patients including 10 patients on thalidomide treatment and 9 patients after HD-CTX. Efficient in vitro generation of DCs (median 7.1×10⁶/100 ml peripheral blood; range 0.1–42.5×10⁶/100 ml peripheral blood) expressing DC-typical surface markers was observed in 23 MM patients (92%), although reduced expression of CD1a, CD40, CD83, and HLA-DR was observed in patients treated with thalidomide. With respect to lymphocyte subsets, MM patients showed significantly (p<0.05) reduced B and CD4+ lymphocytes in the peripheral blood. This effect was most prominent within 6 months of HD-CTX and in patients receiving thalidomide (usually in combination with CTX). CD8+ lymphocytes were significantly increased in MM patients. Thus, despite the well-known deficiencies in their immune system, adequate numbers of DCs can be generated in most myeloma patients. In patients treated with thalidomide, however, it remains to be seen whether the reduced expression of co-stimulatory molecules has functional relevance.The study was supported by Internes Forschungsförderungsprogramm Essen (IFORES) and Förderverein Essener Tumorklinik e. V.     

Impaired esophageal function in patients with irritable bowel syndrome

The aim of the study was to determine prevalence of the signs and symptoms related to esophageal dysfunction in irritable bowel syndrome (IBS) patients, and to investigate sensorimotor function impairment based on the esophageal manometry study, thus to determine the correlation between them. The study included 30 patients with IBS, 14 of them with diarrhea (IBSd) and 16 with constipation (IBSc) as a predominant discomfort. Control group consisted of 30 healthy subjects. The patients were included in the study on the basis of the Rome criteria for IBS. In addition to thorough history and physical examination patient underwent esophagogastroduodenoscopy and esophageal manometry. The values of esophageal manometry obtained in healthy subjects served as controls in manometry studies. The patients with IBS suffered a great number of both colonic and extracolonic signs and symptoms, however, there was no statistically significant difference in the prevalence of particular symptoms between the two patient subgroups. In comparison with healthy subjects, the patients suffering from IBS showed pathologically altered values in the majority of parameters of esophageal motility. Comparison of the two subgroups of IBS patients according to esophageal motility characteristics yielded differences in only few of them. The results obtained in the study could explain why the patients with IBS quite commonly complain of the symptoms related to upper gastrointestinal tract, such as heartburn and chest pain of noncardiac genesis. The results also suggest that the IBS might be associated with considerably more extensive smooth muscle or innervation changes than presumed before.     

Bendamustine overcomes resistance to melphalan in myeloma cell lines by inducing cell death through mitotic catastrophe

Melphalan has been a mainstay of multiple myeloma (MM) therapy for many years. However, following treatment with this alkylator, malignant plasma cells usually escape both apoptosis and cell cycle control, and acquire drug-resistance resulting in tumor progression. Bendamustine is being used in MM patients refractory to conventional DNA-damaging agents, although the mechanisms driving this lack of cross-resistance are still undefined. Here, we investigated the molecular pathway of bendamustine-induced cell death in melphalan-sensitive and melphalan-resistant MM cell lines. Bendamustine affected cell survival resulting in secondary necrosis, and prompted cell death primarily through caspase-2 activation. Also, bendamustine blocked the cell cycle in the G2/M phase and induced micronucleation, erratic chromosome spreading and mitotic spindle perturbations in melphalan-resistant MM cells. In these cells, both Aurora kinase A (AURKA) and Polo-like kinase-1 (PLK-1), key components of the spindle-assembly checkpoint, were down-regulated following incubation with bendamustine, whereas levels of Cyclin B1 increased as a consequence of the prolonged mitotic arrest induced by the drug. These findings indicate that, at least in vitro, bendamustine drives cell death by promoting mitotic catastrophe in melphalan-resistant MM cells. Hence, activation of this alternative pathway of cell death may be a novel approach to the treatment of apoptosis-resistant myelomas.     

Impaired oxidation of debrisoquine in patients with perhexiline neuropathy.

The use of perhexiline maleate as an antianginal agent is occasionally associated with side effects, particularly neuropathy and liver damage. The reason why some individuals develop these toxic reactions is not clear, though some evidence suggests that they may result from impaired oxidative metabolism, due to genetic or hepatic factors, and consequential accumulation of the drug in toxic concentrations. Drug oxidation was measured with an oxidation phenotyping procedure in 34 patients treated with perhexiline, 20 of whom had developed neuropathy and 14 of whom had not. Most of the 20 patients with neuropathy, but not the unaffected patients, showed an impaired ability to effect metabolic drug oxidation. This impairment was independent of hepatic function, concurrent drug therapy, or tobacco or alcohol consumption. The fact that the ability to oxidise several drugs is genetically controlled points to a genetic susceptibility to developing neuropathy in response to perhexiline. Routine determination of the drug oxidation phenotype might lead to safer use of perhexiline by predicting patients who may be more at risk of developing a neuropathic reaction associated with its long-term use.     

Impaired synthesis of DHA in patients with X-linked retinitis pigmentosa

Many patients with X-linked retinitis pigmentosa (XLRP) have lower than normal blood levels of the long-chain polyunsaturated omega3 fatty acid docosahexaenoic acid (DHA; 22:6omega3). This clinical trial was designed to test whether down-regulation of DHA biosynthesis might be responsible for these reduced DHA levels. DHA biosynthesis was assessed in five severely affected patients with XLRP and in five age-matched controls by quantifying conversion of [U-(13)C]alpha-linolenic acid (alpha-LNA) to [(13)C]DHA. Following oral administration of [U-(13)C]alpha-LNA, blood samples were collected at designated intervals for 21 days and isotopic enrichment of all omega3 fatty acids was determined by gas chromatography/mass spectroscopy. Activity of each metabolic step in the conversion of alpha-LNA to DHA was determined by comparison of the ratios of the integrated concentration of (13)C-product to (13)C-precursor in plasma total lipid fractions. The ratio of [(13)C]DHA to [(13)C]18:3omega3 (the entire pathway) and that of [(13)C]20:5omega3 to [(13)C]20:4omega3 (Delta(5)-desaturase) were significantly lower in patients versus controls (P = 0.03 and 0.05, respectively). The estimated biosynthetic rates of [(13)C]20:5omega3, [(13)C]22:5omega3, [(13)C]24:5omega3, [(13)C]24:6omega3, and [(13)C]22:6omega3 were significantly lower in XLRP patients (42%, 43%, 31%, 18%, and 32% of control values, respectively; P < 0.04), supporting down-regulation of Delta(5)-desaturase in XLRP. The disappearance of (13)C-labeled fatty acids from plasma was not greater in XLRP patients compared with controls, suggesting that XLRP was not associated with increased rates of fatty acid oxidation or other routes of catabolism.Thus, despite individual variation among both patients and controls, the data are consistent with a lower rate of Delta(5)-desaturation, suggesting that decreased biosynthesis of DHA may contribute to lower blood levels of DHA in patients with XLRP.