Membrane-type 1 matrix metalloproteinase as predictor of survival and candidate therapeutic target in Ewing sarcoma.

BACKGROUND: Ewing sarcoma (ES) is the second most common primary bone malignancy, with an urgent need for new treatments. ES is associated with high rates of progression and relapse, driven by drug-resistant cells capable of migration, self-renewal and single-cell tumorigenesis, termed cancer stem-like cells (CSCs). Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane-bound proteolytic enzyme, which, via direct and indirect mechanisms, digests four of the main types of collagen. This can be hijacked in malignancy for invasion and metastasis, with high expression predicting decreased survival in multiple cancers. In this study, we have examined the hypothesis that MT1-MMP is expressed by ES cells and explored the relationship between expression and outcomes. PROCEDURE: MT1-MMP expression in ES established cell lines, primary patient-derived cultures and daughter ES-CSCs was characterised by RNA sequencing, Western blotting, immunocytochemistry and flow cytometry. Immunohistochemistry was used to detect MT1-MMP in tumour biopsies, and the relationship between expression, event-free and overall survival examined. RESULTS: MT1-MMP was detected at both RNA and protein levels in five of six established cell lines, all primary cultures (n = 25) and all daughter ES-CSCs (n = 7). Immunohistochemistry of treatment-naïve biopsy tissue demonstrated that high MT1-MMP expression predicted decreased event-free and overall survival (p = .017 and .036, respectively; n = 47); this was not significant in multivariate analysis. CONCLUSIONS: MT1-MMP is expressed by ES cells, including ES-CSCs, making it a candidate therapeutic target. The level of MT1-MMP expression at diagnosis may be considered as a prognostic biomarker if validated by retrospective analysis of a larger cohort of clinical trial samples.

Event-free survival of infants and toddlers enrolled in the HR-NBL-1/SIOPEN trial is associated with the level of neuroblastoma mRNAs at diagnosis.

BACKGROUND: The purpose of this study was to evaluate whether levels of neuroblastoma mRNAs in bone marrow and peripheral blood from stage M infants (≤12 months of age at diagnosis, MYCN amplified) and toddlers (between 12 and 18 months, any MYCN status) predict event-free survival (EFS). METHODS: Bone marrow aspirates and peripheral blood samples from 97 infants/toddlers enrolled in the European High-Risk Neuroblastoma trial were collected at diagnosis in PAXgene™ blood RNA tubes. Samples were analyzed by reverse transcription quantitative polymerase chain reaction according to standardized procedures. RESULTS: Bone marrow tyrosine hydroxylase (TH) or paired-like homeobox 2b (PHOX2B) levels in the highest tertile were associated with worse EFS; hazard ratios, adjusted for age and MYCN status, were 1.5 and 1.8 respectively. Expression of both TH and PHOX2B in the highest tertile predicted worse outcome (p = 0.015), and identified 20 (23%) infants/toddlers with 5-year EFS of 20% (95%CI: 4%-44%). Prognostic significance was maintained after adjusting for over-fitting bias (p = 0.038), age and MYCN status. In peripheral blood, PHOX2B levels in the highest tertile predicted a two-fold increased risk of an event (p = 0.032), and identified 23 (34%) infants/toddlers with 5-year EFS of 29% (95%CI: 12%-48%). Time-dependent receiver operating characteristic analysis confirmed the prognostic value of combined TH and PHOX2B in bone marrow and of PHOX2B in peripheral blood during the first year of follow-up. CONCLUSIONS: High levels of bone marrow TH and PHOX2B and of peripheral blood PHOX2B at diagnosis allow early identification of a group of high-risk infant and toddlers with neuroblastoma who may be candidates for alternative treatments. Integration with additional biomarkers, as well as validation in additional international trials is warranted.

Recommendations for the standardization of bone marrow disease assessment and reporting in children with neuroblastoma on behalf of the International Neuroblastoma Response Criteria Bone Marrow Working Group.

