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Supplementary Tables 1-2 from Gene Expression Profiles Associated with Treatment Response in Oligodendrogliomas

Supplementary Table 3 from Gene Expression Profiles Associated with Treatment Response in Oligodendrogliomas

Oligodendrogliomas are a specific subtype of brain tumor of which the majority responds favorably to chemotherapy. In this study, we made use of expression profiling to identify chemosensitive oligodendroglial tumors. Correlation of expression profiles to loss of heterozygosity on 1p and 19q, common chromosomal aberrations associated with response to treatment, identified 376, 64, and 60 differentially expressed probe sets associated with loss of 1p, 19q or 1p, and 19q, respectively. Correlation of expression profiles to the tumors' response to treatment identified 16 differentially expressed probe sets. Because transcripts associated with chemotherapeutic response were identified independent of common chromosomal aberrations, expression profiling may be used as an alternative approach to the tumors' 1p status to identify chemosensitive oligodendroglial tumors. Finally, we correlated expression profiles to survival of the patient after diagnosis and identified 103 differentially expressed probe sets. The observation that many genes are differentially expressed between long and short survivors indicates that the genetic background of the tumor is an important factor in determining the prognosis of the patient. Furthermore, these transcripts can help identify patient subgroups that are associated with favorable prognosis. Our study is the first to correlate gene expression with chromosomal aberrations and clinical performance (response to treatment and survival) in oligodendrogliomas. The differentially expressed transcripts can help identify patient subgroups with good prognosis and those that will benefit from chemotherapeutic treatments. (Cancer Res 2005; 65(24): 11335-44)

Alcohol-free beers have gained popularity in the last few decades because they provide a healthier alternative to alcoholic beers and can be more widely consumed. Consumers are becoming more aware of the benefits of reducing their alcohol consumption, and this has increased the sales of nonalcoholic alternatives. However, there are still many challenges for the brewing industry to produce an alcohol-free beer that resembles the pleasant fruity flavor and overall sensory experience of regular beers. The aim of this review is to give a comprehensive overview of alcohol-free beer focusing on aroma chemistry. The formation of the most important aroma compounds, such as Strecker aldehydes, higher alcohols, and esters, is reviewed, aiming to outline the gaps in current knowledge. The role of ethanol as a direct and indirect flavor-active compound is examined separately. In parallel, the influence of the most common methods to reduce alcohol content, such as physical (dealcoholization) or biological, on the organoleptic characteristics and consumer perception of the final product, is discussed.

The increasing popularity of alcohol-free beers (AFBs) fosters the industry interest in delivering the best possible product. Yet, a remaining sensory defect of AFBs is the over-perception of wort flavour, caused by elevated concentrations of small volatile flavour compounds (i.e. aldehydes). Previously, molecular sieves (hydrophobic ZSM-5 type zeolites) were found most suitable to remove these flavours by adsorption with high selectivity from the AFBs. In this work, a flavour-improved beer is produced at pilot-scale using this novel technology, and its chemical composition, sensory profile and stability are evaluated against a reference. Aldehyde concentrations in the flavour-improved product were found 79–93% lower than in the reference. The distinct difference was confirmed with a trained sensory panel and could be conserved even after three months ageing at 30 °C. Future work will focus on the process design to scale up this technology.

Alcohol-free beers (AFBs) brewed by cold-contact fermentation exhibit a flavor reminiscent of wort which affects consumer acceptability. The aims of this study were to identify the odor-active compounds in AFB and elucidate the contribution of these to the overall aroma and worty character of the beer. Using a sensomics approach, 27 odor-active aroma compounds were identified and quantitated using gas chromatography–mass spectrometry. The most odor-active compound was methional (boiled potato-like aroma), followed by 3-methylbutanal (cocoa-like), (E)-β-damascenone (apple, jam-like), 5-ethyl-3-hydroxy-4-methyl-2(5H)-furanone (curry, spicy-like), and phenylacetaldehyde (floral, honey-like). The important contribution of these flavor compounds to the worty and honey aroma of AFB was determined by sensory assessment of the recombinate in a beer-like matrix with omission tests. The role of 5-ethyl-3-hydroxy-4-methyl-2(5H)-furanone in AFB aroma was reported for the first time. The outcomes from this study are of relevance for the brewing industry to design strategies for the reduction of the wortiness of AFB.

A common sensory deficiency of alcohol-free beers (AFB) is caused by the presence of worty aldehydes. The aim of this study was to develop and proof the concept of a selective adsorption step, facilitating the removal of aldehydes from AFB. Therefore, the performance of 21 adsorbents (amine-functionalized polymers, hydrophobic resins and zeolites) was tested in wort. Among the studied adsorbents, hydrophobic ZSM-5 type zeolites (CBV28014, HiSiv3000 and ZSM-5 P-360) showed the best selectivity due to their 2-dimensional separation characteristics. Consequently, the obtained multicomponent isotherms in unhopped AFB revealed a linear adsorption behavior for all aldehydes, indicating non-competitive adsorption within the design space. The logarithms of the adsorption affinity constants were found to be linearly correlated to the compounds’ hydrophobicity and solubility. The concept was proven at pilot scale of 150 L, resulting in a reduction of aldehydes between 43.7–70.2 %, while conserving bitterness, pH and color of the AFB. Future work will focus on the sensory evaluation of the flavor-improved product.

