Characterization of acute myeloid leukemia based on levels of global hydroxymethylation.

Patients with acute myeloid leukemia (AML) frequently harbor mutations in genes involved in the DNA (hydroxy)methylation pathway (DNMT3A, TET2, IDH1, and IDH2). In this study, we measured 5-hydroxymethylcytosine (5hmC) levels in 206 clinically and molecularly well-characterized younger adult AML patients (≤60 years) included in the European Organization for Research and Treatment of Cancer/Gruppo Italiano Malattie Ematologiche dell'Adulto (EORTC/GIMEMA) AML-12 06991 clinical trial and correlated the 5hmC levels with mutational status and overall survival (OS). In healthy control cells, 5hmC levels were confined to a narrow range (1.5-fold difference), whereas in AML cells, a much wider range was detected (15-fold difference). We identified 3 5hmC subpopulations in our patient cohort (low, intermediate, and high). The low 5hmC group consisted almost entirely of patients with TET2 or IDH mutations. As expected, TET2 and IDH mutated patients had significantly lower levels of 5hmC compared with patients without mutated TET2 and IDH1/2 (both P < .001). Interestingly, high 5hmC levels correlated with inferior OS (high vs intermediate 5hmC: P = .047, hazard ratio [HR] = 1.81). Multivariate analysis revealed that high 5hmC is an independent poor prognostic indicator for OS (high vs intermediate 5hmC: P = .01, HR = 2.10). This trial was registered at www.clinicaltrials.gov as NCT00004128.

Results of three analytical approaches on long-term efficacy of etanercept for psoriasis in daily practice.

BACKGROUND: A problem encountered when analyzing long-term efficacy is that the number of patients in follow-up decreases with time for different reasons. The method used to account for missing observations for the therapy under analysis has a great influence on the inference of efficacy. OBJECTIVE: To describe the long-term efficacy of etanercept for psoriasis in daily practice using 3 analytical approaches. METHODS: Prospective data from a cohort of patients with psoriasis treated with etanercept for at least 24 weeks were analyzed using 3 analytical approaches: as treated analysis, intention-to-treat analysis (ITT) with last observation carried forward (LOCF) and intention-to-treat analysis with modified nonresponder imputation (modified NRI). RESULTS: One hundred thirty-one patients were treated with etanercept during 134 treatment episodes with a mean treatment duration of 2.7 years. The maximum follow-up was 6.0 years. The methodological approach chosen had a great influence. Psoriasis Area and Severity Index (PASI) 75 response rates varied from 60% in the as-treated approach to 34% in LOCF and to 29% in modified NRI at week 264. LIMITATIONS: All analytical methods applied have limitations. Other outcome measures could be used to overcome the bias introduced by each method of analysis, such as drug survival. CONCLUSIONS: The methodological approach chosen to analyze long-term efficacy data has a great influence on the inferences that may be drawn regarding the degree of efficacy. Therefore we support the use of different methods to present long-term efficacy data.

The tetraspanin CD37 orchestrates the α(4)ß(1) integrin-Akt signaling axis and supports long-lived plasma cell survival.

Signaling by the serine and threonine kinase Akt (also known as protein kinase B), a pathway that is common to all eukaryotic cells, is central to cell survival, proliferation, and gene induction. We sought to elucidate the mechanisms underlying regulation of the kinase activity of Akt in the immune system. We found that the four-transmembrane protein CD37 was essential for B cell survival and long-lived protective immunity. CD37-deficient (Cd37(-/-)) mice had reduced numbers of immunoglobulin G (IgG)-secreting plasma cells in lymphoid organs compared to those in wild-type mice, which we attributed to increased apoptosis of plasma cells in the germinal centers of the spleen, areas in which B cells proliferate and are selected. CD37 was required for the survival of IgG-secreting plasma cells in response to binding of vascular cell adhesion molecule 1 to the α(4)ß(1) integrin. Impaired α(4)ß(1) integrin-dependent Akt signaling in Cd37(-/-) IgG-secreting plasma cells was the underlying cause responsible for impaired cell survival. CD37 was required for the mobility and clustering of α(4)ß(1) integrins in the plasma membrane, thus regulating the membrane distribution of α(4)ß(1) integrin necessary for activation of the Akt survival pathway in the immune system.

