Influence of segmental chromosome abnormalities on survival in children over the age of 12 months with unresectable localised peripheral neuroblastic tumours without MYCN amplification.

BACKGROUND: The prognostic impact of segmental chromosome alterations (SCAs) in children older than 1 year, diagnosed with localised unresectable neuroblastoma (NB) without MYCN amplification enrolled in the European Unresectable Neuroblastoma (EUNB) protocol is still to be clarified, while, for other group of patients, the presence of SCAs is associated with poor prognosis. METHODS: To understand the role of SCAs we performed multilocus/pangenomic analysis of 98 tumour samples from patients enrolled in the EUNB protocol. RESULTS: Age at diagnosis was categorised into two groups using 18 months as the age cutoff. Significant difference in the presence of SCAs was seen in tumours of patients between 12 and 18 months and over 18 months of age at diagnosis, respectively (P=0.04). A significant correlation (P=0.03) was observed between number of SCAs per tumour and age. Event-free (EFS) and overall survival (OS) were calculated in both age groups, according to both the presence and number of SCAs. In older patients, a poorer survival was associated with the presence of SCAs (EFS=46% vs 75%, P=0.023; OS=66.8% vs 100%, P=0.003). Moreover, OS of older patients inversely correlated with number of SCAs (P=0.002). Finally, SCAs provided additional prognostic information beyond histoprognosis, as their presence was associated with poorer OS in patients over 18 months with unfavourable International Neuroblastoma Pathology Classification (INPC) histopathology (P=0.018). CONCLUSIONS: The presence of SCAs is a negative prognostic marker that impairs outcome of patients over the age of 18 months with localised unresectable NB without MYCN amplification, especially when more than one SCA is present. Moreover, in older patients with unfavourable INPC tumour histoprognosis, the presence of SCAs significantly affects OS.

Structurally diverse MDM2-p53 antagonists act as modulators of MDR-1 function in neuroblastoma.

BACKGROUND: A frequent mechanism of acquired multidrug resistance in human cancers is overexpression of ATP-binding cassette transporters such as the Multi-Drug Resistance Protein 1 (MDR-1). Nutlin-3, an MDM2-p53 antagonist, has previously been reported to be a competitive MDR-1 inhibitor. METHODS: This study assessed whether the structurally diverse MDM2-p53 antagonists, MI-63, NDD0005, and RG7388 are also able to modulate MDR-1 function, particularly in p53 mutant neuroblastoma cells, using XTT-based cell viability assays, western blotting, and liquid chromatography-mass spectrometry analysis. RESULTS: Verapamil and the MDM2-p53 antagonists potentiated vincristine-mediated growth inhibition in a concentration-dependent manner when used in combination with high MDR-1-expressing p53 mutant neuroblastoma cell lines at concentrations that did not affect the viability of cells when given alone. Liquid chromatography-mass spectrometry analyses showed that verapamil, Nutlin-3, MI-63 and NDD0005, but not RG7388, led to increased intracellular levels of vincristine in high MDR-1-expressing cell lines. CONCLUSIONS: These results show that in addition to Nutlin-3, other structurally unrelated MDM2-p53 antagonists can also act as MDR-1 inhibitors and reverse MDR-1-mediated multidrug resistance in neuroblastoma cell lines in a p53-independent manner. These findings are important for future clinical trial design with MDM2-p53 antagonists when used in combination with agents that are MDR-1 substrates.

Alternatively spliced mdm2 transcripts with loss of p53 binding domain sequences: transforming ability and frequent detection in human cancer.

The mdm2 oncogene encodes a 90-kilodalton nuclear phosphoprotein that binds and inactivates the p53 tumor suppressor protein. Here we report the observation of five alternatively spliced mdm2 gene transcripts in a range of human cancers and their absence in normal tissues. Transfection of NIH 3T3 cells with each of these forms gave foci of morphologically transformed cells. A higher frequency of splice variants lacking p53 binding domain sequences was found in late-stage and high-grade ovarian and bladder carcinomas. Four of the splice variants show loss of p53 binding, consistent with partial deletion of sequences encoding the p53 binding domain, but retain carboxyterminal zinc-finger domains. These observations suggest a reassessment of the transforming mechanisms of mdm2 and its relation to p53.