Although present in concentrations in microgrammes per litre level, aldehydes, in particular those derived from Strecker degradation, are known to majorly contribute to the undesired wort flavour of alcohol-free beers. In order to improve currently available products, one needs to understand the underlying cause for the over-prevalence and identify leverage points and methods to selectively reduce the aldehydes in alcohol-free beers. This work gives a short overview on relevant flavour-active wort flavours identified in alcohol-free beer and on their involved chemical formation pathways. Consequently, aldehyde removal technologies in general and in brewing industry are presented. Adsorptive removal of off-flavours by aldehyde-scavenging groups is already widely exploited in the packaging industry and may achieve reduction of these components to near depletion, depending on the process conditions. Its principles are adaptable to recovering off-flavours before filling. Also, supercritical CO2extraction has been successfully applied to separate flavours from food matrices. In brewing, the focus has been set to biologic conversion by restricted fermentation steps, but the reduction of key components of more than 70% is not achieved. Newer developments focus on thermal separation techniques that not only include non-specific physical dealcoholisation but also more selective technologies such as pervaporation, where aldehydes are reduced to near depletion. However, for most unit operations, selectivity and capacity are not yet investigated. Future research should explore the shortcomings of current techniques and overcome bottlenecks either by developing more specific methods for aldehyde removal and/or a clever combination of unit operations to optimise the separation and process integration.

Detection thresholds are used routinely to determine the odour-active compounds in foods. The composition of a food matrix, such as hydrophobicity or solids content, has an impact on the release of flavour compounds, and thus on thresholds. In the case of beer, thresholds determined in alcoholic beer may not be the same for alcohol-free beer (AFB). Therefore, the aim of this study was to determine detection thresholds for aroma compounds typically found in beer, within a model AFB. The model was designed to match the sugar concentration and pH of an AFB brewed by a cold contact process. Thresholds were measured using a 3-AFC procedure and calculated using either Best Estimate Threshold (BET) method or by logistic regression. Moreover, an algorithm for the removal of false positives was applied to adjust the assessors' raw responses. Retronasal thresholds were generally lower than orthonasal. Those calculated by BET were significantly higher (p

peer-reviewed The full text of this article will not be available in ULIR until the embargo expires on the 20/05/2021 BACKGROUND: Recently, a new process concept has been proposed to selectively adsorb wort offflavours, i.e. aldehydes, from alcohol-free beers with hydrophobic zeolites. RESULTS: In this work, we investigated the uptake of a mixture of wort flavour compounds (2-methylpropanal, 2-methylbutanal, 3methylbutanal, furfural and methional), from a model solution onto binderless, hydrophobic ZSM-5 zeolite granules in order to quantify mass transfer parameters and identify bottlenecks. Subsequently, the homogenous solid diffusion model was employed to regress the effective diffusion coefficients for each molecule and experimental condition, which ranged between 10-15 and 10-13 m2/s, indicating strong intraparticle mass transfer limitation. Furthermore, it was found that the effective diffusion coefficient is inversely correlated to the molecules’ hydrophobicity, expressed as the logD value and its isotherm affinity constant. CONCLUSION: These results give valuable insight to design and improved adsorbent material and an off-flavour removal unit for industrial scale.

Bitcoin, the decentralised peer-to-peer payment system equips us with a new revolutionary financial tool to send codified value across the globe. As a peer-to-peer payment network, however, bitcoin’s decentralised characteristics make it significantly challenging for regulators to influence bitcoin governance. With legacy regulation insufficiently commanding control over the bitcoin network, regulatory debate has shifted towards finding appropriate regulatory solutions for mitigating bitcoin risk crystallisation. Stark academic regulatory analysis on bitcoin exchange risk has created a profound gap in the literature and sets a logical starting point for this thesis. Using a technical risk-based methodology, I analyse bitcoin exchange risk under three broad headings: consumer protection, financial stability, and anti-money laundering and terrorist financing. Assessing objective empirical evidence, I argue that contrary to mainstream regulatory opinion, the most salient bitcoin exchange risk pertains to consumer protection. From theft, fraud, embezzlement, hacking, and various other scandals, the bitcoin exchange marketplace, for the most part, constitutes as a 'wild west' environment. Inadequate wallet storage mechanisms, insecure cyber security, and deficient capital adequacy requirements, to name but a few, consistently engender risk and harm consumers. As a result, I argue that the slow response from European regulators to address these issues calls for a reevaluation of risk appraisal and a need for a more robust consumer oriented regulatory structure. In that manner, I posit for a 'consumer by design' regulatory framework that inter alia emphasises cold wallet infrastructure, holistic cyber security measures, and capital adequacy requirements. The focal point of this approach centers on changing predominantly malevolent and negligent bitcoin exchange behaviors to that of a prudentially minded entity. Hard targeted risk-based rules fabricate this change, which coincides with post 2008 crises consensus for more financial regulation and the proposition that markets benefit from effective regulatory architecture.

