Purpose: Most neuroblastomas initially respond to therapy but many relapse with chemoresistant disease. p53 mutations are rare in diagnostic neuroblastomas, but we have previously reported inactivation of the p53/MDM2/p14 ARF pathway in 9 of 17 (53%) neuroblastoma cell lines established at relapse. Hypothesis: Inactivation of the p53/MDM2/p14 ARF pathway develops during treatment and contributes to neuroblastoma relapse. Methods: Eighty-four neuroblastomas were studied from 41 patients with relapsed neuroblastoma including 38 paired neuroblastomas at different stages of therapy. p53 mutations were detected by automated sequencing, p14 ARF methylation and deletion by methylation-specific PCR and duplex PCR, respectively, and MDM2 amplification by fluorescent in situ hybridization.Results: Abnormalities in the p53 pathway were identified in 20 of 41 (49%) cases. Downstream defects due to inactivating missense p53 mutations were identified in 6 of 41 (15%) cases, 5 following chemotherapy and/or at relapse and 1 at diagnosis, postchemotherapy, and relapse. The presence of a p53 mutation was independently prognostic for overall survival (hazard ratio, 3.4; 95% confidence interval, 1.2-9.9; P = 0.02). Upstream defects were present in 35% of cases: MDM2 amplification in 3 cases, all at diagnosis and relapse and p14 ARF inactivation in 12 of 41 (29%) cases: 3 had p14 ARF methylation, 2 after chemotherapy, and 9 had homozygous deletions, 8 at diagnosis and relapse. Conclusions: These results show that a high proportion of neuroblastomas which relapse have an abnormality in the p53 pathway. The majority have upstream defects suggesting that agents which reactivate wild-type p53 would be beneficial, in contrast to those with downstream defects in which p53-independent therapies are indicated. Clin Cancer Res; 16(4); 1108-18. ©2010 AACR.Neuroblastoma is the most common extracranial pediatric solid tumor. It remains one of the most difficult cancers to cure, with <40% of patients with high-risk disease (stage 4 over 18 months of age or MYCN-amplified disease) becoming long-term survivors. Most high-risk neuroblastomas initially respond to cytotoxic therapy, however, over half relapse with chemoresistant disease and this often correlates with the intensity of therapy (1).The p53 gene is inactivated by mutation in >50% of human malignancies (2). p53 is a key regulator of cell cycle checkpoints and apoptosis, which upon activation by cellular stress, particularly DNA damage, binds DNA in a sequence-specific manner to activate the transcription of a large number of downstream genes, including p21 and MDM2, which results in apoptosis, cell cycle arrest, differentiation, and DNA repair (reviewed in ref.3). MDM2 functions upstream of p53 as a ubiquitin ligase that targets p53 for proteosome-mediated degradation, forming an autoregulatory feedback loop which tightly regulates p53 cellular levels (4). MDM2 amplification has been shown in some tumors and could suppress the activity of p53 by increasing its degradation.The INK4...

The MYC oncogenes are the most commonly amplified loci in medulloblastoma, and have previously been proposed as biomarkers of adverse disease prognosis by us and others. Here, we report focussed and comprehensive investigations of MYCC, MYCN and MYCL in an extensive medulloblastoma cohort (n = 292), aimed to define more precisely their biological significance and optimal clinical application to direct improved disease risk-stratification and individualisation of therapy. MYCC and MYCN expression elevations were multifactorial, associated with high-risk (gene amplification, large-cell/anaplastic pathology (LCA)) and favourable-risk (WNT/SHH molecular subgroups) disease features. Highly variable cellular gene amplification patterns underlay overall MYC copy number elevations observed in tumour biopsies; we used these alternative measures together to define quantitative methodologies and thresholds for amplification detection in routinely collected tumour material. MYCC and MYCN amplification, but not gain, each had independent prognostic significance in non-infants (≥3.0-16.0 years), but MYCC conferred a greater hazard to survival than MYCN when considered across this treatment group. MYCN's weaker group-wide survival relationship may be explained by its pleiotropic behaviour between clinical disease-risk groups; MYCN predicted poor prognosis in clinical high-risk (metastatic (M+) or LCA), but not standard-risk, patients. Extending these findings, survival decreased in proportion to the total number of independently significant high-risk features present (LCA, M+ or MYCC/MYCN amplification). This cumulative-risk model defines a patient group characterised by ≥2 independent risk-factors and an extremely poor prognosis (<15% survival), which can be identified straightforwardly using the reported MYC amplification detection methodologies alongside clinical assessments, enabling targeting for novel/intensified therapies in future clinical studies.

