The cyclin-dependent kinase inhibitor p57(Kip2) is epigenetically regulated in carboplatin resistance and results in collateral sensitivity to the CDK inhibitor seliciclib in ovarian cancer.

BACKGROUND: Carboplatin remains a first-line agent in the management of epithelial ovarian cancer (EOC). Unfortunately, platinum-resistant disease ultimately occurs in most patients. Using a novel EOC cell line with acquired resistance to carboplatin: PEO1CarbR, genome-wide micro-array profiling identified the cyclin-dependent kinase inhibitor p57(Kip2) as specifically downregulated in carboplatin resistance. Presently, we describe confirmation of these preliminary data with a variety of approaches. METHODS: Cytotoxicity testing (MTT) and cell cycle blockade assessed drug responsiveness. Methylation specific PCR and pyrosequencing identified sites of promoter methylation in p57(Kip2). siRNA to p57(Kip2) was used to look at the changes in apoptosis of carboplatin treated EOC cells. EOC tissues (20 cases) were assessed for mRNA levels of p57(Kip2). RESULTS: Carboplatin resistance was reversed using 5-aza-cytidine in vitro. Promoter methylation sites and preferential sensitivity to seliciclib were seen in PEO1CarbR cells. Silencing p57(Kip)2 decreased the apoptotic response to the effects of platinum but produced sensitisation to seliciclib. EOC biopsies indicated an association of high levels of p57(Kip2)mRNA with complete responses to chemotherapy and improved outcome. CONCLUSION: We conclude that p57(Kip2) is a candidate biomarker of platinum sensitivity/resistance in EOC and such cases may show preferential response to the cyclin-dependent kinase inhibitor seliciclib.

Analysis of historical negative control group data from the rat in vivo micronucleus assay.

A database of micronuclei counts for historical negative control data from rat in vivo micronuclei tests performed in 10 different laboratories was established. Data were available from over 4000 negative control rats from 10 laboratories. The mean frequency of micronucleated cells (MN)/1000 cells ranged from 0.44 to 2.22, a 5-fold range. Overall there were no major sex or strain differences in frequency, although there were some small but statistically significant differences within laboratories. There was appreciable variability between experiments compared with variability within experiments in some laboratories. No specific factor was identified which could explain this variability although it was noted that many different vehicles were used in the experiments. It is hoped that these data will help laboratories beginning studies with the rat micronucleus assay and those involved in the assessment of micronucleus assay results.

Reduced adhesion formation following laparoscopic versus open colorectal surgery.

BACKGROUND: Adhesion formation is common after abdominal surgery. This study aimed to compare the extent of adhesion formation following laparoscopic and open colorectal surgery. METHODS: An observational study was undertaken to identify adhesions in patients undergoing laparoscopy after previous laparoscopic or open colectomy. Adhesions were scored according to a system validated for interobserver (median kappa = 0.80) and intraobserver (kappa = 0.82) agreement. The primary endpoint was the overall adhesion score (0-10); a secondary endpoint was the adhesion score at the main incision site (0-6). RESULTS: Forty-six patients were recruited (13 laparoscopic and 33 open colectomy). In most patients (n = 29), laparoscopy was performed for tumour staging before liver resection. The median (interquartile range) overall adhesion score was 7 (5-8) in the open group and 0 (0-3) in the laparoscopic group (P < 0.001). A similar difference was found for the main incision score: 6 (4-6) versus 0 (0-0) (P < 0.001). CONCLUSION: There may be a reduction in adhesion formation following laparoscopic compared with open colectomy, although the small sample size limits this conclusion.

Report of an Expert Panel on the reanalysis by of a 90-day study conducted by Monsanto in support of the safety of a genetically modified corn variety (MON 863).

MON 863, a genetically engineered corn variety that contains the gene for modified Bacillus thuringiensis Cry3Bb1 protein to protect against corn rootworm, was tested in a 90-day toxicity study as part of the process to gain regulatory approval. This study was reanalyzed by Séralini et al. who contended that the study showed possible hepatorenal effects of MON 863. An Expert Panel was convened to assess the original study results as analyzed by the Monsanto Company and the reanalysis conducted by Séralini et al. The Expert Panel concludes that the Séralini et al. reanalysis provided no evidence to indicate that MON 863 was associated with adverse effects in the 90-day rat study. In each case, statistical findings reported by both Monsanto and Séralini et al. were considered to be unrelated to treatment or of no biological or clinical importance because they failed to demonstrate a dose-response relationship, reproducibility over time, association with other relevant changes (e.g., histopathology), occurrence in both sexes, difference outside the normal range of variation, or biological plausibility with respect to cause-and-effect. The Séralini et al. reanalysis does not advance any new scientific data to indicate that MON 863 caused adverse effects in the 90-day rat study.

Genetic variation in the shape of the mouse mandible and its relationship to glucocorticoid-induced cleft palate analyzed by using recombinant inbred lines.