Background An automated multiplex platform using capillary blood can promote greater throughput and more comprehensive studies in celiac disease (CD). Diagnostic accuracy should be improved using likelihood ratios for the post-test probability of ruling-in disease. Methods The Ig_plex™ Celiac Disease Panel on the sqidlite™ automated platform measured IgA and IgG antibodies to tTG and DGP in n = 224 CD serum or plasma samples. Diagnostic accuracy metrics were applied to the combined multiplex test results for several CD populations and compared to conventional single antibody ELISA tests. Results With multiple positive antibody results, the post-test probability for ruling-in untreated and treated CD increased to over 90%. The number of samples positive for more than one antibody also increased in untreated CD to ≥90%. Measurement of all four CD antibodies generate cut-off dependent accuracy profiles that can monitor response to treatment with the gluten-free diet (GFD). Higher positive tTG and DGP antibodies are seen more frequently in confirmed CD without (81%–94%) than with GFD treatment (44%–64%). In CD lacking biopsy confirmation, overall agreement of plasma to serum was ≥98% for all antibodies, and 100% for venous to capillary plasma. Conclusions The Ig_plex Celiac Disease Panel increases the likelihood of confirming CD based on the post-test probability of disease results for multi-reactive markers. Specific positivity profiles and cut-off intervals can be used to monitor GFD treatment and likely disease progression. Using serum, venous and capillary plasma yield comparable and accurate results.

peer-reviewed Flavor-active esters, produced during fermentation, are vital components and important contributors to the aroma of beer. In order to separate trace amounts of esters, their adsorption behavior in the presence of high concentrations of ethanol and their thermodynamic behavior under the influence of temperature needs to be understood. This study reports the influence of temperature on single component adsorption isotherms of four esters (i.e. ethyl acetate, isopentyl acetate, ethyl 4-methylpentanoate, and ethyl hexanoate) on two hydrophobic resins (i.e. Amberlite XAD16N, and Sepabeads SP20SS) and the estimation of heat, entropy, and Gibbs energy of adsorption. Higher heat and entropy of adsorption are obtained for ethyl hexanoate and ethyl 4-methylpentanoate in comparison, due to their higher hydrophobicity, stronger binding, and the exothermic nature of their adsorption. A higher concentration of ethanol (tested from 1 to 30% (v/v)), lowers the activity coefficient of esters in the aqueous phase, and subsequently lowers adsorption and Langmuir affinity parameters. Increase of temperature from 284.15 to 325.15 K shows a reverse influence on maximum adsorption capacity and Langmuir affinity parameters. Langmuir affinity parameters are obtained at various ethanol concentrations and temperatures. The reported parameters and thermodynamic properties in this paper, are essential for designing an industrial scale adsorption step for separation of flavor-active esters under non-isothermal conditions.

GC-MS/O (gas chromatography-mass spectrometry/olfactometry) is an indispensable technique to associate individual volatile odorants to odors perceived by human assessors. Interpretation of GC-MS/O data is, however, hampered in practice by different factors related to the instrumental set-up and by heterogeneity among odor descriptions given by the assessors (olfactometer). In this paper, a novel automated approach is presented, which deals with these GC-MS/O challenges and enables visualization and interpretation of GC-MS/O data. It includes signal warping via COW (correlation optimized warping), synchronizing MS and O data via detection of odor areas and construction of a TOC (total odor count) to visualize odor heterogeneity, respectively. Our approach is implemented in practice, and we successfully associated odors to compounds in data sets of two alcoholic beverages with different flavor compositions. It leads to a faster and less biased association of odors to compounds compared to current practice, reducing the time and effort needed for interpreting GC-MS/O data.

