Biomedical subjects

Susan A Burchill

Publications and source records attributed to Susan A Burchill.

10 recordsLinked to original sources

PubMed2006

Standardisation of operating procedures for the detection of minimal disease by QRT-PCR in children with neuroblastoma: quality assurance on behalf of SIOPEN-R-NET.

The clinical utility of detecting minimal residual disease (MRD) in children with neuroblastoma (NB) by quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) is not clear. This in part reflects the lack of uniform methodology for analysis and reporting. Reference laboratories across Europe have therefore established standard operating procedures (SOPs) for the detection of NB cells by QRT-PCR. Haemopoietic samples are collected into PAXgene blood RNA tubes, which stabilise mRNA for 48 h at room temperature and more than 6 months at -80 degrees C. Tyrosine hydroxylase (TH) was selected as the target for NB cell detection, expression is normalised to beta2-microglobulin and reported using the DeltaDeltaCt method. The sensitivity of QRT-PCR increased from 58% to 90% following the development of SOPs. A robust, transferable, objective method for the detection of NB cells by QRT-PCR has been defined to improve the power and consistency of studies on MRD in children with NB.

Cell Line, Tumor↗

PubMed2006

What do, can and should we learn from models to evaluate potential anticancer agents?

Transfer of new anticancer agents from bench to clinical trial takes in excess of 10 years and costs up to US $500 million. Despite this massive commitment, many more new agents fail in the clinical trials than are successful. The poor performance of many investigational anticancer agents in the clinic implies that the preclinical models used to evaluate them are flawed, inappropriately used or the information they generate is misinterpreted. This article reviews current practice and the range of preclinical models available. The author provides a personal perspective on what information is needed and how in the future this might best be obtained from preclinical models to more effectively inform the transfer of novel, active agents into clinical practice.

Animals↗

PubMed2005

Detecting minimal residual disease in neuroblastoma patients-the present state of the art.

While cyto- and histological screening of bone marrow samples are still accepted as the gold standard for initial staging of neuroblastoma patients, these applications are insufficient during or after therapy because it is not always possible to detect tumour cell infiltration below the level of 1% by morphology alone. For monitoring of minimal residual disease, techniques offering a considerably higher sensitivity have been developed. Immunocytology, RT-PCR and flow cytometry are most frequently used, but differ with regard to targets (single cells, RNA transcripts), measured parameters (tumour cell number, antigen expression, cytomorphology, cytogenetic aberrations, level/number of RNA transcripts), specificity (uni-/multi-parameter analysis) and sensitivity (number of investigated cells). The pros and cons of these methods are reviewed. Precise quantification of residual tumour cells in bone marrow and blood may show a future impact on risk grouping and therapeutic strategies for patients with disseminated disease, but the potential clinical application of these techniques has to be preceded by thorough standardisation and validation in multi-centre studies.

Flow Cytometry↗

PubMed2005

p38MAPK-Dependent sensitivity of Ewing's sarcoma family of tumors to fenretinide-induced cell death.

PURPOSE: There is an urgent need for new therapeutic strategies in Ewing's sarcoma family of tumors (ESFT). In this study, we have evaluated the effect of fenretinide [N-(4-hydroxyphenyl)retinamide] in ESFT models. EXPERIMENTAL DESIGN: The effect of fenretinide on viable cell number and apoptosis of ESFT cell lines and spheroids and growth of s.c. ESFT in nu/nu mice was investigated. The role of the stress-activated kinases p38(MAPK) and c-Jun NH(2)-terminal kinase in fenretinide-induced death was investigated by Western blot and inhibitor experiments. Accumulation of reactive oxygen species (ROS) and changes in mitochondrial transmembrane potential were investigated by flow cytometry. RESULTS: Fenretinide induced cell death in all ESFT cell lines examined in a dose- and time-dependent manner. ESFT cells were more sensitive to fenretinide than the neuroblastoma cell lines examined. Furthermore, fenretinide induced cell death in ESFT spheroids and delayed s.c. ESFT growth in mice. p38(MAPK) was activated within 15 minutes of fenretinide treatment and was dependent on ROS accumulation. Inhibition of p38(MAPK) activity partially rescued fenretinide-mediated cell death in ESFT but not in SH-SY5Y neuroblastoma cells. c-Jun NH(2)-terminal kinase was activated after 4 hours and was dependent on ROS accumulation but not on activation of p38(MAPK). After 8 hours, fenretinide induced mitochondrial depolarization (Deltapsi(m)) and release of cytochrome c into the cytoplasm in a ROS- and p38(MAPK)-dependent manner. CONCLUSIONS: These data show that the high sensitivity of ESFT cells to fenretinide is dependent in part on the rapid and sustained activation of p38(MAPK). The efficacy of fenretinide in preclinical models demands the evaluation of fenretinide as a potential therapeutic agent in ESFT.

Animals↗

PubMed2005

Vascular endothelial growth factor: a therapeutic target for tumors of the Ewing's sarcoma family.