High GATA2 expression is a poor prognostic marker in pediatric acute myeloid leukemia.

In acute myeloid leukemia (AML), aberrant expression and mutations of transcription factors have been correlated with disease outcome. In the present study, we performed expression and mutation screening of GATA2, which is an essential transcription factor for regulation of myeloid lineage determination, in de novo pediatric AML patients. GATA2 mutations were detected in 5 of 230 patients, representing a frequency of 2.2% overall and 9.8% in cytogenetically normal AML. GATA2 expression analysis demonstrated that in 155 of 237 diagnostic samples (65%), GATA2 expression was higher than in normal BM. In complete remission, normalization of GATA2 expression was observed, whereas GATA2 expression levels stayed high in patients with resistant disease. High GATA2 expression at diagnosis was an independent poor prognostic factor for overall survival (hazard ratio [HR] = 1.7, P = .045), event-free survival (HR = 2.1, P = .002), and disease-free survival (HR = 2.3, P = .004). The prognostic impact of GATA2 was particularly evident in specific AML subgroups. In patients with French-American-British M5 morphology, inv(16), or high WT1 expression, significant differences in survival were observed between patients with high versus normal GATA2 expression. We conclude that high GATA2 expression is a novel poor prognostic marker in pediatric AML, which may contribute to better risk-group stratification and risk-adapted therapy in the future.

A novel hemostasis assay for the simultaneous measurement of coagulation and fibrinolysis.

Thrombin and plasmin are the key enzymes involved in coagulation and fibrinolysis. A novel hemostasis assay (NHA) was developed to measure thrombin and plasmin generation in a single well by a fluorimeter. The NHA uses two fluorescent substrates with non-interfering fluorescent excitation and emission spectra. The assay was tested in vitro using modulators like heparin, hirudin, epsilon-aminocaproic acid, gly-pro-arg-pro peptide and reptilase and validated by measurement of prothrombin fragment 1+2 and plasmin-alpha2-antiplasmin levels. Intra- and inter-assay coefficients of variation were < 9% and 6-25%, respectively. Interplay between coagulation and fibrinolysis was demonstrated by the effect of tissue-type plasminogen activator on thrombin generation and by the different responses of activated protein C and thrombomodulin on fibrinolysis. The last responses showed the linkage between coagulation and fibrinolysis by thrombin activatable fibrinolysis inhibitor. In conclusion, this strategy allows detection of coagulation, fibrinolysis and their interplay in a single assay.

The lymphoid chemokine CCL21 triggers LFA-1 adhesive properties on human dendritic cells.

Dendritic cells (DCs) are the most potent APCs, involved in the induction of immunity and tolerance. Recently we showed that during differentiation of human DCs from monocyte precursors, Lymphocyte function-associated antigen-1 (LFA-1)-binding capacity is lost, although integrin expression levels were maintained constant, suggesting a different regulation mechanism of this integrin on different cell types. However, the exact role of LFA-1 in DC adhesion and migration remains obscure. Chemokines are potent regulators of integrin function, influencing migratory and adhesive properties of leukocytes. Here, we show that upon vaccination of cancer patients with human DCs, cells that have migrated in vivo into the lymph nodes upregulated the active form of LFA-1. We further show that exposure of human DCs to the lymphoid chemokine CCL21 specifically restores the high-affinity form of LFA-1 and induces binding to its ligand ICAM-1 under low shear stress. Our data indicate that on DCs LFA-1 may function as an inducible anchor during lymphatic transmigration or within the lymph nodes. A thorough understanding of the adhesive events during the DC life cycle will help to improve the outcome of DC-based antitumor clinical trials.

Differences between ulcerated and non-ulcerated hemangiomas, a retrospective study of 465 cases.