Paraneoplastic neurological syndromes (PNS) are devastating neurological disorders secondary to cancer, associated with onconeural autoantibodies. Such antibodies are directed against neuronal antigens aberrantly expressed by the tumor. The detection of onconeural antibodies in a patient is extremely important in diagnosing a neurological syndrome as paraneoplastic (70% is not yet known to have cancer) and in directing the search for the underlying neoplasm. At present six onconeural antibodies are considered 'well characterized' and recognize the antigens HuD, CDR62 (Yo), amphiphysin, CRMP-5 (CV2), NOVA-1 (Ri), and Ma2. The gold standard of detection is the characteristic immunohistochemical staining pattern on brain tissue sections combined with confirmation by immunoblotting using recombinant purified proteins. Since all six onconeural antibodies are usually analyzed simultaneously and objective cut-off values for these analyses are warranted, we developed a multiplex assay based on Luminex technology. Reaction of serial dilutions of six onconeural standard sera with microsphere-bound antigens showed lower limits of detection than with Western blotting. Using the six standard sera at a dilution of 1:200, the average within-run coefficient of variation (CV) was 4% (range 1.9-7.3%). The average between-run within-day CV was 5.1% (range 2.9-6.7%) while the average between-day CV was 8.1% (range 2.8-11.6%). The shelf-life of the antigen coupled microspheres was at least two months. The sensitivity of the multiplex assay ranged from 83% (Ri) to 100% (Yo, amphiphysin, CV2) and the specificity from 96% (CV2) to 100% (Ri). In conclusion, Luminex-based multiplex serology is highly reproducible with high sensitivity and specificity for the detection of onconeural antibodies. Conventional immunoblotting for diagnosis of onconeural antibodies in the setting of a routine laboratory may be replaced by this novel, robust technology.

This work aims to propose an optimum resin that can be used in industrial adsorption process for tuning flavor-active components or removal of ethanol for producing an alcohol-free beer. A procedure is reported for selective adsorption of volatile aroma components from water/ethanol mixtures on synthetic hydrophobic resins. High throughput 96-well microtiter-plates batch uptake experimentation is applied for screening resins for adsorption of esters (i.e. isoamyl acetate, and ethyl acetate), higher alcohols (i.e. isoamyl alcohol and isobutyl alcohol), a diketone (diacetyl) and ethanol. The miniaturized batch uptake method is adapted for adsorption of volatile components, and validated with column breakthrough analysis. The results of single-component adsorption tests on Sepabeads SP20-SS are expressed in single-component Langmuir, Freundlich, and Sips isotherm models and multi-component versions of Langmuir and Sips models are applied for expressing multi-component adsorption results obtained on several tested resins. The adsorption parameters are regressed and the selectivity over ethanol is calculated for each tested component and tested resin. Resin scores for four different scenarios of selective adsorption of esters, higher alcohols, diacetyl, and ethanol are obtained. The optimal resin for adsorption of esters is Sepabeads SP20-SS with resin score of 87% and for selective removal of higher alcohols, XAD16N, and XAD4 from Amberlite resin series are proposed with scores of 80 and 74% respectively. For adsorption of diacetyl, XAD16N and XAD4 resins with score of 86% are the optimum choice and Sepabeads SP2MGS and XAD761 resins showed the highest affinity towards ethanol.

Autoantibodies are increasingly used as biomarkers in the detection of autoimmune disorders and cancer. Disease specific antibodies are generally detected by their binding to specific antigens. As an alternative approach, we propose to identify specific complementarity determining regions (CDR) of IgG that relate to an autoimmune disorder or cancer instead of the specific antigen(s). In this manuscript, we tested the technical feasibility to detect and identify CDRs of specific antibodies by mass spectrometry. We used a commercial pooled IgG preparation as well as purified serum IgG fractions that were spiked with different amounts of a fully human monoclonal antibody (adalimumab). These samples were enzymatically digested and analyzed by nanoLC Orbitrap mass spectrometry. In these samples, we were able to identify peptides derived from the CDRs of adalimumab. These peptides could be detected at an amount of 110 attomole, 5 orders of magnitude lower than the total IgG concentration in these samples. Using higher energy collision induced dissociation (HCD) fragmentation and subsequent de novo sequencing, we could successfully identify 50% of the detectable CDR peptides of adalimumab. In addition, we demonstrated that an affinity purification with anti-dinitrophenol (DNP) monoclonal antibody enhanced anti-DNP derived CDR detection in a serum IgG background. In conclusion, specific CDR peptides could be detected and sequenced at relatively low levels (attomole-femtomole range) which should allow the detection of clinically relevant CDR peptides in patient samples. Electronic supplementary material The online version of this article (doi:10.1007/s00216-010-4361-9) contains supplementary material, which is available to authorized users.

The Luminex system is a flow cytometry based tool that permits the simultaneous measurement of many analytes from just a single serum sample. The technology uses microspheres, which are available in different colors and can be coated with different kinds of biomolecules. For the immobilisation of His-tagged proteins, two types of beads can be used: chemically activated carboxylated beads or Penta-His beads, which have antibodies against His-tags on their surface. In this study, we compared carboxylated and Penta-His beads. For carboxylated as compared to Penta-His beads, the non-specific background is lower (Median Fluorescence Intensity; MFI > 250, 0% versus 15%), the specific signal intensity is higher (mean MFI 2860 versus 722) and not dependent on the configuration of the protein. Above all, the protein coupled carboxylated beads are useful over longer periods of time. Therefore, we conclude that for developing a multiplex assay for semi-quantitative measurement of antibody responses against His-tagged proteins the best microspheres to use are the carboxylated ones. (C) 2008 Elsevier B.V. All rights reserved.