The role of folate receptor alpha (FRalpha) in the response of malignant pleural mesothelioma to pemetrexed-containing chemotherapy.

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 (FRalpha). METHODS: Cell lines were treated with pemetrexed to determine the concentration that reduced growth to 50% (GI(50)). FRalpha expression was determined by western blotting and that of FRalpha, reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT) by real-time quantitative RT-PCR. Immunohistochemistry for FRalpha 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 microM. No FRalpha protein was detected in any cell line, and there was no relationship between sensitivity and expression of folate transporters. FRalpha was detected in 39% of tumour samples, generally in a small percentage of cells. There was no correlation between the presence of FRalpha 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 FRalpha.

Multicenter study to assess potential hazards from exposure to lipid peroxidation products in soya bean oil from Trilucent breast implants.

In response to a Hazard Notice by the Medical Devices Agency of the UK in 2000 regarding the Trilucent breast implant (TBI), an expert panel was convened to implement a research program to determine whether genotoxic compounds were formed in the soybean oil filler (SOF) of TBIs and whether these could be released to produce local or systemic genotoxicity. The panel established a research program involving six laboratories. The program recruited 47 patients who had received TBIs (9 patients had received silicone implants previously). A reference group (REBI) of 34 patients who had exchanged either silicone (17 patients) implants (REBI-E) or patients (17) who were to receive primary implantation augmentation with silicone (REBI-PIA), and who were included as needed to increase either the pre- or post-explantation sample number. Of the 17 REBI-E patients, 5 had silicone implants and 12 had saline implants previously (prior to the last exchange). Investigation was undertaken before and after replacement surgery in the TBI patients and before and after replacement or augmentation surgery in the REBI patients. The pre- to post-operative sample interval was 8-12 weeks. Pre-operative samples were collected within 7 days prior to the operation. Information on a variety of demographic and behavioral features was collected. Biochemical and biological endpoints relating to genotoxic lipid peroxidation (LPO) products potentially formed in the SOF, and released locally or distributed systemically, were measured. The SOF of explanted TBIs was found to have substantial levels of LPO products, particularly malondialdehyde (MDA), and low levels of trans-4-hydroxy-2-nonenal (HNE) not found in unused implants. Mutagenicity of the SOF was related to the levels of MDA. Capsules that formed around TBIs were microscopically similar to those of reference implants, but MDA-DNA adducts were observed in capsular macrophages and fibroblasts of only TBI capsules. These cell types are not progenitors of breast carcinoma (BCa) and the location of the implants precludes LPO products reaching the mammary epithelial cells which are progenitors of BCa. Blood levels of LPO products were not increased in TBI patients compared to REBI patients and did not change with explantation. In TBI patients, white blood cells did not show evidence of increased levels of LPO-related aldehyde DNA adducts. In conclusion, based on a number of measured parameters, there was no evident effect that would contribute to breast or systemic cancer risk in the TBI patients, and the recommended treatment of TBI patients involving explantation was judged appropriate.

The predictive value of p53 and p33(ING1b) in patients with Dukes'C colorectal cancer.

OBJECTIVE: Identification of biological markers that may predict response to chemotherapy would allow the individualization of treatment by enabling selection of patients most likely to benefit from chemotherapy. The aims of this study were to determine whether p53 mutation status and p53 and p33(ING1b) protein expression can predict which patients with Dukes' C colorectal cancer following curative surgical resection respond to adjuvant chemotherapy with 5-fluorouracil (5-FU). METHOD: Patients with Dukes'C colorectal cancer (n = 41) were studied. DNA was extracted and analysed for p53 mutation using PCR-based direct DNA sequencing. Tumours were analysed for p53 protein expression by immunohistochemistry using DO-7 monoclonal antibody and for p33(ING1b) protein expression using GN1 monoclonal antibody. RESULTS: There was a significant association between p53 mutation status analysed by gene sequencing and overall and metastasis-free survival (P = 0.03 and 0.004, respectively, log-rank test). By contrast, no significant correlation was found between p53 and p33(ING1b) protein expression and overall or metastasis-free survival. CONCLUSION: In patients with Dukes'C colorectal cancer who underwent curative surgical resection of the primary tumour, followed by 5-FU-based adjuvant chemotherapy, p53 mutation status as assessed by gene sequencing is a significant predictor of overall and metastasis-free survival.