Our purpose was to get better insight into the ulceration of hemangiomas, by comparing patient characteristics of non-ulcerated hemangiomas with hemangiomas with active or past ulceration. A retrospective analysis was performed of files of patients who visited the Radboud University Medical Centre Nijmegen (UMCN), the Netherlands, between 1997 and 2007 for one or more infantile hemangiomas. The medical records of 465 patients were reviewed. Twenty three percent of the patients were diagnosed with ulceration. The size of ulcerated hemangiomas was significantly larger (28.6 cm2 vs. 6.0 cm2, p < 0.05). Predilection areas for ulceration were the head-neck region and the anogenital region. Ulceration was significantly most frequently seen in hemangiomas with a superficial (epidermal) component (98.5%, p < 0.05) and a segmental distribution (29.3%, p < 0.05). Ulceration most frequently took place during the proliferation phase of the hemangioma (83.1%). In the whole study population the male to female ratio was 1:2 compared to a tendency to more girls (1:3) for the group with ulcerated hemangiomas (p = 0.08). We conclude that larger, more superficial hemangiomas in areas more susceptible to trauma and contamination were more likely to ulcerate. This study contributes to the possibility of assessing the likelihood of ulceration in an individual patient.

Cardiovascular risk factors in high-need psoriasis patients and its implications for biological therapies.

BACKGROUND: The associations between psoriasis and cardiovascular risk factors are reported to be stronger as psoriasis severity increases. This makes studying cardiovascular risk factors in high-need psoriasis patients, eligible for biological therapy, interesting. OBJECTIVE: To survey the prevalence of cardiovascular risk factors in high-need psoriasis patients and to compare these data to patients with other dermatological diseases. Furthermore, the implications of these findings for treatment with biologics were outlined. METHODS: The prevalence of cardiovascular risk factors was investigated in a high-need psoriatic patient cohort and compared to patients with other skin diseases who filled out a questionnaire about the presence of cardiovascular risk factors. RESULTS: A significantly higher prevalence of obesity, smoking, and hypertension was found for the high-need psoriatic patients' cohort compared with non-psoriatic controls. Striking differences were found with respect to body mass index and obesity, as 35.5% of all high-need psoriatic patients were obese. CONCLUSIONS: High-need psoriatic patients show a high prevalence of cardiovascular risk factors, and may consequently be predisposed to cardiovascular diseases. As this is relevant for therapy management in daily clinical practice, especially biologics, cardiovascular risk should be evaluated for each high-need psoriasis patient before and during systemic treatment.

Clinical characteristics of cutaneous melanoma and second primary malignancies in a dutch hospital-based cohort of cutaneous melanoma patients.

The increasing number of living cutaneous melanoma patients and the increased risk of developing a second primary tumour incited us to analyse the clinical characteristics of cutaneous melanoma and define the frequency, site, and type of second primary cancers in cutaneous melanoma patients. We collected data on patients who visited the Department of Dermatology at the Radboud University Nijmegen Medical Centre and were newly diagnosed with cutaneous melanoma or metastasis of melanoma with unknown primary localization between 2002 and 2006. A total of 194 cases were included; eleven patients developed a subsequent melanoma, 24 had at least one basal cell carcinoma, three had at least one squamous cell carcinoma, and 21 patients had a second non-cutaneous primary malignancy. In conclusion, 48 patients developed a subsequent malignancy. As nonmelanoma skin cancer is the most frequent second malignancy, our results subscribe to the necessity of follow-up by a dermatologist.

Reduced CD26bright expression of peripheral blood CD8+ T-cell subsets in psoriatic patients.

BACKGROUND: T cells have been shown to be highly relevant in psoriasis. CD26 is a novel T-cell activation marker involved in various T-cell functions, e.g. (i) co-stimulation, (ii) migration and (iii) T-cell memory response. In particular, CD26bright peripheral blood T cells have been shown to be altered in several autoimmune diseases. OBJECTIVE: To characterize CD26-expression of T-cell subsets in psoriatic patients compared to healthy subjects. METHODS: Peripheral blood was obtained from 15 untreated patients with severe psoriasis and from nine healthy subjects. The presence of specific CD26-related T-cell subsets was assessed by flow cytometry. RESULTS: The CD26bright expression of CD8+ lymphocytes revealed a statistically significant (P<0.05) decrease in psoriatic patients. The majority of CD4+CD26bright cells are CD45RO+, whereas the minority of CD8+CD26bright cells are CD45RO+ in both groups. CONCLUSIONS: The present study demonstrates that the CD8CD26bright T-cell population is markedly decreased in peripheral blood of psoriatic patients. Moreover, CD26 expression did not show a restriction to memory T cells. As CD26 is of relevance for T-cell functions, future investigations should focus on elucidating these functions in psoriasis. It is attractive to speculate that the reduction in the CD8CD26bright subpopulation may represent a biomarker for recompartimentalization of activated T cells and a reduced CD8CD26bright count may correlate with increased responsiveness to T-cell targeted treatments.