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein abundantly expressed in malignant gliomas. We have constructed a novel oncolytic adenovirus, Ad5-gfa2(B)3-E1, for treatment of these tumors. In this construct, the E1 region is under control of the tissue-specific GFAP promoter (gfa2) with three additional copies of the glial specific ‘B’ enhancer. Infection of a GFAP-positive cell line with Ad5-gfa2(B)3-E1 resulted in E1A and E1B expression at 75% and 30% of the levels obtained after wtAd5 infection. Q-PCR showed that Ad5-gfa2(B)3-E1 replicated 4.5 times more efficiently in the GFAP-positive than in the GFAP-negative cell lines. Cell viability assays showed efficient elimination of GFAP-positive cells by Ad5-gfa2(B)3-E1, in some cell lines as efficiently as wtAd5, while the elimination was attenuated in GFAP-negative cell lines. When tested in human tumor xenografts in nude mice, Ad5-gfa2(B)3-E1 effectively suppressed the growth of GFAP-positive SNB-19 glial tumors but not of GFAP-negative A549 lung tumors. In Ad5-gfa2(B)3-E1, the E3 region was deleted to create space for future insertion of heterologous therapeutic genes. Experiments with dl7001, an E3-deleted variant of wtAd5, confirmed that the specificity of Ad5-gfa2(B)3-E1 replication was based on the promoter driving E1 and not on the E3 deletion. Strategies to further improve the efficacy of Ad5-gfa2(B)3-E1 for the treatment of malignant gliomas include the insertion of therapeutic genes in E3 or retargeting to receptors that are more abundantly expressed on primary glioma cells than CAR. Copyright © 2007 John Wiley & Sons, Ltd.

Lurtotecan (GI147211; LRT) is a semisynthetic and water-soluble analogue of the topoisomerase I inhibitor camptothecin. To determine whether the therapeutic efficacy of LRT in patients could be improved, the drug was encapsulated in liposomes (NX211; Gilead Sciences). In order to allow accurate description of the pharmacokinetic behavior of NX211 in cancer patients, we have developed sensitive RP-HPLC assays with fluorescence detection (lambdaex=378 nm; lambdaem=420 nm) for the determination of total LRT levels in human plasma and urine. Sample pretreatment involved deproteinization with 10% (w/v) aqueous perchloric acid-acetonitrile (2:1, v/v), and chromatographic separations were achieved on an Inertsil-ODS 80A analytical column. The lower limit of quantitation (LLQ) was established at 1.00 ng/ml in plasma (200-microl sample) and at 100 ng/ml in urine (200 microl of 40-fold diluted sample). The within-run and between-run precisions were7.5%. LRT concentrations in urine of100 ng/ml were determined by a modified procedure comprising a single solvent extraction with n-butanol-diethyl ether (3:4, v/v). In this assay, the fluorescence signal of LRT was increased 14-fold prior to detection by post-column exposure to UV light (254 nm) in a photochemical reaction unit. The LLQ of this assay was 0.500 ng/ml (150-microl sample) and the within-run and between-run precisions were10%.

Cremophor EL (CrEL) is a polyoxyethylated castor oil surfactant used in the intravenous formulation of the anticancer drug paclitaxel (Taxol). Quantitative determination of CrEL in patient samples can be achieved by complexation of the compound with the Coomassie brilliant blue G-250 dye in protein-free extracts [Sparreboom, A., Loos, W. J., Verweij, J., De Vos, A. I., Van der Burg, M. E. L., Stoter, G., and Nooter, K., Anal. Biochem. 255, 171-175 (1998)]. A disadvantage of this method of CrEL determination is that the assay plot of absorbance at 595 nm, the peak wavelength of the CrEL-dye complex, versus the concentration of the surfactant is not linear. The present study shows that the nonlinearity is associated with a decrease in the free dye concentration and a reduction in complex formation by increasing the CrEL concentration. By measurement of the ratio of absorbances at the maxima of the red (450 nm) and blue charge forms (595 nm) of Coomassie brilliant blue G-250, a full-scale linear relationship can be obtained over the entire range studied (0.500 to 10.0 microliter/mL). Validation data revealed that transformation of the detection procedure exhibits significantly improved specificity, accuracy(/= 6.33% relative error), and precision (10.0%) compared to our previous assay. The modified method was successfully applied to the measurement of CrEL in plasma of 11 cancer patients treated with a 1-h infusion of paclitaxel.