Self-perpetuating mechanisms of immunoglobulin G aggregation in rheumatoid inflammation.

When human IgG is exposed to free radical generating systems such as ultraviolet irradiation, peroxidizing lipids, or activated human neutrophils, characteristic auto-fluorescent monomeric and polymeric IgG is formed (excitation [Ex], 360 nm, emission [Em], 454 nm). 1 h ultraviolet irradiation of IgG results in the following reductions in constituent amino acids; cysteine (37.0%), tryptophan (17.0%), tyrosine (10.5%), and lysine (3.6%). The fluorescent IgG complexes, when produced in vitro, can stimulate the release of superoxide from normal human neutrophils. In the presence of excess unaltered IgG, further fluorescent damage to IgG occurs. Measurement and isolation of fluorescent monomeric and polymeric IgG by high performance liquid chromatography, from in vitro systems and from fresh rheumatoid sera and synovial fluid, indicates that identical complexes are present in vivo; all these fluorescent complexes share the property of enhancing free radical production from neutrophils. The results described in this study support the hypothesis that fluorescent monomeric and aggregated IgG may be formed in vivo by oxygen-centered free radicals derived from neutrophils, and that in rheumatoid inflammation this reaction may be self-perpetuating within the inflamed joint.

Evidence for the development of p53 mutations after cytotoxic therapy in a neuroblastoma cell line.

p53 mutations are rare in neuroblastomas at diagnosis perhaps accounting for their initial response to therapy, but advanced neuroblastoma frequently relapses, and it is possible that p53 mutations develop later. Two neuroblastoma cell lines derived from the same patient before [SKNBE(1n)] and after [SKNBE(2c)] cytotoxic therapy were analyzed for the presence of chromosome 17 and p53 genes by fluorescent in situ hybridization, p53 mutations by DNA sequencing, and p53 function after irradiation by studying the transcription of p53-regulated genes, cell cycle arrest, and induction of apoptosis. The SKNBE(1n) cell line was wild-type for p53, had two p53 genes, two copies of chromosome arm 17p and showed functional p53 after irradiation. The SKNBE(2c) cell line derived from the same patient 5 months later at relapse had loss of an entire chromosome 17, resulting in hemizygosity for the p53 locus on 17p and a missense p53 mutation in exon 5, and p53 was not functional after irradiation. The appearance of a p53 mutation in a cell line derived from a relapsed neuroblastoma suggests that this may be a mechanism of resistance to therapy. If p53 mutations develop frequently in relapsed neuroblastoma, cytotoxic agents more sensitive to mutant p53 might be more effective at relapse.

Prognostic significance of HER2 and HER4 coexpression in childhood medulloblastoma.

Recent in vitro studies of the epidermal growth factor receptor (EGFR) family have revealed complex signaling interactions involving the production of ligand-mediated heterodimers synergistic for the transformation of cells in vitro. In a series of 70 patients with childhood medulloblastoma, we have used immunohistochemistry and Western blotting analysis to investigate the expression patterns of all four EGFR family members (EGFR, HER2, HER3, and HER4) and heregulin-alpha, a ligand for the HER3 and HER4 receptors. The majority of cases expressed two or more receptor proteins; coexpression of the HER2 and HER4 receptors occurred in 54%. Expression of the ligand heregulin-alpha was detected in 31% of tumors. To investigate whether coexpression results in receptor heterodimerization, we have also performed immunoprecipitation analysis of protein extracts from primary tumors, and we demonstrate various patterns of receptor interaction including between HER2 and HER4. In multivariate 25-year survival analysis with clinicopathological disease features, no individual receptor or heregulin-alpha achieved significance. In contrast, when considered together in the multivariate model, coexpression of HER2 and HER4 demonstrated independent prognostic significance (P = 0.006). These data suggest the hypothesis that HER2-HER4 receptor heterodimerization is of particular biological significance in this disease, and this report is the first to demonstrate potential clinical significance of EGFR family heterodimerization in human cancer. Finally, we have also analyzed expression of the AP-2 transcription factor implicated in the positive regulation of HER2 and HER3 gene transcription in malignant cells and reveal an association between AP-2 expression and not only HER2 and HER3, but also HER4 levels in medulloblastoma primary tumors.