BACKGROUND: The current study was conducted to expedite international standardized reporting of bone marrow disease in children with neuroblastoma and to improve equivalence of care. METHODS: A multidisciplinary International Neuroblastoma Response Criteria Bone Marrow Working Group was convened by the US National Cancer Institute in January 2012 with representation from Europe, North America, and Australia. Practical transferable recommendations to standardize the reporting of bone marrow disease were developed. RESULTS: To the authors' knowledge, the current study is the first to comprehensively present consensus criteria for the collection, analysis, and reporting of the percentage area of bone marrow parenchyma occupied by tumor cells in trephine-biopsies. The quantitative analysis of neuroblastoma content in bone marrow aspirates by immunocytology and reverse transcriptase-quantitative polymerase chain reaction are revised. The inclusion of paired-like homeobox 2b (PHOX2B) for immunohistochemistry and reverse transcriptase-quantitative polymerase chain reaction is recommended. Recommendations for recording bone marrow response are provided. The authors endorse the quantitative assessment of neuroblastoma cell content in bilateral core needle biopsies-trephines and aspirates in all children with neuroblastoma, with the exception of infants, in whom the evaluation of aspirates alone is advised. It is interesting to note that 5% disease is accepted as an internationally achievable level for disease assessment. CONCLUSIONS: The quantitative assessment of neuroblastoma cells is recommended to provide data from which evidence-based numerical criteria for the reporting of bone marrow response can be realized. This is particularly important in the minimal disease setting and when neuroblastoma detection in bone marrow is intermittent, where clinical impact has yet to be validated. The wide adoption of these harmonized criteria will enhance the ability to compare outcomes from different trials and facilitate collaborative trial design. Cancer 2017;123:1095-1105. © 2016 American Cancer Society.

Bevacizumab, Irinotecan, or Topotecan Added to Temozolomide for Children With Relapsed and Refractory Neuroblastoma: Results of the ITCC-SIOPEN BEACON-Neuroblastoma Trial.

PURPOSE: Outcomes for children with relapsed and refractory high-risk neuroblastoma (RR-HRNB) remain dismal. The BEACON Neuroblastoma trial (EudraCT 2012-000072-42) evaluated three backbone chemotherapy regimens and the addition of the antiangiogenic agent bevacizumab (B). MATERIALS AND METHODS: Patients age 1-21 years with RR-HRNB with adequate organ function and performance status were randomly assigned in a 3 × 2 factorial design to temozolomide (T), irinotecan-temozolomide (IT), or topotecan-temozolomide (TTo) with or without B. The primary end point was best overall response (complete or partial) rate (ORR) during the first six courses, by RECIST or International Neuroblastoma Response Criteria for patients with measurable or evaluable disease, respectively. Safety, progression-free survival (PFS), and overall survival (OS) time were secondary end points. RESULTS: One hundred sixty patients with RR-HRNB were included. For B random assignment (n = 160), the ORR was 26% (95% CI, 17 to 37) with B and 18% (95% CI, 10 to 28) without B (risk ratio [RR], 1.52 [95% CI, 0.83 to 2.77]; P = .17). Adjusted hazard ratio for PFS and OS were 0.89 (95% CI, 0.63 to 1.27) and 1.01 (95% CI, 0.70 to 1.45), respectively. For irinotecan ([I]; n = 121) and topotecan (n = 60) random assignments, RRs for ORR were 0.94 and 1.22, respectively. A potential interaction between I and B was identified. For patients in the bevacizumab-irinotecan-temozolomide (BIT) arm, the ORR was 23% (95% CI, 10 to 42), and the 1-year PFS estimate was 0.67 (95% CI, 0.47 to 0.80). CONCLUSION: The addition of B met protocol-defined success criteria for ORR and appeared to improve PFS. Within this phase II trial, BIT showed signals of antitumor activity with acceptable tolerability. Future trials will confirm these results in the chemoimmunotherapy era.

Identification of reference microRNAs and suitability of archived hemopoietic samples for robust microRNA expression profiling.

