High STEAP1 expression is associated with improved outcome of Ewing's sarcoma patients.

BACKGROUND: Ewing's sarcoma (ES) is the second most common bone or soft-tissue sarcoma in childhood and adolescence and features a high propensity to metastasize. The six-transmembrane epithelial antigen of the prostate 1 (STEAP1) is a membrane-bound mesenchymal stem cell marker highly expressed in ES. Here, we investigated the role of STEAP1 as an immunohistological marker for outcome prediction in patients with ES. PATIENTS AND METHODS: Membranous STEAP1 immunoreactivity was analyzed using immunohistochemistry in 114 primary pre-chemotherapy ES of patients diagnosed from 1983 to 2010 and compared with clinical parameters and patient outcome. Median follow-up was 3.85 years (range 0.43-17.51). RESULTS: A total of 62.3% of the ES samples displayed detectable STEAP1 expression with predominant localization of the protein at the plasma membrane. High membranous STEAP1 immunoreactivity was found in 53.5%, which correlated with better overall survival (P=0.021). Accordingly, no or low membranous STEAP1 expression was identified as an independent risk factor in multivariate analysis (hazard ratio 2.65, P=0.036). CONCLUSION: High membranous STEAP1 expression predicts improved outcome and may help to define a specific subgroup of ES patients, who might benefit from adapted therapy regimens.

Stable interference of EWS-FLI1 in an Ewing sarcoma cell line impairs IGF-1/IGF-1R signalling and reveals TOPK as a new target.

BACKGROUND: Ewing sarcoma is a paradigm of solid tumour -bearing chromosomal translocations resulting in fusion proteins that act as deregulated transcription factors. Ewing sarcoma translocations fuse the EWS gene with an ETS transcription factor, mainly FLI1. Most of the EWS-FLI1 target genes still remain unknown and many have been identified in heterologous model systems. METHODS: We have developed a stable RNA interference model knocking down EWS-FLI1 in the Ewing sarcoma cell line TC71. Gene expression analyses were performed to study the effect of RNA interference on the genetic signature of EWS-FLI1 and to identify genes that could contribute to tumourigenesis. RESULTS: EWS-FLI1 inhibition induced apoptosis, reduced cell migratory and tumourigenic capacities, and caused reduction in tumour growth. IGF-1 was downregulated and the IGF-1/IGF-1R signalling pathway was impaired. PBK/TOPK (T-LAK cell-originated protein kinase) expression was decreased because of EWS-FLI1 inhibition. We showed that TOPK is a new target gene of EWS-FLI1. TOPK inhibition prompted a decrease in the proliferation rate and a dramatic change in the cell's ability to grow in coalescence. CONCLUSION: This is the first report of TOPK activity in Ewing sarcoma and suggests a significant role of this MAPKK-like protein kinase in the Ewing sarcoma biology.

Suppression of KCMF1 by constitutive high CD99 expression is involved in the migratory ability of Ewing's sarcoma cells.

High CD99 expression levels and rearrangements of the EWS gene with ETS transcription factor genes characterize the Ewing's sarcoma family of tumors (ESFT). CD99 is a cell surface glycoprotein whose engagement has been implicated in cell proliferation as well as upregulation and transport of several transmembrane proteins in hematopoietic cells. In ESFT, antibody ligation of CD99 induces fast homotypic cell aggregation and cell death although its functional role in these processes remains largely unknown. Here, using an RNAi approach, we studied for the first time the consequences of modulated CD99 expression in six different ESFT cell lines, representing the most frequent variant forms of EWS gene rearrangement. CD99 suppression resulted in growth inhibition and reduced migration of ESFT cells. Among genes whose expression changes in response to CD99 modulation, the potassium-channel modulatory factor KCMF1 was consistently upregulated. In a series of 22 primary ESFT, KCMF1 expression levels inversely correlated with CD99 abundancy. Cells forced to express ectopic KCMF1 showed a similar reduction in migratory ability as CD99 silenced ESFT cells. Our results suggest that in ESFT, high CD99 expression levels contribute to the malignant properties of ESFT by promoting growth and migration of tumor cells and identify KCMF1 as a potential metastasis suppressor gene downregulated by high constitutive CD99 expression in ESFT.