Bone marrow mononuclear cells of MDS patients are characterized by in vitro proliferation and increased apoptosis independently of stromal interactions.

Enhanced proliferation of MDS progenitors is abrogated by increased apoptosis of their progeny in vivo. We investigated whether bone marrow mononuclear cells (BMMNC) of MDS patients also showed enhanced proliferation and apoptosis in vitro in comparison with acute myeloid leukemia (AML) and normal BM (NBM). NBM showed a decrease in the number of clusters in time due to apoptosis of clusters and due to development of clusters into colonies with low apoptotic level. In MDS patients, about two-fold more clusters have developed at day 4, and in contrast with NBM, the total number of clusters at day 7 remained high in spite of an increasing percentage of apoptotic clusters (from 52 to 76%) in combination with more colony formation. The number of clusters and colonies showed a sharp decrease at day 10 because of persistently high apoptosis at cluster level and increasing apoptosis in colonies. BMMNC of AML patients showed a decreased proliferation with enhanced apoptosis at cluster level in contrast to a relatively low apoptotic levels in the colony-forming cells. This data show that increased proliferation is abrogated by enhanced apoptosis in MDS, whereas AML showed decreased proliferation with a low level of apoptosis in colony-forming cells. These growth profiles of BMMNC are independent of stromal influences and may represent intrinsic features of the MDS progenitors and accessory cell interactions.

Etanercept and efalizumab treatment for high-need psoriasis. Effects and side effects in a prospective cohort study in outpatient clinical practice.

BACKGROUND: Since the beginning of 2005, etanercept and efalizumab are officially registered and reimbursed for the treatment of recalcitrant psoriasis in The Netherlands. OBJECTIVE: The evaluation of the efficacy, safety and adverse events of etanercept and efalizumab treatment in daily practice. METHODS: A prospective cohort study was carried out for patients treated with etanercept or efalizumab between February 2005 and March 2006. RESULTS: Over the past 13 months 45 individuals were treated with etanercept and 17 subjects were treated with efalizumab. The cohort represented a high-need population. At week 12, 82% of the subjects treated with 2 x 50 mg etanercept/week and 71% of the subjects treated with 2 x 25 mg etanercept/week reached a PASI-50. Efficacy of etanercept treatment was comparable to the results of clinical trials. For efalizumab, efficacy in responding patients was also comparable to clinical trial data, but the percentage of dropouts was substantial. During biologic treatment, safety was preserved and mainly mild adverse events were reported. CONCLUSION: Etanercept and efalizumab are effective and safe treatments of psoriasis, even in a high-need population. Etanercept was able to sustain the clinical improvement throughout 24 weeks, whereas efalizumab was not in 47% of subjects.

Heparan sulfate on activated glomerular endothelial cells and exogenous heparinoids influence the rolling and adhesion of leucocytes.