In many cancers, including neuroblastoma, microRNA (miRNA) expression profiling of peripheral blood (PB) and bone marrow (BM) may increase understanding of the metastatic process and lead to the identification of clinically informative biomarkers. The quality of miRNAs in PB and BM samples archived in PAXgene™ blood RNA tubes from large-scale clinical studies and the identity of reference miRNAs for standard reporting of data are to date unknown. In this study, we evaluated the reliability of expression profiling of 377 miRNAs using quantitative polymerase chain reaction (qPCR) in PB and BM samples (n=90) stored at -80 °C for up to 5 years in PAXgene™ blood RNA tubes. There was no correlation with storage time and variation of expression for any single miRNA (r < 0.50). The profile of miRNAs isolated as small RNAs or co-isolated with small/large RNAs was highly correlated (r=0.96). The mean expression of all miRNAs and the geNorm program identified miR-26a, miR-28-5p, and miR-24 as the most stable reference miRNAs. This study describes detailed methodologies for reliable miRNA isolation and profiling of PB and BM, including reference miRNAs for qPCR normalization, and demonstrates the suitability of clinical samples archived at -80 °C into PAXgene™ blood RNA tubes for miRNA expression studies.

The promise of telomere length, telomerase activity and its regulation in the translocation-dependent cancer ESFT; clinical challenges and utility.

The Ewing's sarcoma family of tumours (ESFT) are diagnosed by EWS-ETS gene translocations. The resulting fusion proteins play a role in both the initiation and maintenance of these solid aggressive malignant tumours, suppressing cellular senescence and increasing cell proliferation and survival. EWS-ETS fusion proteins have altered transcriptional activity, inducing expression of a number of different target genes including telomerase. Up-regulation of hTERT is most likely responsible for the high levels of telomerase activity in primary ESFT, although telomerase activity and expression of hTERT are not predictive of outcome. However levels of telomerase activity in peripheral blood may be useful to monitor response to some therapeutics. Despite high levels of telomerase activity, telomeres in ESFT are frequently shorter than those of matched normal cells. Uncertainty about the role that telomerase and regulators of its activity play in the maintenance of telomere length in normal and cancer cells, and lack of studies examining the relationship between telomerase activity, regulators of its activity and their clinical significance in patient samples have limited their introduction into clinical practice. Studies in clinical samples using standardised assays are critical to establish how telomerase and regulators of its activity might best be exploited for patient benefit.

Detection of Circulating and Disseminated Neuroblastoma Cells Using the ImageStream Flow Cytometer for Use as Predictive and Pharmacodynamic Biomarkers.

PURPOSE: Circulating tumor cells (CTCs) serve as noninvasive tumor biomarkers in many types of cancer. Our aim was to detect CTCs from patients with neuroblastoma for use as predictive and pharmacodynamic biomarkers. EXPERIMENTAL DESIGN: We collected matched blood and bone marrow samples from 40 patients with neuroblastoma to detect GD2 +/CD45- neuroblastoma CTCs from blood and disseminated tumor cells (DTCs) from bone marrow using the Imagestream Imaging flow cytometer (ISx). In six cases, circulating free DNA (cfDNA) extracted from plasma isolated from the CTC sample was analyzed by high-density single-nucleotide polymorphism (SNP) arrays. RESULTS: CTCs were detected in 26 of 42 blood samples (1-264/mL) and DTCs in 25 of 35 bone marrow samples (57-291,544/mL). Higher numbers of CTCs in patients with newly diagnosed, high-risk neuroblastoma correlated with failure to obtain a complete bone marrow (BM) metastatic response after induction chemotherapy (P < 0.01). Ex vivo Nutlin-3 (MDM2 inhibitor) treatment of blood and BM increased p53 and p21 expression in CTCs and DTCs compared with DMSO controls. In five of six cases, cfDNA analyzed by SNP arrays revealed copy number abnormalities associated with neuroblastoma. CONCLUSIONS: This is the first study to show that CTCs and DTCs are detectable in neuroblastoma using the ISx, with concurrently extracted cfDNA used for copy number profiling, and may be useful as pharmacodynamic biomarkers in early-phase clinical trials. Further investigation is required to determine whether CTC numbers are predictive biomarkers of BM response to first-line induction chemotherapy.

Ploidy and karyotype complexity are powerful prognostic indicators in the Ewing's sarcoma family of tumors: a study by the United Kingdom Cancer Cytogenetics and the Children's Cancer and Leukaemia Group.