Microsatellite instability in Ewing tumor is not associated with loss of mismatch repair protein expression.

Only few clinical factors predict the prognosis of patients with Ewing tumors. Unfavorable outcome is associated with primary metastatic disease, age > 15 years, tumor volume above 200 ml, and the histological response to chemotherapy. The aim of this study was to elucidate the prevalence and clinical impact of microsatellite instability (MSI) together with the relation between MSI and mismatch repair protein expression in Ewing tumors. DNA from 61 primary Ewing tumors and 11 Ewing tumor cell lines was extracted and microsatellite analysis for the detection of instability or loss of heterozygosity was performed for the five markers of the Bethesda panel BAT25, BAT26, D5S346, D2S123, and D17S250, which represents the established marker panel for the analysis of hereditary non-polyposis colorectal carcinoma (HNPCC) patients. In addition, single nucleotide repeat regions of the two tumor genes BAX and transforming growth factor receptor II (TGFBR2) were also included. All of the 61 samples were suitable for LOH analysis and 55 for the determination of MSI-status. LOH of these microsatellite markers was detected in 9 of the 61 patients (14.8%). Over all, genetic instability, i.e. MSI and/or LOH, was detected in 17 tumors (27.9%). One out of the 11 tumor cell lines (STA ET1) was characterized by instability of all the five Bethesda markers, while from primary tumor samples, only one showed MSI in more than one microsatellite marker (D5S346 and D17S250, MSI-high). Eight of the fifty-five patients (14.5%) showed instability of one microsatellite locus (MSI-low). No instability was detected in BAT26, D2S123, BAX and TGFBR2. There was no significant correlation between MSI and loss of expression of mismatch repair proteins MLH1, MSH2, or MSH6. The impairment of the p53 signaling pathway (expression of TP53 and/or MDM2 by immunohistochemistry) was significantly associated with reduced overall survival (15 of 49 patients (30.6%), P = 0.0410, log-rank test). We conclude that MSI is not prevalent in Ewing tumor and that the nature of instability differs from the form observed in colorectal carcinoma, the model tumor of MSI. This is documented by the different pattern of MSI (no BAT26 instability) in Ewing tumors and the lack of a strict correlation between MSI-high and loss of expression of MSH2, MSH6 and MLH1.

Alternative lengthening of telomeres is associated with chromosomal instability in osteosarcomas.

Telomere maintenance is regarded as a key mechanism in overcoming cellular senescence in tumor cells and in most cases is achieved by the activation of telomerase. However there is at least one alternative mechanism of telomere lengthening (ALT) which is characterized by heterogeneous and elongated telomeres in the absence of telomerase activity (TA). We evaluated the prevalence of TA, gene expression of telomerase subunits and ALT in relation to telomere morphology and function in matrix producing bone tumors and in osteosarcoma cell lines and present evidence of a direct association of ALT with telomere dysfunction and chromosomal instability. Telomere fluorescence in situ hybridization (T-FISH) in ALT cells revealed elongated and shortened telomeres, partly in unusual configurations and loci, dicentric marker chromosomes and signal-free chromosome ends. Free ends give rise to end-to-end associations and may induce breakage-fusion-bridge cycles resulting in an increased number of complex chromosomal rearrangements, as detected by multiplex-FISH (M-FISH). We propose that ALT cannot be seen as an equivalent to telomerase activity in telomere maintenance. Its association with telomere dysfunction and chromosomal instability may have major implications for tumor progression.

Telomerase activity and telomerase subunits gene expression patterns in neuroblastoma: a molecular and immunohistochemical study establishing prognostic tools for fresh-frozen and paraffin-embedded tissues.