Paraneoplastic neurological syndromes (PNS) are severe immune mediated effects of cancer. The presence of IgG autoantibodies against onconeural antigens in serum is a hallmark of the disease. Multiple paraneoplastic antibodies have been described, including antibodies against HuD, Yo, amphiphysin and CV2. In this study, we test the hypothesis that primary amino-acid structures of the antigen binding part of antibodies from various individuals share common sequences that are specific for each auto-antigen. We selected 60 patients with PNS, associated with antibodies against HuD, Yo, Amp or CV2. Affinity purified IgG was separated using SDS-PAGE and IgG heavy chains were excised, trypsinized and subjected to tandem mass spectrometry. We selected masses that uniquely identified a PNS autoantibody group, and used MS/MS fragmentation spectra to obtain information on peptide sequences. Out of 19,173 unique masses, 28 immunoglobulin-derived peptides were found exclusively in samples from a single autoantibody defined PNS group. Our results confirm that specific peptide structures exist in the antigen binding site of IgG that are shared between individuals harboring autoantibodies against the same onconeural antigen. Thus, the immune response in these patients followed converging paths during the rearrangement, selection and maturation of immunoglobulin sequences. The identified peptides can be applied in the diagnosis of PNS, but these data also indicate that a similar approach in a variety of other diseases involving an immune response would have an appealing outlook.

Clinical trials in malignant glioma have demonstrated excellent safety of recombinant adenovirus type 5 (Ad5) but lack of convincing efficacy. The overall low expression levels of the Coxsackie and Adenovirus receptor and the presence of high anti-Ad5-neutralizing antibody (NAb) titers in the human population are considered detrimental for consistency of clinical results. To identify an adenoviral vector better suited to infect primary glioma cells, we tested a library of fiber-chimeric Ad5-based adenoviral vectors on 12 fresh human glioma cell suspensions. Significantly improved marker gene expression was obtained with several Ad5-chimeric vectors, predominantly vectors carrying fiber molecules derived from B-group viruses (Ad11, Ad16, Ad35 and Ad50). We next tested Ad35 sero prevalence in sera derived from 90 Dutch cancer patients including 30 glioma patients and investigated the transduction efficiency of this vector in glioma cell suspensions. Our results demonstrate that the sero prevalence and the titers of NAb against Ad35 are significantly lower than against Ad5. Also, recombinant Ad35 has significantly increased ability to transfer a gene to primary glioma cells compared to Ad5. We thus conclude that Ad35 represents an interesting candidate vector for gene therapy of malignant glioma.

The overall median survival of patients with a malignant glioma is1 y. Because malignant gliomas rarely metastasize outside the skull, locoregional treatment strategies, such as gene therapy, are under investigation. Recently, convection-enhanced delivery (CED) has been presented as a method to improve delivery of large molecules. The goal of this study was to evaluate whether CED improves intratumoral delivery of adenoviral vectors and compare it with single injection (SI) and multiple injection (4x, MI).A replication-deficient adenoviral vector encoding the herpes simplex virus thymidine kinase (HSV-tk) and the human somatostatin receptor subtype 2 (sst(2)) was administered into nude mice bearing subcutaneous U87 xenografts. Tumors were injected with 1.5 x 10(9) plaque-forming units of Ad5.tk.sstr by CED, SI, or MI. Three days later, [(99m)Tc-N(4)(0-1),Asp(0),Tyr(3)]octreotate ((99m)Tc-Demotate 2) was injected intravenously to monitor the virus-induced sst(2) expression. gamma-Camera imaging was performed for in vivo imaging, and the tumor uptake of (99m)Tc-Demotate 2 was determined by gamma-counter. Furthermore, the tumor was sectioned and ex vivo autoradiography was performed. After decay of radioactivity, adjacent sections were submitted to in vitro autoradiography with (125)I-DOTA-Tyr(3)-octreotate, which was used to calculate the transduced areas.Transfected xenograft tissues showed high sst(2) expression and were clearly visualized with a gamma-camera. Accumulation of radioactivity was 2-fold higher in the tumors that were injected with MI compared with CED and SI (P = 0.01). CED and SI resulted in equal uptake of radioactivity in the tumors. The measured areas of transduction in ex vivo and in vitro autoradiographs showed a high concordance (r(2) = 0.89, P0.0001). The maximum area of transfection was significantly larger after MI than after CED (P0.05) or SI (P = 0.05). Also, the measured volume of distribution was twice as high after administration of Ad5.tk.sstr by MI (56.6 mm(3)) compared with SI (25.3 mm(3)) or CED (26.4 mm(3)).CED does not increase adenoviral vector distribution in a glioma xenograft model compared with SI. Therefore, in the clinic MI is probably the most effective delivery method for the large adenoviral particle (70 nm) in malignant gliomas.