Ewing's sarcoma family tumors (ESFT) are characterized by the presence of EWSR1-ETS fusion genes. Secondary chromosome changes are frequently described, although their clinical significance is not clear. In this study, we have collected and reviewed abnormal karyotypes from 88 patients with primary ESFT and a rearrangement of 22q12. Secondary changes were identified in 80% (70/88) of tumors at diagnosis. Multivariate analysis showed a worse overall and relapse free survival (RFS) for those with a complex karyotype (overall survival, P = 0.005; RFS, P = 0.04), independent of metastatic disease. Univariate survival analysis showed that a chromosome number above 50 or a complex karyotype was associated with a worse overall survival (>50 chromosomes, P = 0.05; complex karyotype, P = 0.04). There was no association between type of cytogenetic abnormality and the presence of metastatic disease at diagnosis. Univariate and multivariate survival analysis of a small subgroup with trisomy 20 indicated that trisomy 20 was associated with a worse overall and RFS. There was no difference in outcome associated with other recurrent trisomies (2, 5, 7, 8, or 12) or the common recurrent secondary structural rearrangements (deletions of 1p36, 9p12, 17p13, and 16q, and gain of 1q), although numbers were small. These data demonstrate the continued value of cytogenetics as a genome-wide screen in ESFT and illustrates the potential importance of secondary chromosome changes for stratification of patients for risk. Specifically, karyotype complexity appears to be a powerful predictor of prognosis, and the presence of trisomy 20 may be a marker of a more aggressive subset of this group.

Macrophage-Derived IL1ß and TNFα Regulate Arginine Metabolism in Neuroblastoma.

Neuroblastoma is the most common childhood solid tumor, yet the prognosis for high-risk disease remains poor. We demonstrate here that arginase 2 (ARG2) drives neuroblastoma cell proliferation via regulation of arginine metabolism. Targeting arginine metabolism, either by blocking cationic amino acid transporter 1 (CAT-1)-dependent arginine uptake in vitro or therapeutic depletion of arginine by pegylated recombinant arginase BCT-100, significantly delayed tumor development and prolonged murine survival. Tumor cells polarized infiltrating monocytes to an M1-macrophage phenotype, which released IL1ß and TNFα in a RAC-alpha serine/threonine-protein kinase (AKT)-dependent manner. IL1ß and TNFα established a feedback loop to upregulate ARG2 expression via p38 and extracellular regulated kinases 1/2 (ERK1/2) signaling in neuroblastoma and neural crest-derived cells. Proteomic analysis revealed that enrichment of IL1ß and TNFα in stage IV human tumor microenvironments was associated with a worse prognosis. These data thus describe an immune-metabolic regulatory loop between tumor cells and infiltrating myeloid cells regulating ARG2, which can be clinically exploited. SIGNIFICANCE: These findings illustrate that cross-talk between myeloid cells and tumor cells creates a metabolic regulatory loop that promotes neuroblastoma progression.

Expression analysis of the MCPH1/BRIT1 and BRCA1 tumor suppressor genes and telomerase splice variants in epithelial ovarian cancer.

AIMS: The aim of this study was to explore the correlation of hTERT splice variant expression with MCPH1/BRIT1 and BRCA1 expression in epithelial ovarian cancer (EOC) samples. BACKGROUND: Telomerase activation can contribute to the progression of tumors and the development of cancer. However, the regulation of telomerase activity remains unclear. MCPH1 (also known as BRIT1, BRCT-repeat inhibitor of hTERT expression) and BRCA1 are tumor suppressor genes that have been linked to telomerase expression. METHODS: qPCR was used to investigate telomerase splice variants, MCPH1/BRIT1 and BRCA1 expression in EOC tissue and primary cultures. RESULTS: The wild type α+/ß+ hTERT variant was the most common splice variant in the EOC samples, followed by α+/ß- hTERT, a dominant negative regulator of telomerase activity. EOC samples expressing high total hTERT demonstrated significantly lower MCPH1/BRIT1 expression in both tissue (p = 0.05) and primary cultures (p = 0.03). We identified a negative correlation between MCPH1/BRIT1 and α+/ß+ hTERT (p = 0.04), and a strong positive association between MCPH1/BRIT1 and both α-/ß+ hTERT and α-/ß- hTERT (both p = 0.02). A positive association was observed between BRCA1 and α-/ß+ hTERT and α-/ß- hTERT expression (p = 0.003 and p = 0.04, respectively). CONCLUSIONS: These findings support a regulatory effect of MCPH1/BRIT1 and BRCA1 on telomerase activity, particularly the negative association between MCPH1/BRIT1 and the functional form of hTERT (α+/ß+).