Human lymphoblastoid cells with acquired resistance to C2-desamino-C2-methyl-N10-propargyl-5,8-dideazafolic acid: a novel folate-based thymidylate synthase inhibitor.

We describe the characterization of human lymphoblastoid cell lines with acquired resistance (greater than 20,000-fold) to a novel folate-based thymidylate synthase (TS) (EC 2.1.1.45) inhibitor, C2-desamino-C2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI198583). This acquired resistance was associated with a 64-fold amplification of the TS gene, a similar elevation in the corresponding mRNA, and an approximately 200-fold increase in both TS activity and TS protein. This amplification was maintained when the cells were grown in the absence of the selective agent, ICI198583, for 340 generations. TS isolated from one of the resistant cell lines, W1-L2:C1, displayed inhibition kinetic parameters similar to those of TS isolated from the parent W1-L2 cell line. It thus appears unlikely that resistance is due to an altered TS enzyme having a lower affinity for ICI198583. The resistant cell line, W1-L2:C1, was cross-resistant to other folate-based TS inhibitors but was as sensitive as the parent cell line, W1-L2, to 5-fluorodeoxyuridine. The W1-L2:C1 cell line was collaterally sensitive to the classical dihydrofolate reductase (EC 1.5.1.3) inhibitor methotrexate as well as to the lipophilic dihydrofolate reductase inhibitors metoprine and 2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazolin e glucuronic acid salt (also called trimetrexate). When the W1-L2 and W1-L2:C1 cell lines were exposed to 1 microM ICI198583 for 24 h they accumulated the same concentration of total cellular ICI198583 polyglutamates despite the fact that the latter cell line accumulated a 300-fold greater concentration of ICI198583 monoglutamate. As polyglutamates, the tetra- and pentaglutamate forms predominated in the W1-L2 cell line, whereas the diglutamate form predominated in the W1-L2:C1 cell line, with few higher polyglutamates being detected. The lack of tri- and higher polyglutamates of ICI198583 (i.e., the more active species) in the W1-L2:C1 cell line may also contribute to the observed resistance. These findings may have important implications in light of the rapid onset of resistance to antifolates in the clinic.

Expression of the ErbB-neuregulin signaling network during human cerebellar development: implications for the biology of medulloblastoma.

The four receptor tyrosine kinase I receptors, ErbB-1, ErbB-2, ErbB-3, and ErbB-4, which have been implicated in the development of a variety of normal and malignant tissues, are activated through ligand mediated homo- and heterodimerization. We have previously reported the frequent coexpression, heterodimerzation, and prognostic significance of ErbB-2 and ErbB-4 in childhood medulloblastoma, an embryonal tumor of the cerebellar external granule cell layer (EGL). In the present study, we have used immunohistochemistry and Western blotting analysis to analyze the expression of the ErbB receptors and neuregulin (NRG) 1-alpha and NRG1-beta ligands during normal human cerebellar development. We demonstrate that ErbB-1, ErbB-3, ErbB-4, and NRG1-beta display specific temporal and topographical distribution in the cerebellum during intrauterine and postnatal life, and that normal ErbB-NRG signaling in the EGL multiplying zone is likely to be mediated by ErbB-4 and NRG1-beta. In contrast, ErbB-2, which is expressed in 86% of medulloblastomas, could not be detected at any stage of cerebellar development. Therefore, we propose that positive deregulation of ErbB-2 expression in the cerebellar EGL, leading to the formation of a NRG41-beta-driven ErbB-2/ErbB-4 autocrine loop, is an important factor in medulloblastoma tumorigenesis. In further support of this hypothesis, we provide evidence using reverse transcription-PCR analysis that expression of the ErbB-2 and ErbB-4 receptors, but not ErbB-1 or ErbB-3, is deregulated in medulloblastoma compared with normal developing cerebellum. We also demonstrate NRG1-beta expression in 87% (n = 46 of 48) of medulloblastoma primary tumors, with the greatest expression levels occurring in tumors with high ErbB-2 and ErbB-4 receptor coexpression. Furthermore, the expression of all three components of the proposed autocrine loop (ie., ErbB-2, ErbB-4, and NRG1-beta) was significantly related to the presence of metastases at diagnosis (P < 0.05).