PURPOSE: We have recently demonstrated that telomerase activity (TA) is an independent prognostic factor in neuroblastomas. In the present study, the prognostic impact of TA and gene expression of the three major telomerase subunits is evaluated by molecular and immunohistochemical techniques in fresh-frozen and paraffin-embedded tissues. PATIENTS AND METHODS: One hundred thirty-three neuroblastomas of all stages were analyzed for TA. The TA levels of 75 neuroblastoma cases were correlated with gene expression of telomerase subunits hTRT, human telomerase RNA (hTR), and telomerase protein 1 (TP1) by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), using an innovative approach on the LightCycler instrument (Roche Diagnostics, Mannheim, Germany). For selected cases, the applicability of RT-PCR and immunohistochemistry for hTRT expression analysis was investigated in paraffin-embedded tissues. TA and subunit expression patterns were correlated with traditional prognostic indicators and disease outcome. RESULTS: TA was present in a total of 39 (29.3%) of 133 neuroblastomas and in 31 (29.8%) of 104 initial neuroblastomas without cytotoxic pretreatment. TA was significantly correlated with both event-free and overall survival (P <.0001). Furthermore, we found a significant correlation between expression levels of TA and hTRT (P <.0001) as well as hTR (P <.001). Multivariate analysis revealed only TA and tumor stage but not serum lactate dehydrogenase, MYCN amplification, or age at diagnosis as independent prognostic factors. CONCLUSION: The significant correlation with clinical outcome strongly recommends that analysis of TA be incorporated into the clinical investigation of each individual neuroblastoma at the time of diagnosis. Because the mere presence or absence of TA without further quantification is sufficient basis for predicting disease outcome, the telomeric repeat amplification protocol assay could be complemented with but not replaced by analysis of hTRT or hTR expression.

Properties of cochlear nucleus neurons in primary culture.

Dissociated primary cell cultures were derived from the cochlear nuclei (CN) of postnatal rats using standard techniques. Cultured cells differentiated morphologically, but their dendritic profiles were generally less specialized than those of CN cells in vivo. Physiologically, cultured cells could be divided into three classes: tonic, phasic and non-spiking cells, which differed in many of their fundamental biophysical properties. The percentage of cultured cells that spiked repetitively increased over time to a maximum of 85% at 6 days. However, the percentage of cells that produced action potentials decreased with time in culture, from 91% during the first 8 days to less than 40% after 9 days. CN cells were successfully cultured in both serum-supplemented and serum-free (Neurobasal) media. More neurons survived at low plating densities in Neurobasal than in medium containing serum, although neuronal survival was similar at higher densities. Few neurons raised in the serum-free medium were spontaneously active; other response properties were similar to those of cells grown in the presence of serum. Although differentiation of CN cells in culture did not completely mirror the in vivo developmental pattern, these experiments demonstrate that primary culture represents a viable method for the in vitro study of CN neurons.

Genetic imbalances in benign bone tumors revealed by comparative genomic hybridization.

There have been no reports on choromosomal aberrations of benign bone tumors revealed by comparative genomic hybridization (CGH). CGH analysis of benign tumors may be useful in understanding the mechanism of tumorigenesis with comparisons to malignant tumors. There were 4 tumors (2 enchondromas, one chondromyxoid fibroma, and one osteoid osteoma) and 8 tumor-like conditions (4 aneurysmal bone cysts (ABCs), one eosinophilic granuloma, one fibrous dysplasia, one solitary bone cyst, and one Rosai-Dorfman disease) available for analysis. One of 2 enchondromas and one of 4 ABCs exhibited rapid growth. Six lesions showed chromosomal aberrations, while 6 others did not. The most frequent aberrations were the loss of a whole chromosome-19 in 6 cases, the loss of chromosome-arm 22q in 4 cases, and the loss of chromosome-arm 17p in 3 cases. Gains were seen in 13q21 in 2 cartilaginous tumors and at 12q15-q21 in eosinophilic granulomas. Therefore, in benign bone tumors or tumor-like lesions, chromosomal aberrations are not frequent; however, some clear tendencies of clustering of aberrations can be observed.

Enteric bacterial protein AvrA promotes colonic tumorigenesis and activates colonic beta-catenin signaling pathway.