BACKGROUND: Proliferative glomerulonephritides are characterized by the influx of leucocytes. Heparan sulfate (HS) plays an important role in the recruitment, rolling and firm adhesion of leucocytes to activated endothelium. Recently, we have shown the importance of HS on activated mouse glomerular endothelial cells (mGEnC-1) for the firm adhesion of leucocytes in a static adhesion assay. In the present study, we evaluated the role of HS on glomerular endothelial cells and the effect of adding heparinoids on the leucocyte-glomerular endothelium interaction under dynamic flow conditions. METHODS: The number of rolling and firmly adhering leucocytes, and the rolling velocity of leucocytes was determined on a monolayer of unactivated or TNF-alpha-activated mGEnC-1 under dynamic flow conditions using physiological relevant shear stress rates in a flow chamber system. Furthermore, the effects of removal of HS on TNF-alpha-activated mGEnC-1 by heparinase III treatment, and of different concentrations of heparin, tinzaparin and HS, on the rolling and adhesion of leucocytes were evaluated. RESULTS: At the calculated physiological shear stress rate of 0.8 dynes/cm2 the number of rolling and firmly adhering leucocytes to mGEnC-1 increased 2-fold after activation with TNF-alpha, whereas the rolling velocity of the leucocytes decreased 2-fold. Addition of heparin, tinzaparin or HS, and the removal of HS on mGEnC-1 reduced the number of leucocytes rolling and adhering to activated mGEnC-1 about 2-3-fold, while the rolling velocity increased more than 2-fold. CONCLUSIONS: HS on activated glomerular endothelial cells is important for the interaction with leucocytes under flow conditions, while exogenous heparinoids interfere with this interaction. These results suggest that supplementary treatment of proliferative glomerulonephritides with heparinoids is an interesting option to pursue.

Isolation of FRET-positive cells using single 408-nm laser flow cytometry.

BACKGROUND: Flow cytometry may be used to isolate large amounts of living, fluorescently labeled cells. Certain fluorescent labels, like enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), allow the assessment of direct protein-protein interaction in situ, by fluorescence resonance energy transfer (FRET). However, current flow cytometric methods either require elaborate technical adaptations or, using a single laser protocol, are hampered by background signal. We optimized a single 408-nm laser protocol to detect FRET between ECFP/EYFP-tagged proteins. METHODS: Cell lines stably expressing ECFP and/or EYFP or an EYFP-ECFP fusion protein were used to design the settings for the flow cytometer to detect FRET-positive cells using a single 408-nm laser. Using these settings, interactions between the subunits of the transcription factor NF-Y were studied. RESULTS: Flow cytometric analysis of the cells expressing an EYFP-ECFP fusion protein yielded a discrete FRET-positive population. Using the same settings, in cells expressing NF-YB-CFP and NF-YC-YFP fusion proteins, FRET could also be detected. These cells were sorted and FRET was confirmed by confocal microscopy. CONCLUSION: FRET-positive cells, expressing ECFP- and EYFP-tagged proteins, can be detected using single 408-nm laser excitation, with low background signal. This allows high-throughput analysis and isolation of viable FRET-positive and -negative cells for subsequent biological experiments.

Magnetic resonance tracking of dendritic cells in melanoma patients for monitoring of cellular therapy.

The success of cellular therapies will depend in part on accurate delivery of cells to target organs. In dendritic cell therapy, in particular, delivery and subsequent migration of cells to regional lymph nodes is essential for effective stimulation of the immune system. We show here that in vivo magnetic resonance tracking of magnetically labeled cells is feasible in humans for detecting very low numbers of dendritic cells in conjunction with detailed anatomical information. Autologous dendritic cells were labeled with a clinical superparamagnetic iron oxide formulation or (111)In-oxine and were co-injected intranodally in melanoma patients under ultrasound guidance. In contrast to scintigraphic imaging, magnetic resonance imaging (MRI) allowed assessment of the accuracy of dendritic cell delivery and of inter- and intra-nodal cell migration patterns. MRI cell tracking using iron oxides appears clinically safe and well suited to monitor cellular therapy in humans.

Programmed cell death is an intrinsic feature of MDS progenitors, predominantly found in the cluster-forming cells.