Surgical outcome and patterns of recurrence for retroperitoneal sarcoma at a single centre.

INTRODUCTION: Retroperitoneal sarcoma is a surgically managed condition that can recur locally following macroscopically complete resection. Owing to the low incidence of the condition, advances in treatment are reported infrequently but complete compartmental resection and adjuvant or neoadjuvant radiotherapy are areas under investigation. Given the practical difficulty of randomised trials, observational data can highlight advantages from progressive treatment approaches. METHODS: A retrospective database of consecutive retroperitoneal sarcoma resections performed at a single referral centre between March 1997 and March 2013 was interrogated. Histological, radiological and clinical data were collected. Univariate and multivariate analyses for disease free and overall survival were performed to establish independent predictors of disease recurrence and patient survival. RESULTS: A total of 79 patients underwent 90 resections (63 primary). The mean five-year overall and disease free survival rates were 55.3% and 24.8% respectively. Higher patient age, high tumour grade, presence of extraretroperitoneal disease and invasive tumour phenotype were found to significantly predict survival following multivariate analysis. Half (50%) of the tumours displayed invasive behaviour on histopathology and 42% of locoregional recurrence was intraperitoneal. CONCLUSIONS: Retroperitoneal sarcoma is commonly an infiltrative tumour and often recurs outside of the retroperitoneum. These features limit the therapeutic impact of interventions that focus on gaining local control such as complete compartmental resection and radiotherapy. It seems likely that future advances in the management of this cancer will involve new systemic agents to treat this frequently systemic disease.

Investigation of co-amplification of the candidate genes ornithine decarboxylase, ribonucleotide reductase, syndecan-1 and a DEAD box gene, DDX1, with N-myc in neuroblastoma. United Kingdom Children's Cancer Study Group.

Although N-myc amplification is strongly associated with a poor prognosis, not all patients with neuroblastomas having N-myc amplification fare badly. To investigate whether genes other than N-myc are responsible for contributing to the prognosis, we examined seven cell lines and 87 primary tumours for co-amplification of candidate genes known to be present near the normal N-myc locus: ornithine decarboxylase (ODC), ribonucleotide reductase (RRM2), syndecan-1 and a DEAD box protein gene, DDX1. Sequence analysis of the pG21 cDNA clone previously reported to represent an expressed gene frequently co-amplified with N-myc, showed this to be from the DDX1 gene. No co-amplification with the first three genes was found in any of the cell lines or tumour samples. DDX1, however was found to be amplified along with N-myc in 4/6 (67%) cell lines and 6/16 (38%) of the N-myc amplified tumours. Co-amplification of DDX1 and N-myc was found more frequently in stage 4 or 4S tumours than lower stage (1-3) tumours. With the exclusion of a single 4S case, there was a highly significant reduction in the mean disease-free interval from 24.4 +/- 4.7 (SE, n = 10) months for cases with co-amplification of N-myc and DDX1 compared with 9.2 +/- 1.8 (SE, n = 5) months for those cases showing amplification of N-myc alone (P = 0.0056, Welch's unpaired t-test). No amplification of DDX1, ODC, RRM2, or syndecan-1 was found in the absence of N-myc amplification. These observation indicate that the N-myc amplicon is of varied size and/or position relative to the N-myc gene, with DDX1 representing at least one other gene frequently co-amplified with N-myc. Further studies are required to confirm the biological and prognostic significance of DDX1 co-amplification and to elucidate the role that DDX1 plays in tumour genesis and progression.

Fluorescence changes in human gamma-globulin induced by free-radical activity.

Low-intensity ultraviolet irradiation was used to generate free-radical activity in lipid-free human gamma-globulin. The changes were monitored by fluorescence spectroscopy and gel filtration chromatography. The pattern and the effect of thiol compounds, free-radical scavengers and antioxidants point to a free-radical-mediated process. Initial energy uptake was probably in the region of aromatic amino acid residues, followed by complex intramolecular rearrangements. Irradiation led to the formation of molecular species and complexes whose fluorescence characteristics were different from those of native gamma-globulin and indistinguishable from those observed in inflammatory exudates.

