Online focus groups as a tool to collect data in hard-to-include populations: examples from paediatric oncology

Contains fulltext : 81501.pdf (publisher's version ) (Open Access)BACKGROUND: The purpose of this article is to describe and evaluate the methodology of online focus group discussions within the setting of paediatric oncology. METHODS: Qualitative study consisting of separate moderated asynchronous online discussion groups with 7 paediatric cancer patients (aged 8-17), 11 parents, and 18 survivors of childhood cancer (aged 8-17 at diagnosis). RESULTS: All three participant groups could be actively engaged over a one-week period. Respondents highly valued the flexibility and convenience of logging in at their own time and place to join the discussion. Adolescent patients and survivors emphasized that the anonymity experienced made them feel comfortable to express their views in detail. The findings indicate a strong preference for online group discussions across all participant groups. CONCLUSION: The findings show that online focus group methodology is a feasible tool for collecting qualitative data within the setting of paediatric oncology, and may offer new opportunities to collect data in other hard-to-include populations. The evaluations seem to indicate that the online group discussions have given participants an opportunity to articulate their experiences and views in a way they might not have done in a traditional group discussion.9 p

Addition of PTK787/ZK 222584 can lower the dosage of amsacrine to achieve equal amounts of acute myeloid leukemia cell death

Acute myeloid leukemia (AML) is a disease with a poor prognosis. It has been demonstrated that AML cells express the vascular endothelial growth factors, VEGFA and VEGFC, as well as kinase insert domain-containing receptor (VEGFR2), the main receptor for downstream effects, resulting in an autocrine pathway for cell survival. This study investigates the role of the VEGFR inhibitor PTK787/ZK 222584 in leukemic cell death, and the possibility of an additional effect on cell death by a chemotherapeutic drug, amsacrine. In three AML cell lines and 33 pediatric AML patient samples, we performed total cell-kill assays to determine the percentages of cell death achieved by PTK787/ZK 222584 and/or amsacrine. Both drugs induced AML cell death. Using a response surface analysis, we could show that, in cell lines as well as in primary AML blasts, an equal magnitude of leukemic cell death could be obtained when lower doses of the more toxic amsacrine were combined with low dosages of the less toxic VEGFR inhibitor. This study shows that PTK787/ ZK 222584 might have more clinical potential in AML when combined with a chemotherapeutic drug such as amsacrine. In future, it will be interesting to study whether the complications and the long-term effects of chemotherapy can be reduced by lowering the dosages of amsacrine, and by replacing it with other drugs with lower toxicity profiles, such as PTK787/ZK 222584

Detection of minimal residual disease identifies differences in treatment response between T-ALL and precursor B-ALL

We performed sensitive polymerase chain reaction-based minimal residual disease (MRD) analyses on bone marrow samples at 9 follow-up time points in 71 children with T-lineage acute lymphoblastic leukemia (T-ALL) and compared the results with the precursor B-lineage ALL (B-ALL) results (n = 210) of our previous study. At the first 5 follow-up time points, the frequency of MRD-positive patients and the MRD levels were higher in T-ALL than in precursor-B-ALL, reflecting the more frequent occurrence of resistant disease in T-ALL. Subsequently, patients were classified according to their MRD level at time point 1 (TP1), taken at the end of induction treatment (5 weeks), and at TP2 just before the start of consolidation treatment (3 months). Patients were considered at low risk if TP1 and TP2 were MRD negative and at high risk if MRD levels at TP1 and TP2 were 10(-3) or higher; remaining patients were considered at intermediate risk. The relative distribution of patients with T-ALL (n = 43) over the MRD-based risk groups differed significantly from that of precursor B-ALL (n = 109). Twenty-three percent of patients with T-ALL and 46% of patients with precursor B-ALL were classified in the low-risk group (P =.01) and had a 5-year relapse-free survival (RFS) rate of 98% or greater. In contrast, 28% of patients with T-ALL were classified in the MRD-based high-risk group compared to only 11% of patients with precursor B-ALL (P =.02), and the RFS rates were 0% and 25%, respectively (P =.03). Not only was the distribution of patients with T-ALL different over the MRD-based risk groups, the prognostic value of MRD levels at TP1 and TP2 was higher in T-ALL (larger RFS gradient), and consistently higher RFS rates were found for MRD-negative T-ALL patients at the first 5 follow-up time points