Variation in mandible shape has been investigated in a set of recombinant inbred (RI) lines of mice, the C57BL/6J X A/J (BXA;AXB) RI lines. Considerable genetic variation was detected between the RI lines, but most lines were intermediate in shape when compared with the parent lines. Variation in mandible shape could not be explained by any single gene differences known between the parent lines including the H-2 locus. Some RI lines had mandible shapes unlike either parent, and one in particular, line BXA1, had an unusual shape with a pronounced condyloid process. It was concluded that mandible shape has a complex inheritance involving a number of genes, each with small effects. In some cases, recombination of the genes can produce bone shapes quite different from those of the original parent line.--There was no evidence that the variability in steroid-induced cleft palate incidence in the BXA;AXB RI lines is related to the variation in adult mandible shape as detected in this study.

Gleason scoring varies among pathologists and this affects clinical risk in patients with prostate cancer.

AIMS: To investigate whether our practice of specialist review of all diagnostic biopsies was necessary to prevent misgrading of referred prostate cancer patients, and whether this misclassification, if any, would have resulted in misclassification of clinical risk grouping (Seattle Risk Grouping [SRG]) and subsequent treatment strategy and prognosis. MATERIALS AND METHODS: Important prognostic indicators for prostate cancer include the presenting prostate-specific antigen (PSA), clinical stage and Gleason sum of the tumour. These three variables are incorporated into the SRG cohorts to establish treatment strategy. Patients with prostate cancer referred for brachytherapy had their prostate biopsies reviewed by a reference pathologist (PD) with a special interest in prostate cancer. We compared the agreement between the scoring of the referring pathologists with that of PD, and evaluated if any differences changed the SRG and therefore the clinical risk and treatment strategy for the patients. RESULTS: In only 52% (43/83) of cases, was there total agreement between the two sets of pathologists. The inter-rater agreement was statistically 'fair' (unweighted kappa statistic 0.27). In 90% (36/40) of cases with disagreement, PD assigned higher Gleason sums. In 40% (16/40) of cases with disagreement, the change in Gleason sum altered the SRG; in one out of 16 cases, the SRG was downgraded from 'intermediate' to 'low' risk disease; in six out of 16 cases, it was upgraded from 'low' to 'intermediate' risk, and, in nine out of 16, from 'intermediate' to 'high' risk. CONCLUSION: Our findings confirm previous reports of only limited correlation between pathologists in reporting Gleason sums. In this study, 19% (16/83) of cases had their grading changed to a level that altered clinical risk, almost always (94%; 15/16) to one that worsened prognosis. This would have significantly affected treatment strategy for these patients, and thus we recommend that all centres ensure accurate Gleason grading by the use of pathologists with special interests in prostate cancer.

Genetic variation in the metabolism of coumarin in mouse liver.

The metabolism of 50 microM [3-14C] coumarin to polar products separated by high performance liquid chromatography (HPLC) and covalently bound metabolites in liver microsomes was compared in a series of inbred strains of mice. Coumarin metabolism to total polar products was higher in female than male mice. In all strains, the coumarin 3,4-epoxidation pathway was the major route of metabolism with o-hydroxyphenylacetaldehyde (o-HPA) as the major metabolite. However, in females, there was a major strain difference in the degree of metabolism to coumarin 7-hydroxylase with DBA/2 and 129 having high 7-hydroxycoumarin formation, CBA/Ca having intermediate levels and the other strains low levels. The differences between the strains was much less pronounced in the male mice. There was also evidence for strain variation in metabolism in the quantities of a number of other coumarin metabolites as detected by HPLC analysis of incubate extracts. However, this variation was of a quantitative nature and relatively small. The metabolism of B6C3F1 hybrid mice, in which coumarin had been identified as carcinogenic in a long-term cancer bioassay, was qualitatively similar to that of the other genotypes. The DBA/2 mouse has been suggested as a model for the metabolism of coumarin in humans. The pattern of metabolism found in this strain is different from most other strains. However, the pattern found for all the mouse strains, including DBA/2, differed appreciably from the profiles for other species including humans in the extent of 7-hydroxylation.

Population genetics of induced mutations.