Salmonella infections can become chronic and increase the risk of cancer. The mechanisms by which specific Salmonella organisms contribute to cancer, however, are still unknown. Live and attenuated Salmonella are used as vectors to target cancer cells, but there have been no systematic studies of the oncogenic potential of chronic Salmonella infections in cancer models. AvrA, a pathogenic product of Salmonella, is inserted into host cells during infection and influences eukaryotic cell pathways. In the current study, we colonized mice with Salmonella AvrA-sufficient or AvrA-deficient Salmonella typhimirium strains and induced inflammation-associated colon cancer by azoxymethane/dextran sulfate sodium (AOM/DSS). We confirmed Salmonella persisted in the colon for up to 45 weeks. Salmonella was identified not only in epithelial cells on the colonic luminal surface and base of the crypts but also in invading tumors. Tumor incidence in the AvrA+infected group was 100% compared with 51.4% in the AOM/DSS group without bacterial gavage and 56.3% in mice infected with the AvrA- strain. Infection with AvrA+ strain also altered tumor distribution from the distal to proximal colon that might reflect changes in the microbiome. AvrA-expressing bacteria also upregulated beta-catenin signaling as assessed by decreased beta-catenin ubiquitination, increased nuclear beta-catenin and increased phosphorylated-beta-catenin (Ser552), a marker of proliferating stem-progenitor cells. Other ß-catenin targets increased by AvrA included Bmi1, a cancer stem cell marker, matrix metalloproteinase-7, and cyclin D1. In summary, AvrA-expressing Salmonella infection activates ß-catenin signals and enhances colonic tumorigenesis. Our findings provide important new mechanistic insights into how a bacterial protein targets proliferating stem-progenitor cells and contributes to cancer development. Our observations also raise a note of caution regarding the use of mutant Salmonella organisms as vectors for anti-cancer therapy. Finally, these studies could suggest biomarkers (such as AvrA level in gut) to assess cancer risk in susceptible individuals and infection-related dysregulation of ß-catenin signaling in cancer.

1q gain and CDT2 overexpression underlie an aggressive and highly proliferative form of Ewing sarcoma.

Despite extensive characterization of the role of the EWS-ETS fusions, little is known about secondary genetic alterations and their clinical contribution to Ewing sarcoma (ES). It has been demonstrated that the molecular structure of EWS-ETS lacks prognostic value. Moreover, CDKN2A deletion and TP53 mutation, despite carrying a poor prognosis, are infrequent. In this scenario identifying secondary genetic alterations with a significant prevalence could contribute to understand the molecular mechanisms underlying the most aggressive forms of ES.We screened a 67 ES tumor set for copy number alterations by array comparative genomic hybridization. 1q gain (1qG), detected in 31% of tumor samples, was found markedly associated with relapse and poor overall and disease-free survival and demonstrated a prognostic value independent of classical clinical parameters. Reanalysis of an expression dataset belonging to an independent tumor set (n=37) not only validated this finding but also led us to identify a transcriptomic profile of severe cell cycle deregulation in 1qG ES tumors. Consistently, a higher proliferation rate was detected in this tumor subset by Ki-67 immunohistochemistry. CDT2, a 1q-located candidate gene encoding a protein involved in ubiquitin ligase activity and significantly overexpressed in 1qG ES tumors, was validated in vitro and in vivo proving its major contribution to this molecular and clinical phenotype. This integrative genomic study of 105 ES tumors in overall renders the potential value of 1qG and CDT2 overexpression as prognostic biomarkers and also affords a rationale for the application of already available new therapeutic compounds selectively targeting the protein-ubiquitin machinery.

Efficacy of and resistance to anti-IGF-1R therapies in Ewing's sarcoma is dependent on insulin receptor signaling.

Identification of patient selection criteria and understanding of the potential mechanisms involved in the development of resistance are crucial for an appropriate and successful design of clinical trials with anti-insulin-like growth factor (IGF)-1R therapies. Few Ewing's sarcomas are highly sensitive to IGF-1R targeting and understanding the reason why, may hold the secret to improve successful treatments. In this paper, we show that a major mechanism of resistance to highly specific inhibitors of IGF-1R, either antibodies or tyrosine kinase inhibitors may involve enhanced insulin receptor (IR)-A homodimer formation and IGF-2 production. Resistant cells are able to switch from IGF-1/IGF-1R to IGF-2/IR-A dependency to maintain sustained activation of AKT and ERK1/2, proliferation, migration and metastasis. These cells also showed higher proliferative response to insulin, in keeping with a switch towards insulin pathways sustaining proliferation and malignancy, rather than metabolism. Our findings demonstrate a role for IR-A in eliciting intrinsic and adaptive resistance to anti-IGF-1R therapies. Thus, we indicate that tumors with low IGF-1R:IR ratio are unlikely to greatly benefit from anti-IGF-1R therapies and that the efficacy of anti-IGF-1R therapies should be evaluated in relationship to the IR-A:IGF-1R ratio in cancer cells. Moreover, we provide evidences supporting IR-A as an important target in sarcoma therapy.