PURPOSE: We have reported previously that intratumoral microvessel density (MVD) is a significant prognostic indicator of event-free survival in the Ewing's sarcoma family of tumors (ESFT). Here, the angiogenic growth factor expression profile and its relationship with MVD has been investigated in ESFT. EXPERIMENTAL DESIGN AND RESULTS: Using ESFT model systems, the potential of these factors as therapeutic targets has been evaluated. A significant correlation (P = 0.02) was observed between vascular endothelial growth factor (VEGF) expression and MVD, consistent with the hypothesis that VEGF regulates the development of microvessels in ESFT. There was no correlation between MVD and any of the other growth factors studied. All six ESFT cell lines studied produced and secreted VEGF; five of six cell lines also secreted placental growth factor, one cell line (A673) at high levels. Tumor conditioned medium induced proliferation of human umbilical vein endothelial cells. Expression of VEGF receptors Flt-1 and Flk-1/KDR was heterogeneous across the cell lines. Both receptor tyrosine kinase inhibitors SU6668 (targets Flk-1/KDR, platelet-derived growth factor receptor-beta, and fibroblast growth factor receptor 1) and SU5416 (targets Flk-1/KDR) as well as anti-VEGF agents rhuMAb-VEGF (bevacizumab) and VEGF Trap delayed s.c. growth of ESFT in mice compared with untreated groups: SU6668 (100 mg/kg/d), SU5416 (25 mg/kg/d), rhuMAb-VEGF (10 mg/kg twice weekly), and VEGF Trap (2.5 or 25 mg/kg twice weekly). CONCLUSIONS: These data suggest that VEGF is the single most important regulator of angiogenesis in ESFT and may be exploited for therapeutic advantage.

Adolescent↗

PubMed2004

The use of real-time reverse transcription-PCR for prostate-specific antigen mRNA to discriminate between blood samples from healthy volunteers and from patients with metastatic prostate cancer.

PURPOSE: A clinical role for nonquantitative reverse transcription-PCR (RT-PCR) using prostate-specific antigen in blood samples from patients with prostate cancer remains undefined. Assay variation and detection of prostate-specific antigen mRNA illegitimate transcription may explain inconsistent results between studies. Defining levels of prostate-specific antigen mRNA expression in blood samples from healthy volunteers and patients with prostate cancer would allow cutoffs to be established to distinguish the two groups. EXPERIMENTAL DESIGN: Quantitative real-time RT-PCR for prostate-specific antigen mRNA was established and levels of prostate-specific antigen mRNA measured in bloods samples from healthy volunteers (n=21) and patients with localized (n=27) and metastatic (n=40) prostate cancer. RESULTS: Levels of prostate-specific antigen mRNA were significantly higher in blood samples from patients with metastatic prostate cancer than in blood samples from patients with localized prostate cancer (P <0.001) or in blood samples from healthy volunteers (P <0.01); levels between patients with localized prostate cancer and healthy volunteers were no different. Assay sensitivity to detect patients with metastatic prostate cancer was 68% with specificity of 95%. In patients with newly diagnosed metastatic prostate cancer, monitoring response to hormonal therapy was possible with this assay. No correlation between levels of prostate-specific antigen mRNA and serum prostate-specific antigen protein levels was found, suggesting that prostate-specific antigen mRNA and serum prostate-specific antigen protein levels reflect different features of prostate cancer, i.e., circulating tumor cells and total tumor bulk, respectively. CONCLUSIONS: Quantitative RT-PCR discriminates patients with metastatic prostate cancer from healthy volunteers and patients with localized prostate cancer but cannot discriminate patients with localized prostate cancer from healthy volunteers. A role for quantitative RT-PCR has been identified in the assessment and monitoring of patients with metastatic prostate cancer.

Cell Line, Tumor↗

PubMed2004

Basic fibroblast growth factor-induced cell death is effected through sustained activation of p38MAPK and up-regulation of the death receptor p75NTR.

Basic fibroblast growth factor (bFGF) induces cell death in cells of the Ewing's sarcoma family of tumors in vivo and in vitro. In this study we demonstrate that this is dependent on the rapid and sustained activation of p38(MAPK), in contrast to the transient activation of p38(MAPK) associated with bFGF-induced cell proliferation. Stem cell factor-induced survival of TC-32 cells was also associated with transient activation of p38(MAPK). Inhibition of p38(MAPK) by SB202190 and p38(MAPK) small interfering RNA reduces bFGF-induced death in TC-32 cells, consistent with the hypothesis that activation of p38(MAPK) is essential for induction of death by bFGF. This appears to be dependent on sustained activation of p38(MAPK), demonstrated by inhibition of bFGF-induced cell death following addition of SB202190 to TC-32 cells 5 min after exposure to bFGF (20 ng/ml) and activation of p38(MAPK). Prolonged activation of p38(MAPK) is accompanied by a rapid and sustained phosphorylation of Ras and ERK; inhibition of ERK phosphorylation using the MEK-1 inhibitor PD98059 rescued approximately 30% of cells from bFGF-induced death suggesting ERK plays a secondary role in the induction of death. This hypothesis is supported by observations in the A673 cell line; bFGF induced sustained activation of ERK and transient activation of p38(MAPK), which was not associated with cell death. These data demonstrate that sustained activation of p38(MAPK) is essential for activation of the death cascade following exposure of Ewing's sarcoma family of tumors cells to bFGF and provide evidence that activation of p38(MAPK) results in an up-regulation of the death receptor p75(NTR).