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.

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.

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.

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.

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.

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.

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.

Neuroblastoma mRNAs predict outcome in children with stage 4 neuroblastoma: a European HR-NBL1/SIOPEN study.

PURPOSE: To evaluate the hypothesis that detection of neuroblastoma mRNAs by reverse transcriptase quantitative polymerase chain reaction (RTqPCR) in peripheral blood (PB) and bone marrow aspirates (BM) from children with stage 4 neuroblastoma are clinically useful biomarkers of risk. METHODS: RTqPCR for paired-like homeobox 2b (PHOX2B), tyrosine hydroxylase (TH), and doublecortin (DCX) mRNA in PB and BM of children enrolled onto the High-Risk Neuroblastoma Trial-1 of the European Society of Pediatric Oncology Neuroblastoma Group (HR-NBL1/SIOPEN) was performed at diagnosis and after induction therapy. RESULTS: High levels of TH, PHOX2B, or DCX mRNA in PB or BM at diagnosis strongly predicted for worse event-free survival (EFS) and overall survival (OS) in a cohort of 290 children. After induction therapy, high levels of these mRNAs predicted worse EFS and OS in BM but not in PB. Combinations of mRNAs in BM did not add to the predictive power of any single mRNA. However, in the original (n = 182) and validation (n = 137) PB cohorts, high TH (log10TH > 0.8) or high PHOX2B (log10PHOX2B > 0.28) identify 19% of children as ultrahigh risk, with 5-year EFS and OS rates of 0%; OS rate was 25% (95% CI, 16% to 36%) and EFS rate was 38% (95% CI, 28% to 49%) in the remaining children. The magnitude of reduction in mRNA level between diagnosis and postinduction therapy in BM or PB was not of additional predictive value. CONCLUSION: High levels of TH and PHOX2B mRNA in PB at diagnosis objectively identify children with ultrahigh-risk disease who may benefit from novel treatment approaches. The level of TH, PHOX2B, and DCX mRNA in BM and/or PB at diagnosis might contribute to an algorithm to improve stratification of children for treatment.

Heterogeneous role of the glutathione antioxidant system in modulating the response of ESFT to fenretinide in normoxia and hypoxia.

Glutathione (GSH) is implicated in drug resistance mechanisms of several cancers and is a key regulator of cell death pathways within cells. We studied Ewing's sarcoma family of tumours (ESFT) cell lines and three mechanistically distinct anticancer agents (fenretinide, doxorubicin, and vincristine) to investigate whether the GSH antioxidant system is involved in the reduced sensitivity to these chemotherapeutic agents in hypoxia. Cell viability and death were assessed by the trypan blue exclusion assay and annexin V-PI staining, respectively. Hypoxia significantly decreased the sensitivity of all ESFT cell lines to fenretinide-induced death, whereas the effect of doxorubicin or vincristine was marginal and cell-line-specific. The response of the GSH antioxidant system in ESFT cell lines to hypoxia was variable and also cell-line-specific, although the level of GSH appeared to be most dependent on de novo biosynthesis rather than recycling. RNAi-mediated knockdown of key GSH regulatory enzymes γ-glutamylcysteine synthetase or glutathione disulfide reductase partially reversed the hypoxia-induced resistance to fenretinide, and increasing GSH levels using N-acetylcysteine augmented the hypoxia-induced resistance in a cell line-specific manner. These observations are consistent with the conclusion that the role of the GSH antioxidant system in modulating the sensitivity of ESFT cells to fenretinide is heterogeneous depending on environment and cell type. This is likely to limit the value of targeting GSH as a therapeutic strategy to overcome hypoxia-induced drug resistance in ESFT. Whether targeting the GSH antioxidant system in conjunction with other therapeutics may benefit some patients with ESFT remains to be seen.