Hsa-mir-145 is the top EWS-FLI1-repressed microRNA involved in a positive feedback loop in Ewing's sarcoma.

EWS-FLI1 is a chromosome translocation-derived chimeric transcription factor that has a central and rate-limiting role in the pathogenesis of Ewing's sarcoma. Although the EWS-FLI1 transcriptomic signature has been extensively characterized on the mRNA level, information on its impact on non-coding RNA expression is lacking. We have performed a genome-wide analysis of microRNAs affected by RNAi-mediated silencing of EWS-FLI1 in Ewing's sarcoma cell lines, and differentially expressed between primary Ewing's sarcoma and mesenchymal progenitor cells. Here, we report on the identification of hsa-mir-145 as the top EWS-FLI1-repressed microRNA. Upon knockdown of EWS-FLI1, hsa-mir-145 expression dramatically increases in all Ewing's sarcoma cell lines tested. Vice versa, ectopic expression of the microRNA in Ewing's sarcoma cell lines strongly reduced EWS-FLI1 protein, whereas transfection of an anti-mir to hsa-mir-145 increased the EWS-FLI1 levels. Reporter gene assays revealed that this modulation of EWS-FLI1 protein was mediated by the microRNA targeting the FLI1 3'-untranslated region. Mutual regulations of EWS-FLI1 and hsa-mir-145 were mirrored by an inverse correlation between their expression levels in four of the Ewing's sarcoma cell lines tested. Consistent with the role of EWS-FLI1 in Ewing's sarcoma growth regulation, forced hsa-mir-145 expression halted Ewing's sarcoma cell line growth. These results identify feedback regulation between EWS-FLI1 and hsa-mir-145 as an important component of the EWS-FLI1-mediated Ewing's sarcomagenesis that may open a new avenue to future microRNA-mediated therapy of this devastating malignant disease.

Antisense RNA control of gene expression in bacteriophage P22. I. Structures of sar RNA and its target, ant mRNA.

Sar RNA is an antisense RNA that is partly responsible for the negative regulation of antirepressor synthesis during development of bacteriophage P22 (Liao SM et al., 1987, Genes & Dev 1:197-203; Wu Th, Liao SM, McClure WR, Susskind MM, Genes & Dev 1:204-212). The structures of sar RNA and its target, ant mRNA, were probed using limited RNase digestion as a function of Mg2+ concentration. Sar RNA forms two hairpins that are present at all Mg2+ concentrations (Mg2+-independent hairpins). One of the hairpins contains three tandem U x U base pairs. Ant RNA forms three Mg2+-independent hairpins and one Mg2+-dependent hairpin. In addition, many nucleotides in sar RNA and ant RNA appear to be involved in tertiary interactions. The effects of RNA structure on the pairing reaction are considered in the accompanying paper (Schaefer KL, McClure WR, 1997, RNA 3:157-174).

Antisense RNA control of gene expression in bacteriophage P22. II. Kinetic mechanism and cation specificity of the pairing reaction.

The bacteriophage P22 sar RNA-ant mRNA pairing reaction was characterized kinetically. The pairing reaction proceeds by a three-step pathway. First, reversible base pairs form between complementary hairpin loops in sar RNA and ant RNA (Kd = 270 nM). Next, stable duplex formation initiates between single-stranded nucleotides in sar RNA and ant RNA; the ant RNA nucleotides are at the bottom of a hairpin stem that is partially accessible. Concomitant unwinding of one sar RNA hairpin and the complementary ant RNA hairpin then occurs, to form a partially paired intermediate (k2 = 12 min(-1). Finally, a complete duplex forms after unwinding of the other sar RNA hairpin and the complementary ant RNA hairpin (k3 = 7 min(-1). Experiments with sar RNA sequence and length variants demonstrate that the precise structures of both sar RNA hairpins affect the kinetic parameters. The pairing reaction is Mg2+-dependent, and shows high specificity for the required cation. Maximal pairing rates are achieved when more than one Mg2+ ion is bound. The cation-dependence and specificity indicate a requirement for Mg2+-dependent tertiary structure.