The significance of PTEN and AKT aberrations in pediatric T-cell acute lymphoblastic leukemia

textabstractBackground PI3K/AKT pathway mutations are found in T-cell acute lymphoblastic leukemia, but their overall impact and associations with other genetic aberrations is unknown. PTEN mutations have been proposed as secondary mutations that follow NOTCH1-activating mutations and cause cellular resistance to γ-secretase inhibitors. Design and Methods The impact of PTEN, PI3K and AKT aberrations was studied in a genetically well-characterized pediatric T-cell leukemia patient cohort (n=146) treated on DCOG or COALL protocols. Results PTEN and AKT E17K aberrations were detected in 13% and 2% of patients, respectively. Defective PTEN-splicing was identified in incidental cases. Patients without PTEN protein but lacking exon-, splice-, promoter mutations or promoter hypermethylation were present. PTEN/AKTmutations were especially abundant in TAL- or LMO-rearranged leukemia but nearly absent in TLX3-rearranged patients (P=0.03), the opposite to that observed for NOTCH1- activating mutations. Most PTEN/AKT mutant patients either lacked NOTCH1-activating mutations (P=0.006) or had weak NOTCH1-activating mutations (P=0.011), and consequently expressed low intracellular NOTCH1, cMYC and MUSASHI levels. T-cell leukemia patients without PTEN/AKT and NOTCH1-activating mutations fared well, with a cumulative incidence of relapse of only 8% versus 35% for PTEN/AKT and/or NOTCH1-activated patients (P=0.005). Conclusions PI3K/AKT pathway aberrations are present in 18% of pediatric T-cell acute lymphoblastic leukemia patients. Absence of strong NOTCH1-activating mutations in these cases may explain cellular insensitivity to γ-secretase inhibitors

Evidence Based Selection of Housekeeping Genes

For accurate and reliable gene expression analysis, normalization of gene expression data against housekeeping genes (reference or internal control genes) is required. It is known that commonly used housekeeping genes (e.g. ACTB, GAPDH, HPRT1, and B2M) vary considerably under different experimental conditions and therefore their use for normalization is limited. We performed a meta-analysis of 13,629 human gene array samples in order to identify the most stable expressed genes. Here we show novel candidate housekeeping genes (e.g. RPS13, RPL27, RPS20 and OAZ1) with enhanced stability among a multitude of different cell types and varying experimental conditions. None of the commonly used housekeeping genes were present in the top 50 of the most stable expressed genes. In addition, using 2,543 diverse mouse gene array samples we were able to confirm the enhanced stability of the candidate novel housekeeping genes in another mammalian species. Therefore, the identified novel candidate housekeeping genes seem to be the most appropriate choice for normalizing gene expression data

A New Perspective on Transcriptional System Regulation (TSR): Towards TSR Profiling

It has been hypothesized that the net expression of a gene is determined by the combined effects of various transcriptional system regulators (TSRs). However, characterizing the complexity of regulation of the transcriptome is a major challenge. Principal component analysis on 17,550 heterogeneous human microarray experiments revealed that 50 orthogonal factors (hereafter called TSRs) are able to capture 64% of the variability in expression in a wide range of experimental conditions and tissues. We identified gene clusters controlled in the same direction and show that gene expression can be conceptualized as a process influenced by a fairly limited set of TSRs. Furthermore, TSRs can be linked to biological functions, as we demonstrate a strong relation between TSR-related gene clusters and biological functionality as well as cellular localization, i.e. gene products of similarly regulated genes by a specific TSR are located in identical parts of a cell. Using 3,934 diverse mouse microarray experiments we found striking similarities in transcriptional system regulation between human and mouse. Our results give biological insights into regulation of the cellular transcriptome and provide a tool to characterize expression profiles with highly reliable TSRs instead of thousands of individual genes, leading to a >500-fold reduction of complexity with just 50 TSRs. This might open new avenues for those performing gene expression profiling studies

The diagnostic value of CRP, IL-8, PCT, and sTREM-1 in the detection of bacterial infections in pediatric oncology patients with febrile neutropenia

In this study, we evaluated C-reactive protein (CRP), interleukin (IL)-8, procalcitonin (PCT), and soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) as predictors for bacterial infection in febrile neutropenia, plus their usefulness in febrile neutropenia during chemotherapy-induced gastrointestinal mucositis. Plasma was obtained from pediatric oncology patients at presentation with febrile neutropenia (n = 43) and 24-48 h later (n = 17). The patients were classified as having or not having a bacterial infection. Plasma was also obtained of patients in the absence and in the presence of mucositis (n = 26). At presentation with febrile neutropenia, median IL-8 and PCT levels were significantly increased in patients with a bacterial infection, in contrast to CRP and sTREM-1. IL-8 was the most sensitive marker for the early detection of bacterial infection, in combination with clinical parameters or PCT the sensitivity reached 100%. After 24-48 h, only PCT was significantly elevated during bacterial infection. IL-8 levels were significantly increased during mucositis. Mucositis did not cause considerable changes in PCT levels. IL-8 is the most useful marker for the early detection of bacterial infections, compared with CRP, PCT, and sTREM-1. IL-8 in combination with clinical parameters or PCT might be even more useful. Gastrointestinal mucositis alone does not affect PCT levels, in contrast to IL-8 levels, and therefore, PCT might be more useful for the detection of bacterial infections during mucositis than IL-8