Induction of thymidylate synthase as a 5-fluorouracil resistance mechanism.

Thymidylate synthase (TS) is a key enzyme in the de novo synthesis of 2'-deoxythymidine-5'-monophosphate (dTMP) from 2'-deoxyuridine-5'-monophosphate (dUMP), for which 5,10-methylene-tetrahydrofolate (CH(2)-THF) is the methyl donor. TS is an important target for chemotherapy; it is inhibited by folate and nucleotide analogs, such as by 5-fluoro-dUMP (FdUMP), the active metabolite of 5-fluorouracil (5FU). FdUMP forms a relatively stable ternary complex with TS and CH(2)THF, which is further stabilized by leucovorin (LV). 5FU treatment can induce TS expression, which might bypass dTMP depletion. An improved efficacy of 5FU might be achieved by increasing and prolonging TS inhibition, a prevention of dissociation of the ternary complex, and prevention of TS induction. In a panel of 17 colon cancer cells, including several variants with acquired resistance to 5FU, sensitivity was related to TS levels, but exclusion of the resistant variants abolished this relation. For antifolates, polyglutamylation was more important than the intrinsic TS level. Cells with low p53 levels were more sensitive to 5FU and the antifolate raltitrexed (RTX) than cells with high, mutated p53. Free TS protein down-regulates its own translation, but its transcription is regulated by E2F, a cell cycle checkpoint regulator. Together, this results in low TS levels in stationary phase cells. Although cells with a low TS might theoretically be more sensitive to 5FU, the low proliferation rate prevents induction of DNA damage and 5FU toxicity. TS levels were not related to polymorphisms of the TS promoter. Treatment with 5FU or RTX rapidly induced TS levels two- to five-fold. In animal models, 5FU treatment resulted in TS inhibition followed by a two- to three-fold TS induction. Both LV and a high dose of 5FU not only enhanced TS inhibition, but also prevented TS induction and increased the antitumor effect. In patients, TS levels as determined by enzyme activity assays, immunohistochemistry and mRNA expression, were related to a response to 5FU. 5FU treatment initially decreased TS levels, but this was followed by an induction, as seen with an increased ratio of TS protein over TS-mRNA. The clear retrospective relation between TS levels and response now forms the basis for a prospective study, in which TS levels are measured before treatment in order to determine the treatment protocol.

Quality assessment of genetic markers used for therapy stratification.

PURPOSE: Therapy stratification based on genetic markers is becoming increasingly important, which makes commitment to the highest possible reliability of the involved markers mandatory. In neuroblastic tumors, amplification of the MYCN gene is an unequivocal marker that indicates aggressive tumor behavior and is consequently used for therapy stratification. To guarantee reliable and standardized quality of genetic features, a quality-assessment study was initiated by the European Neuroblastoma Quality Assessment (ENQUA; connected to International Society of Pediatric Oncology) Group. MATERIALS AND METHODS: One hundred thirty-seven coded specimens from 17 tumors were analyzed in 11 European national/regional reference laboratories using molecular techniques, in situ hybridization, and flow and image cytometry. Tumor samples with divergent results were re-evaluated. RESULTS: Three hundred fifty-two investigations were performed, which resulted in 23 divergent findings, 17 of which were judged as errors after re-evaluation. MYCN analyses determined by Southern blot and in situ hybridization led to 3.7% and 4% of errors, respectively. Tumor cell content was not indicated in 32% of the samples, and 11% of seemingly correct MYCN results were based on the investigation of normal cells (eg, Schwann cells). Thirty-eight investigations were considered nonassessable. CONCLUSION: This study demonstrated the importance of revealing the difficulties and limitations for each technique and problems in interpreting results, which are crucial for therapeutic decisions. Moreover, it led to the formulation of guidelines that are applicable to all kinds of tumors and that contain the standardization of techniques, including the exact determination of the tumor cell content. Finally, the group has developed a common terminology for molecular-genetic results.

The impact of p53 status on cellular sensitivity to antifolate drugs.