Animals↗

PubMed2004

A systematic review of molecular and biological tumor markers in neuroblastoma.

PURPOSE: The aim of this study was to conduct a systematic review, and where possible meta-analyses, of molecular and biological tumor markers described in neuroblastoma, and to establish an evidence-based perspective on their clinical value for the screening, diagnosis, prognosis, and monitoring of patients. EXPERIMENTAL DESIGN: A well-defined, reproducible search strategy was used to identify the relevant literature from 1966 to February 2000. RESULTS: A total of 428 papers studying the use of 195 different tumor markers in neuroblastoma were identified. Small sample sizes, poor statistical reporting, large heterogeneity across studies (e.g., in cutoff levels), and publication bias limited meta-analysis to the area of prognosis only; MYCN, chromosome 1p, DNA index, vanillylmandelic acid:homovanillic acid ratio, CD44, Trk-A, neuron-specific enolase, lactate dehydrogenase, ferritin, and multidrug resistance were all identified as potentially important prognostic tools. CONCLUSIONS: This systematic review forms a knowledge base of the tumor markers studied thus far in neuroblastoma, and has identified some of the most important prognostic markers, which should be considered in future research and treatment strategies. Importantly, the review has also highlighted some general problems across primary tumor marker studies, in particular poor and heterogeneous reporting. These need to be addressed to allow better clinical interpretation and enable more appropriate evidence-based reviews in the future. In particular, collaboration of cancer research groups is needed to enable bigger sample sizes, standardize methods of analysis and reporting, and facilitate the pooling of individual patient data.

Biomarkers, Tumor↗

PubMed2003

Myogenin--a more specific target for RT-PCR detection of rhabdomyosarcoma than MyoD1.

BACKGROUND: MyoD1 and myogenin are differentially expressed in early myogenesis and have been identified in rhabdomyosarcoma (RMS). This study evaluates reverse transcriptase-polymerase chain reaction (RT-PCR) for MyoD1 and myogenin mRNA as diagnostic markers of RMS, and the potential application of this method for the detection of small volume disease in bone marrow (BM) and peripheral blood (PB). PROCEDURE: Expression of MyoD1 and myogenin mRNA was examined by RT-PCR in RMSs (9 alveolar RMS, 10 embryonal RMS, 1 pleomorphic RMS), and 21 other paediatric tumor samples (10 neuroblastoma, 10 Ewing sarcomas, and 1 Sarcoma (not otherwise specified) (S(NOS)). BM (n = 19) and PB (n = 22) samples from the same RMS study population were also examined for MyoD1 and myogenin mRNA expression. RESULTS: Positive expression of both markers was demonstrated in adult muscle, but not in normal PB. Myogenin mRNA was expressed in 16/18 and MyoD1 mRNA in 12/12 RMSs studied. Myogenin was not expressed in 10/10 neuroblastomas, but was present in 2/10 Ewing sarcomas. However, MyoD1 mRNA was detected in 10/10 Ewing sarcomas and 7/10 neuroblastomas. Myogenin mRNA was detected in two BM samples from children with histologically negative BM and in 1/22 PB samples. Detection of MyoD1 mRNA in BM and PB was compromised by the amplification of a similar sized, non-specific product. CONCLUSIONS: Myogenin mRNA is a more specific marker than MyoD1 for the diagnosis of RMS. Myogenin mRNA is potentially a useful target for the assessment of small volume disease in RMS.

Adolescent↗

PubMed2002

Basic fibroblast growth factor (bFGF)-induced cell death is mediated through a caspase-dependent and p53-independent cell death receptor pathway.

The mechanism of bFGF-induced cell death in tumours of the Ewing's sarcoma family (ESFT) has been investigated. bFGF-induces phosphorylation of FGFr 1 and activation of Ras/ERK in ESFT cells that die when exposed to bFGF. Induction of cell death was associated with activation of both initiator (caspases-2, -8 and -10) and effector (caspases-3, -6 and -7) caspases. Moreover, the general caspase inhibitor Z-VAD-FMK protected cells from bFGF-induced cell death. After treatment with bFGF, a loss of mitochondrial transmembrane potential was accompanied by down-regulation of Bcl-2. However, the observed cell death was not associated with release of cytochrome c from the mitochondria. Furthermore, expression of wild-type p53 was not required for bFGF-induced cell death. These observations suggest that bFGF-induced cell death may be mediated through a cell death receptor mechanism, supported by up-regulation of the p75 neurotrophin receptor. bFGF-induced cell death was associated with up-regulation of p21 and p53, down-regulation of PCNA and cyclin A and a decrease in active pRb1, changes consistent with accumulation of cells in G1. These data demonstrate that bFGF-induced cell death is effected through a caspase-dependent and p53-independent mechanism, that may be mediated through a cell death receptor pathway.

Animals↗