Oligodendrogliomas are a specific subtype of brain tumor of which the majority responds favorably to chemotherapy. In this study, we made use of expression profiling to identify chemosensitive oligodendroglial tumors. Correlation of expression profiles to loss of heterozygosity on 1p and 19q, common chromosomal aberrations associated with response to treatment, identified 376, 64, and 60 differentially expressed probe sets associated with loss of 1p, 19q or 1p, and 19q, respectively. Correlation of expression profiles to the tumors' response to treatment identified 16 differentially expressed probe sets. Because transcripts associated with chemotherapeutic response were identified independent of common chromosomal aberrations, expression profiling may be used as an alternative approach to the tumors' 1p status to identify chemosensitive oligodendroglial tumors. Finally, we correlated expression profiles to survival of the patient after diagnosis and identified 103 differentially expressed probe sets. The observation that many genes are differentially expressed between long and short survivors indicates that the genetic background of the tumor is an important factor in determining the prognosis of the patient. Furthermore, these transcripts can help identify patient subgroups that are associated with favorable prognosis. Our study is the first to correlate gene expression with chromosomal aberrations and clinical performance (response to treatment and survival) in oligodendrogliomas. The differentially expressed transcripts can help identify patient subgroups with good prognosis and those that will benefit from chemotherapeutic treatments. (Cancer Res 2005; 65(24): 11335-44)

Nonlinear disposition of paclitaxel (Taxol) in cancer patients has been described in several studies, but the underlying mechanism is still a matter of speculation. Previously, we have shown in vitro that the paclitaxel formulation vehicle, Cremophor EL (CrEL), alters the blood distribution of paclitaxel as a result of entrapment of the compound in circulating CrEL micelles, thereby reducing the free drug fraction available for cellular partitioning. Based on these findings, we prospectively re-evaluated the linearity of paclitaxel disposition in patients using whole blood and plasma analysis, and sought to define a new pharmacokinetic model to describe the data. Seven patients with solid tumors were treated with paclitaxel infused over 3 h, each at consecutive 3-weekly dose levels of 225, 175 and 135 mg/m2 (CrEL dose level, 18.8, 14.6, and 11.3 ml/m2, respectively). Patient samples were collected up to 24 h after the start of infusion, and analyzed by high-performance liquid chromatography. Paclitaxel peak levels and areas under the curve in whole blood increased linearly with dose, whereas plasma levels showed substantial deviation from linearity. This was shown to be caused by a CrEL concentration-dependent decrease in paclitaxel uptake in blood cells, as reflected by the blood:plasma concentration ratios which altered significantly from 0.83 +/- 0.11 (at 135 mg/m2) to 0.68 +/- 0.07 (at 225 mg/m2). It is concluded that the nonlinear disposition of paclitaxel is related to paclitaxel dose-related levels of the formulation vehicle CrEL, leading to a disproportionate drug accumulation in the plasma fraction. The pharmacokinetic model developed accurately described the data, and will help guide future development and refinement of clinical protocols, especially in defining the exposure measure best linked to paclitaxel effects and toxicities.

Gender-dependent differences in the clinical pharmacokinetic behavior of various drugs have been documented previously. Most commonly, these differences are associated with differences in body composition, renal elimination, drug absorption or hepatic metabolism. Gender-dependent differences in the pharmacokinetics of topotecan (Hycamtin®) have not yet been described. In this report, pharmacokinetic data of the lactone and carboxylate forms of topotecan were derived from clinical studies in which topotecan was administered either orally or i.v. to a total of 55 males and 37 females. A significant difference (p=0.0082) of 38% was found between the apparent clearance of topotecan lactone after oral administration in males (237±105 I/h) and females (163±62.5 I/h). When adjusted for body surface area, this difference remained significant (p=0.031). Similarly, differences were noted in the percentage of topotecan in the lactone form (37.1±5.32 versus 41.7±6.51%, p=0.0076). Statistical analysis revealed that individual hematocrit values, which were consistently lower in females (p

PURPOSE: In in vitro studies, synergism and sequence-dependent effects were reported for the combination of topotecan and cisplatin. Recently, an oral formulation of topotecan became available. This phase I study was performed to assess the feasibility of the combination of oral topotecan and cisplatin, the pharmacokinetic interaction, and sequence-dependent effects. PATIENTS AND METHODS: Topotecan was administered orally (PO) daily for 5 days in escalating doses and cisplatin was given intravenously (IV) at a fixed dose of 75 mg/m2 either before topotecan administration on day 1 (sequence CT) or after topotecan administration on day 5 (sequence TC) once every 3 weeks. Patients were treated in a randomized cross-over design. RESULTS: Forty-nine patients were entered onto the study; one patient was not eligible. Sequence CT induced significantly more severe myelosuppression than did sequence TC, and the maximum-tolerated dosage of topotecan in sequence CT was 1.25 mg/m2/d × 5. In sequence TC, the maximum-tolerated dosage of topotecan was 2.0 mg/m2/d × 5. Dose-limiting toxicity consisted of myelosuppression and diarrhea. Pharmacokinetics of topotecan and cisplatin were linear over the dose range studied; no sequence-dependent effects were observed. In addition, topotecan did not influence the protein binding of cisplatin or the platinum-DNA adduct formation in peripheral leukocytes in either sequence. CONCLUSION: The recommended dosages for phase II studies involving patients like the patients in our study are topotecan 1.25 mg/m2/d PO × 5 preceded by cisplatin 75 mg/m2 IV day 1 once every 3 weeks, and topotecan 2.0 mg/m2/d PO followed by cisplatin 75 mg/m2 IV day 5. No pharmacokinetic interaction could be discerned in our study. The antitumor efficacy of both schedules should be evaluated in a randomized phase II study.