The impact of p53 status on cellular sensitivity to antifolate drugs has been examined in seven human cell lines (A549, MCF7, T-47D, CCRF-CEM, COR-L23, A2780, and HCT-116) and p53 nonfunctional counterparts of two of the cell lines (HCT-116/N7 and A2780/CP70). p53 status was determined by sequencing and functional assays. The sensitivities of the cell lines to growth inhibition (sulphorhodamine B assay) produced by four antifolate drugs (Alimta, methotrexate, raltitrexed, and lometrexol) were studied. There was no clear relationship between functional p53 status and sensitivity to methotrexate or lometrexol, whereas a functional p53 status was possibly associated with resistance to Alimta- and raltitrexed-induced growth inhibition. In contrast, in the two pairs of related human tumor cell lines (HCT-116 and HCT-116/N7 and A2780 and A2780/CP70) cells with functional p53 were more sensitive to Alimta- and raltitrexed-induced growth inhibition (P = 0.002). Detailed studies were performed with the A2780 cell lines, and in the parental cells sensitivity to Alimta- and raltitrexed-induced cytotoxicity (clonogenic assay) was similar to the sensitivity determined in the sulphorhodamine B assay. However, in A2780/CP70 cells, 1 microM of drug resulted in only 40-60% growth inhibition yet > or = 85% cytotoxicity. After Alimta and raltitrexed exposure for < or = 72 h, there were no differences between the A2780 and A278/CP70 cell lines in cell cycle phase distribution, absolute cell number, or the induction of apoptosis. However, the cellular protein content of the A2780/CP70 cells was 3-6-fold higher than in A2780 cells after Alimta and raltitrexed treatment, suggesting that cells without functional p53 can maintain protein synthesis in the absence of cell division (unbalanced cell growth). In conclusion, the apparent impact of functional p53 status on sensitivity to antifolate drugs may depend upon the phenotypic/genotypic background as well as the assay used to measure cellular sensitivity.

TP53 accumulation predicts improved survival in patients resistant to systemic cisplatin-based chemotherapy for muscle-invasive bladder cancer.

To examine retrospectively the prognostic significance of TP53 immunoreactivity for both tumor response and patient survival in 83 patients with nonmetastatic muscle-invasive bladder cancer treated with a single transurethral resection (TUR) of tumor and combined cisplatin-based systemic chemotherapy followed by repeat TUR, paraffin-embedded sections of a bladder tumor obtained at TUR before chemotherapy (1 T2, 52 T3, and 30 T4) were immunostained for TP53 using monoclonal PAb1801 and DO-7 antibodies. For the entire cohort, TP53 immunopositivity (PAb1801 or DO-7) did not predict complete response (CR), complete or partial response (PR), progressive disease, or time to death from bladder cancer. There was a highly significant correlation between PAb1801 and DO-7 nuclear immunoreactivity (r = 0.8242; P<0.0001). In 76 patients in which complete clinical data were available, tumor stage (T2/T3; P = 0.0499), CR and PR (P = 0.0016) and CR (P<0.0001) were associated with patient survival. In a multivariate model, CR (P<0.0001) was the only independent predictor of improved survival. In complete responders, neither TP53 immunostaining nor clinicopathological factors stratified patients into prognostic groups. However, in the subset of patients (n = 38) who were chemoresistant (PR or progressive disease), improved survival was associated with > or =20% TP53 immunoreactivity (PAb1801; P = 0.0191) and tumor stage (T2/T3; P = 0.0358). TP53 immunopositivity (PAb1801 or DO-7) did not predict overall survival or response to systemic chemotherapy in patients with nonmetastatic but predominantly clinical stage > or =T3 bladder cancer, but it had prognostic significance within the chemoresistant subgroup.

Prognostic significance of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 in voided urine samples from patients with transitional cell carcinoma of the bladder.