Angiotensin II receptor antagonists: the prototype losartan.

OBJECTIVE: To describe a new class of antihypertensive agents, the angiotensin II receptor antagonists, with emphasis on the prototype losartan. Pharmacokinetic data and clinical trials are reviewed, as well as adverse reactions, drug interactions, and dosing guidelines. DATA SOURCES: A MEDLINE search of English-language literature published from 1966 through 1995 was performed. In addition, Merck and Co. provided bibliographic data on file for losartan. STUDY SELECTION: Emphasis was placed on clinical and pharmacokinetic studies in humans. Controlled, double-blind studies were evaluated to assess the efficacy and adverse effect profile of losartan. DATA SYNTHESIS: Losartan is a nonpeptide, competitive antagonist of the type I angiotensin II receptor. In comparative clinical trials, losartan appears to have antihypertensive efficacy similar to that of the angiotensin-converting enzyme (ACE) inhibitors. Losartan is well tolerated, with an adverse effect profile similar to that of placebo and a reduced incidence of cough versus that with ACE inhibitors. A combination product consisting of losartan 50 mg and hydrochlorothiazide 12.5 mg has also received approval for the treatment of hypertension. The combination product is not indicated for initial therapy, but is recommended for patients who do not respond adequately to losartan monotherapy. The angiotensin II receptor antagonists are also being investigated for beneficial effects in patients with ventricular hypertrophy, renal disease, and heart failure. CONCLUSIONS: Losartan, the first angiotensin II receptor antagonist to receive approval for use in the US, appears to be an effective new antihypertensive agent with an adverse effect profile similar to that of placebo. Losartan may be an alternative for patients who cannot tolerate ACE inhibitors. However, the effect of losartan on mortality remains to be evaluated. The role of the angiotensin II receptor antagonists in areas such as ventricular hypertrophy, renal function, and heart failure has yet to be determined.

Alterations in CD4-binding regions of the MHC class II molecule I-Ek do not impede CD4+ T cell development.

The T cell coreceptors CD4 and CD8 enhance T cell responses to TCR signals by participating in complexes containing TCR, coreceptor, and MHC molecules. These ternary complexes are also hypothesized to play a seminal role during T cell development, although the precise timing, frequency, and consequences of TCR-coreceptor-MHC interactions during positive selection and lineage commitment remain unclear. To address these issues, we designed transgenic mice expressing mutant I-Ek molecules with reduced CD4-binding capability. These transgenic lines were crossed to three different lines of I-Ek-specific TCR transgenic mice, and the efficiency of production of CD4+ lineage cells in the doubly transgenic progeny was assessed. Surprisingly, replacing wild-type I-Ek molecules with these mutant molecules did not affect the production of CD4+CD8- thymocytes or CD4+ peripheral T cells expressing any of the three TCRs examined. These data, when considered together with other experiments addressing the role of coreceptor during development, suggest that not all MHC class II-specific thymocytes require optimal and simultaneous TCR-CD4-MHC interactions to mature. Alternatively, it is possible that these particular alterations of I-Ek do not disrupt the CD4-MHC interaction adequately, potentially indicating functional differences between I-A and I-E MHC class II molecules.

Multiple-dose pharmacokinetics of pentoxifylline and its metabolites during renal insufficiency.