Background:Thiothymidine (S4TdR) can be incorporated into DNA and sensitise cells to DNA damage and cell death following exposure to UVA light. Studies were performed to determine if the combination of S4TdR and UVA could be an effective treatment for bladder cancer.Methods:Uptake and incorporation of S4TdR was determined in rat and human bladder tumour cell lines. Measures of DNA crosslinking and apoptosis were also performed. In vivo activity of the combination of S4TdR and UVA was investigated in an orthotopic model of bladder cancer in rats.Results:Thiothymidine (200 μ) replaced up to 0.63% of thymidine in rat and tumour bladder cancer cells. The combination of S4TdR (10–200 μ) and UVA (1–5 kJ m−2) caused apoptosis and cell death at doses that were not toxic alone. Addition of raltitrexed (Astra Zeneca, Alderley Edge, Cheshire, UK) increased the incorporation of S4TdR into DNA (up to 20-fold at IC5) and further sensitised cells to UVA. Cytotoxic effect was associated with crosslinking of DNA, at least partially to protein. Intravenous administration of S4TdR, in combination with UVA delivered directly to the bladder, resulted in an antitumour effect in three of five animals treated.Conclusion:These data indicate that the combination of S4TdR and UVA has potential as a treatment for bladder cancer, and give some insight into the mechanism of action. Further work is necessary to optimise the delivery of the two components.

These studies highlight the histological similarities between tumours from MYCN transgenic mice and human neuroblastomas, and reaffirm their role as a valuable model to study the biology of aggressive human neuroblastoma.

OBJECTIVETo investigate the feasibility of transcript profiling in diagnostic formalin‐fixed and paraffin‐embedded (FFPE) biopsies for prostate cancer.MATERIALS AND METHODSLaser‐capture microdissection (LCM) was used to microdissect glandular epithelium as well as stromal tissue in archival prostate needle biopsies. Optimized RNA extraction, reverse transcription and real‐time PCR (QPCR) protocols were used to detect transcript expression. RNA degradation effects were assessed using hydrolysed cell line RNA and matched xenograft FFPE and frozen tumours.RESULTSLCM and RNA extraction was achieved in all biopsies from a pilot cohort of five patients. cDNA produced was successfully used to detect expression of glyceraldehyde‐3‐phosphate dehydrogenase, RPL13, prostate‐specific antigen, vimentin, inhibitor of differentiation/DNA binding 1 (Id‐1) and polycomb group protein enhancer of zeste homolog 2 (EZH2) transcripts. In the cell line and xenograft models, we investigated the effect of RNA degradation on transcript quantification by QPCR. In both models normalization of transcript quantity with a housekeeping gene resulted in restored expression in all degraded samples to within a 50% difference of control samples. Using an extended cohort of 29 biopsies, we tested application in detecting differences in EZH2 and Id‐1 expression between malignant and benign epithelium. The results confirmed that our technique was capable of quantifying significant differences in expression between malignant and benign epithelium consistent with the reported trends.CONCLUSIONThis study reports the use of standard FFPE needle biopsies for transcript profiling and supports the concept of molecular prognostic studies in tissue acquired at diagnosis in prostate cancer.

BACKGROUND: The standard treatment of choice for malignant pleural mesothelioma is chemotherapy with pemetrexed and platinum, but the clinical outcome is poor. This study investigates the response to pemetrexed in a panel of eight mesothelioma cell lines and the clinical outcome for patients treated with pemetrexed in relation to folate receptor alpha (FRa). METHODS: Cell lines were treated with pemetrexed to determine the concentration that reduced growth to 50% (GI 50 ). FRa expression was determined by western blotting and that of FRa, reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT) by real-time quantitative RT -PCR. Immunohistochemistry for FRa was carried out on 62 paraffin-embedded samples of mesothelioma from patients who were subsequently treated with pemetrexed. RESULTS: A wide range of GI 50 values was obtained for the cell lines, H2452 cells being the most sensitive (GI 50 22 nM) and RS5 cells having a GI 50 value greater than 10 mM. No FRa protein was detected in any cell line, and there was no relationship between sensitivity and expression of folate transporters. FRa was detected in 39% of tumour samples, generally in a small percentage of cells. There was no correlation between the presence of FRa and the outcome of pemetrexed treatment, and no significant difference between histological subtypes. CONCLUSION: Response to treatment with pemetrexed does not depend on the presence of FRa.

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(2009) Chromosome 1q gain is not associated with a poor outcome in childhood medulloblastoma: Requirements for the validation of potential prognostic biomarkers,