PURPOSE: To study the role of urinary matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in bladder cancer and their relationship to tumor progression. EXPERIMENTAL DESIGN: MMP-1 and TIMP-1 were measured by ELISA in urine samples from 131 patients with bladder tumors (7 cis, 74 Ta, 29 T1, and 21 T2-T4; 46 G(1), 41 G(2), and 37 G(3)), 5 patients with prostate cancer, 33 patients with benign lower urinary tract disorders, and 36 healthy volunteers. Complete clinical data were available for 100 patients with bladder cancer with a median follow-up time of 24 months (range: 4-39 months). RESULTS: MMP-1 was detected in urine samples from 21 of 131 (16%) patients with bladder cancer but was undetectable in samples from all other groups (P < 0.0001). Urinary MMP-1 was detected in a higher percentage of patients with T2-T4 tumors and G(3) tumors than patients with cis/Ta/T1 or G(1)-G(2) tumors (P = 0.04 and P = 0.0074, respectively). Patients with detectable concentrations of urinary MMP-1 had higher rates of disease progression (P = 0.04) and death from bladder cancer (P = 0.02) than patients with undetectable urinary MMP-1. All patient groups had higher urinary TIMP-1 concentrations than healthy volunteers (P = 0.02). Patients with muscle-invasive tumors had higher concentrations of urinary TIMP-1 than patients with cis/Ta/T1 tumors (P = 0.037), but there was no association between TIMP-1 and tumor grade. Urinary TIMP-1 levels strongly correlated with tumor size (P = 0.0002). Progression-free survival rates were lower for patients with urinary TIMP-1 concentrations above the median (1.8 ng/ml, P = 0.04), but urinary TIMP-1 levels were not related to disease-specific survival. Patients with T2-T4 tumors and G(3) tumors had significantly lower urinary MMP-1:TIMP-1 ratios than patients with Ta/T1 bladder tumors (P = 0.039) or G(1)-G(2) tumors (P = 0.0415). CONCLUSIONS: Where urinary MMP-1 is detectable, the patient is more likely to have a bladder tumor of advanced stage or grade and may be at increased risk of disease progression and death of bladder cancer. The relationship between urinary TIMP-1, muscle-invasion, and disease progression in bladder cancer is at variance with its role as an inhibitor of MMPs and warrants additional evaluation.

Impact of polyglutamation on sensitivity to raltitrexed and methotrexate in relation to drug-induced inhibition of de novo thymidylate and purine biosynthesis in CCRF-CEM cell lines.

The aim of this study was to investigate the influence of folylpolyglutamyl synthetase (FPGS) activity on the cellular pharmacology of the classical antifolates raltitrexed and methotrexate (MTX) using two human leukemia cell lines, CCRF-CEM and CCRF-CEM:RC2Tomudex. Cell growth inhibition and drug-induced inhibition of de novo thymidylate and purine biosynthesis were used as measures of the cellular effects of the drugs. CCRF-CEM:RC2Tomudex cells had <11% of the FPGS activity of CCRF-CEM cells, whereas MTX uptake and TS activity were equivalent. In CCRF-CEM:RC2Tomudex cells, MTX polyglutamate formation was undetectable after exposure to 1 microM [3H]MTX for 24 h. After exposure to 0.1 microM raltitrexed, levels of total intracellular raltitrexed-derived material in CCRF-CEM:RC2Tomudex cells were 30- to 50-fold lower than in the CCRF-CEM cell line. CCRF-CEM: RC2Tomudex cells were >1000-fold resistant to raltitrexed and 6-fold resistant to lometrexol but sensitive to MTX and nolatrexed when exposed to these antifolates for 96 h. After 6 h of exposure, CCRF-CEM cells retained sensitivity to MTX and raltitrexed but were less sensitive to lometrexol-mediated growth inhibition. In contrast, CCRF-CEM: RC2Tomudex cells were markedly insensitive to raltitrexed, lometrexol, and to a lesser degree, MTX. Simultaneous measurement of de novo thymidylate and purine biosynthesis revealed 90% inhibition of TS activity by 100 nM MTX in both cell lines, whereas inhibition of de novo purine synthesis was only observed in CCRF-CEM cells, and only after exposure to 1000 nM MTX. Ten nM raltitrexed induced >90% inhibition of TS activity in CCRF-CEM cells, whereas in CCRF-CEM:RC2Tomudex cells, there was no evidence of inhibition after exposure to 1000 nM raltitrexed. These studies demonstrate that polyglutamation is a critical determinant of the cellular pharmacology of both raltitrexed and MTX, markedly influencing potency in the case of raltitrexed and locus of action in the case of MTX.