OBJECTIVE: Bone marrows (BM) of myelodysplastic syndrome (MDS) patients show increased proliferation and premature programmed cell death (PCD) in vivo as well as in vitro. We explored the proliferative capacity and apoptotic propensity of CD34+ progenitor cells of MDS patients excluding accessory cell interference. MATERIALS AND METHODS: CD34+/CD3-/CD19- cells of 5 MDS patients and 5 normal BM were sorted as single cells into single wells and were cultured in liquid medium. Wells were evaluated on days 4, 7, 10, and 14. PCD was determined by staining with annexin V-FITC. Growth rate and cell doubling time (Td) were calculated for each colony-forming cell. RESULTS: Normal BM CD34+ cells formed clusters and colonies and both showed increasing PCD in time, although within colonies the degree of apoptosis was twice as high (about 25%) as compared with clusters at all time points. In MDS increased cluster formation was observed at all evaluation points when compared to normal BM, whereas the number of colonies was markedly reduced (1/7 of normal). These colonies were also smaller, usually smaller than 100 cells. Significantly enhanced levels of PCD of clusters (53-79%) in combination with longer cell doubling times explain this slower formation of smaller colonies. Surprisingly, these colonies showed considerably lower levels of PCD (7-32%) as compared to normal (1-48%, median values). CONCLUSIONS: In the absence of stromal influences and accessory cells, this study in MDS patients showed intrinsically enhanced proliferation and apoptosis of cluster-forming cells, as the opposite was true for colony-forming cells.

Functional characterization of beta1-integrin-positive epidermal cell populations.

Epidermal keratinocytes are heterogeneous and can be divided into stem cells (strong beta1-integrin expression) with unlimited clonogenic potential, transient amplifying cells (weaker beta1-integrin expression) with restricted proliferative capacity and terminally differentiated cells (no beta1-integrin expression) that have lost the capacity to divide. We tested the hypothesis that cell kinetic characteristics of the epidermal subpopulations differ. Single cell suspensions from small human skin punch biopsies were sorted flow cytometrically into a beta1-integrin weakly positive (dim) and strongly positive (bright) subpopulation and the clonogenic potential was compared in cell culture experiments. Image analysis was used to determine growth characteristics of the colonies. We found that cell size in the beta1-integrin bright subpopulation increased when colonies aged, whereas this was constant in the dim subpopulation. The total number of colonies formed and the growth rate of the colonies were higher in the beta1-integrin dim cells than in the bright subpopulation. Experimental data from this study confirm the hypothesis that cell kinetic characteristics of beta1-integrin dim and bright cells are different. Combining flow cytometric sorting, cell culture and image analysis provides powerful means for phenotypical and functional characterization of epidermal subpopulations.

Systemic treatment of psoriatic patients with bexarotene decreases epidermal proliferation and parameters for inflammation, and improves differentiation in lesional skin.

BACKGROUND: Bexarotene, a novel synthetic retinoid X receptor (RXR)-selective retinoid, has been reported to have antiproliferative and apoptotic stimulating effects in cutaneous T-cell lymphoma. In benign, hyperproliferative, and retinoid sensitive disorders, such as psoriasis, bexarotene has not been evaluated so far and no information on these parameters is available. OBJECTIVE: In the present study, immunohistochemical parameters for proliferation, differentiation, inflammation, and apoptosis were investigated in a group of bexarotene-treated psoriatic patients. METHODS: Twenty-nine patients with plaque-type psoriasis were treated for 12 weeks with oral bexarotene in four dose-defined treatment panels. Treatment was initiated in the following consecutive order: 1.0 mg/kg/day, 2.0 mg/kg/day, 0.5 mg/kg/day, and 3.0 mg/kg/day. Biopsies for immunohistochemical analysis were taken at the baseline and after 12 weeks of treatment. RESULTS: Significant reductions in Ki-67, keratin 16, transglutaminase, dermal CD4, epidermal CD8, and inflammation scores were seen after bexarotene treatment in combination with a significant increase in keratin 10. No induction of keratin 13 and 19 and no alterations in apoptosis associated p53 expression were observed. Apart from a weak significant dose-response effect for Ki-67, no other significant dose-response effects were seen. CONCLUSION: We have demonstrated efficacy of oral bexarotene in psoriasis in doses up to 3.0 mg/kg/day during 12 weeks of treatment for proliferation, differentiation, and inflammation parameters. Studies investigating higher doses of bexarotene in a larger number of patients are necessary to reveal potentially dose-related immunohistochemical effects of this new rexinoid and to elucidate the role of RXR-signaling in retinoid-associated keratin expression.