Cremophor EL (CrEL) is a castor oil surfactant used as a vehicle for formulation of a variety of poorly water-soluble agents, including paclitaxel. Recently, we found that CrEL can influence the in vitro blood distribution of paclitaxel by reducing the free drug fraction, thereby altering drug accumulation in erythrocytes. The purpose of this study was to investigate the clinical pharmacokinetics of CrEL, and to examine inter-relationships of paclitaxel disposition, infusion duration and CrEL kinetics. The CrEL plasma clearance, studied in 17 patients for a total of 28 courses, was time dependent and increased significantly with prolongation of the infusion duration from 1 to 3 to 24 h (p

PURPOSE: To investigate the pharmacokinetics and pharmacodynamics of irinotecan and cisplatin administered once every 3 weeks in a dose-escalating study in patients with solid tumors. PATIENTS AND METHODS: Fifty-two cancer patients were treated with irinotecan administered as a 90-minute infusion at doses ranging from 175 to 300 mg/m2 followed by cisplatin administered as a 3-hour intravenous infusion at doses ranging from 60 to 80 mg/m2. After reaching the maximum-tolerated dose, the sequence of drug administration was revised. For pharmacokinetic analysis, serial plasma samples were obtained on days 1 through 3 of the first cycle. Forty-five patients were assessable for irinotecan pharmacokinetics, and 46 were assessable for cisplatin pharmacokinetics. RESULTS: Irinotecan and cisplatin demonstrated linear pharmacokinetics comparable to that observed with single-agent administration, which suggests an absence of pharmacokinetic interaction. SN-38G constituted the major plasma metabolite of irinotecan, whereas 7-ethyl-10-[4-N-(1-piperidino)1-amino]-carbonyloxycamptothecine (NPC) was only a minor metabolite in plasma, possibly indicating a rapid conversion of NPC to SN-38. The terminal elimination phases of SN-38 and SN-38G were similar and relatively delayed when compared with the elimination of irinotecan. Maximal DNA adduct formation did not significantly differ from that observed with single-agent administration. The percentage decrease in WBC was significantly related to the areas under the plasma concentration-time curve (AUCs) of the lactone form of irinotecan (P = .0245) and SN-38 (P = .0123). The severity of diarrhea was not significantly related to the AUCs of irinotecan and SN-38, nor to the systemic glucuronidation rate of SN-38. CONCLUSION: There was no apparent pharmacokinetic interaction between irinotecan and cisplatin in this study. Reversion of the administration sequence of the drugs did not seem to have any influence on the pharmacokinetics. The incidence and severity of delayed-type diarrhea was not related to any of the studied parameters.

9-Amino-20(S)-camptothecin (9-AC) is a water-insoluble topoisomerase I inhibitor with evident schedule-dependent antitumor activity in preclinical studies. The pharmacokinetic behavior of 9-AC given as a bolus i.v. infusion (1.0 mg/m2 over 5 min) was recently characterized in 12 patients in a bioavailability study. Remarkable rebound concentrations of 9-AC total drug (i.e. lactone plus carboxylate forms) were observed at about 2-3 h after dosing. In vitro experiments indicated that this phenomenon was associated with a substantial uptake of 9-AC lactone by erythrocytes immediately after dosing and its subsequent release followed by accumulation of 9-AC carboxylate in the plasma compartment mediated by a pH-dependent hydrolysis of the lactone form, which is unable to diffuse across cell membranes. The preferential binding of 9-AC carboxylate to human serum albumin shifts the equilibrium between the lactone and carboxylate forms of 9-AC to the pharmacological inactive carboxylate form.

Although successful in many rodent models of malignant gliomas, results of clinical adenoviral gene therapy trials using replication deficient vectors have been disappointing. One of the explanations of the discrepancy between results in animal models and clinical trials is the low expression of the Coxsackie Adenovirus receptor (CAR), the main high affinity receptor for binding of adenovirus serotype 5, on primary glioma cells as opposed to established glioma cell lines. To obtain improved adenoviral vectors, we made use of the differential tropism of the naturally occurring 51 human adenoviral serotypes to test the hypothesis that adenoviral vectors, binding to other receptors than CAR, would result in improved infection of primary glioma cells.