OBJECTIVE: To characterize pentoxifylline (PTF) and metabolite disposition after multiple oral doses given two and three times a day to patients with renal dysfunction. DESIGN: An open-label, randomized, crossover, parallel group design. SETTING: Community-based clinical research center. PATIENT POPULATIONS: Subjects with renal function stratified based on 24-hour urinary creatine clearance (Clcr): group I = Clcr > 80 mL/min (n = 9); group II = Clcr 30-80 mL/min (n = 6); and group III = Clcr < 30 mL/min (n = 10). METHODS: PTF 400 mg bid or tid was administered on days 1-7 and 400 mg bid or tid was given on days 14-20 with a 1-week washout. Timed blood samples were taken on days 1, 7, and 20. Blood samples were analyzed for PTF and its metabolites (M-I, M-IV, M-V) by gas-liquid chromatography. MAIN OUTCOME MEASURES: Maximum plasma concentrations (Cmax), time to maximum concentration (tmax), average steady-state plasma concentration (CavgSS), and area under the plasma concentration-time curve at steady-state (AUCSS) were determined by visual and model independent methods. ANOVA, paired t-test, and linear regression were used with significance level set at p < 0.05. RESULTS: The ratio of PTF AUCSS (tid):AUCSS (bid) and M-I AUCSS (bid and tid) were not significantly different between the groups. Significant differences were found in M-IV and M-V Cmax, AUCSS, CavgSS, and AUCSS ratios (M-IV:PTF and M-V:PTF) between renal function groups (p < 0.05 for all). A change in dosage regimen from tid to bid resulted in significant changes in M-IV and M-V CavgSS for subjects with normal renal function and in those with moderate dysfunction, although not in subjects with severe renal dysfunction. CONCLUSIONS: Renal dysfunction did not cause significant accumulations of PTF or M-I after multiple bid and tid dosing, however, M-IV and M-V had significant accumulation in patients with renal impairment. Dosage reduction to 400 mg bid for patients with moderate renal impairment and 200-400 mg/d for severe renal impairment, as well as close clinical monitoring, seem prudent until the complex pharmacologic interactions of PTF and its metabolites can be further delineated.

Monoclonality in normal epithelium and in hyperplastic and neoplastic lesions of the breast.

The clonal nature of neoplastic lesions such as invasive breast cancer and ductal carcinoma in situ (DCIS) has been widely proven by several proliferative, genetic or other malignancy-associated markers. The aim of this study is to clarify whether benign hyperplastic lesions such as ductal hyperplasia of usual type (DH) and papilloma can be distinguished from neoplastic lesions such as DCIS by X-chromosome inactivation analysis. Clonal analysis was performed using a polymerase chain reaction-based assay for non-random X-chromosome inactivation of the human androgen receptor gene (HUMARA). Formalin-fixed and paraffin-embedded archival tissue of ten DCIS, sixteen DH, nine papillomas, and seven normal terminal ductal lobular units (TDLUs) was laser-microdissected to avoid contamination with surrounding tissue. All of the cases analysed revealed a monoclonal origin. Furthermore, in one of these cases, opposite X chromosomes were inactivated within the same breast. X-linked inactivation analysis clearly demonstrates that, at least in the breast, monoclonality is not restricted to neoplastic processes. The data support the hypothesis that the mammary gland is organized into distinct stem cell-derived monoclonal patches and that TDLUs are monoclonal in origin. Any proliferative lesion arising within such a pre-existing clonal patch should therefore be clonal, irrespective of whether it originates from one or more patch cells. Thus, X-chromosome inactivation analysis cannot be considered a valid method for distinguishing between neoplastic and hyperplastic breast lesions.

Traditional and emerging molecular markers in neuroblastoma prognosis: the good, the bad and the ugly.

BACKGROUND: Neuroblastomas (NB) are a heterogeneous group of childhood tumours with a wide range of likelihood for tumour progression. As traditional parameters do not ensure completely accurate prognostic grouping, new molecular markers are needed for assessing the individual patient's prognosis more precisely. PATIENTS AND METHODS: 133 NB of all stages were analysed in blind-trial fashion for telomerase activity (TA), expression of surviving, and MYCN status. These data were correlated with other traditional prognostic indicators and disease outcome. RESULTS AND CONCLUSIONS: TA is a powerful independent prognostic marker for all stages and is capable of differentiating between good and poor outcome in putative "favourable" clinical or biological subgroups of NB patients. High surviving expression is associated with an adverse outcome, but is more difficult to interprete than TA because survivin expression needs to be accurately quantified to be of predictive value. We propose an extended progression model for NB including emerging prognostic markers, with emphasis on telomerase activity.