The contribution of induced mutations to the burden of genetic disease in the context of population genetics is considered. A clear distinction is made between the effects of genetic disease and mutational events. Much of the existing burden of genetic disease is a consequence of mutations that occurred in the past. The problem of distinguishing between spontaneous and induced mutations is discussed. Molecular genetics techniques are blurring the definitions of these terms. Classical population genetics shows that the frequency of affected individuals will reach an equilibrium depending on the mutation rate and the selective pressure against affected individuals. Increasing the mutation rate or reducing the selective pressures would result in a new equilibrium with an increase in the frequency in subsequent generations of affected individuals with dominant and X-linked mutant alleles. The increase in the number of recessive mutant alleles would be much slower and take many generations to reach the new equilibrium level. One assumption behind such equilibria is random mating. Changes in human demography with a rapid increase in population size, the breakup of small, relatively inbred subpopulations, and relaxed selective pressures will lead to a new equilibrium for recessive genes at probably higher frequencies. These factors will be the major contributors to increasing the burden of recessive genetic disease by increasing the total numbers of cases. The proportion of the population with a genetic disease will also continue to grow as a greater proportion of the population survives to late middle age and succumbs to diseases associated with old age, such as cancer, circulatory disease, dementias, and diabetes, each of which is likely to have a genetic component.(ABSTRACT TRUNCATED AT 250 WORDS)

Report and summary of the major conclusions from statistics in genotoxicity testing working group from the International Workshop on Genotoxicity Test Procedures (IWGTP), March 1999.

A working group of five statisticians experienced in the use of statistical methods in mutagenicity reviewed aspects of the statistical analysis of genotoxicity test procedures. Issues discussed included methods for integrating biological importance and statistical significance, the relationship of the experimental unit to the experimental design, and the impact of new developments in statistics and computing. Three major recommendations were made relating to the need for: (1) the effective use of statistical advice in designing interlaboratory and intralaboratory investigations; (2) the development of appropriate experimental designs for new assays; and (3) education and training in the use of statistical methodology in mutagenicity testing. Environ. Mol. Mutagen. 35:260-263, 2000 Published 2000 Wiley-Liss, Inc.

Variation in the hepatotoxic effects of carbon disulphide in the rat: evidence for polygenic inheritance.

The hepatotoxic effects of carbon disulphide have been examined in rats obtained from a series of crosses between two inbred strains which differ widely in their susceptibility. The distribution of susceptibility in the F2 generation indicates that the variation cannot be explained by a simple genetic model. A polygenic model is a more likely explanation.

Screening for possible human carcinogens and mutagens. False positives, false negatives: statistical implications.

A screening method aimed at identifying potential human carcinogens using either animal cancer bioassays or short-term genotoxic assays has 4 possible results: true positive, true negative, false positive and false negative. Such a categorisation is superficially similar to the results of hypothesis testing in a statistical analysis. In this latter case the false positive rate is determined by the significance level of the test and the false negative rate by the statistical power of the test. Although the two types of categorisation appear somewhat similar, different statistical issues are involved in their interpretation. Statistical methods appropriate for the analysis of the results of a series of assays include the use of Bayes' theorem and multivariate methods such as clustering techniques for the selection of batteries of short-term test capable of a better prediction of potential carcinogens. The conclusions drawn from such studies are dependent upon the estimates of values of sensitivity and specificity used, the choice of statistical method and the nature of the data set. The statistical issues resulting from the analysis of specific genotoxicity experiments involve the choice of suitable experimental designs and appropriate analyses together with the relationship of statistical significance to biological importance. The purpose of statistical analysis should increasingly be to estimate and explore effects rather than for formal hypothesis testing.

Dose-response and threshold-mediated mechanisms in mutagenesis: statistical models and study design.

The objective of this paper is to review the use, in mutagenesis, of various mathematical models to describe the dose-response relationship and to try to identify thresholds. It is often taken as axiomatic that genotoxic carcinogens could damage DNA at any level of exposure, leading to a mutation, and that this could ultimately result in tumour development. This has led to the assumption that for genotoxic chemicals, there is no discernible threshold. This assumption is increasingly being challenged in the case of aneugens. The distinction between 'absolute' and 'pragmatic' thresholds is made and the difficulties in determining 'absolute' thresholds using hypothesis testing approaches are described. The potential of approaches, based upon estimation rather than statistical significance for the characterization of dose-response relationships, is stressed. The achievement of a good fit of a mathematical model to experimental data is not proof that the mechanism supposedly underlying this model is operating. It has been argued, in the case of genotoxic chemicals, that any effects produced by a genotoxic chemical which augments that producing a background incidence in unexposed individuals will lead to a dose-response relationship that is non-thresholded and is linear at low doses. The assumptions underlying this presumption are explored in the context of the increasing knowledge of the mechanistic basis of mutagenicity and carcinogenicity. The possibility that exposure to low levels of genotoxic chemicals may induce and enhance defence and repair mechanisms is not easily incorporated into many of the existing mathematical models and should be an objective in the development of the next generation of biologically based dose-response (BB-DR) models. Studies aimed at detecting or characterizing non-linearities in the dose-response relationship need appropriate experimental designs with careful attention to the choice of biomarker, number and selection of dose levels, optimum allocation of experimental units and appropriate levels of replication within and repetition of experiments. The characterization of dose-response relationships with appropriate measures of uncertainty can help to identify 'pragmatic' thresholds based upon biologically relevant criteria which can help in the regulatory process.

The use of a battery of strains of mice in a factorial design to study the induction of dominant lethal mutations.

The induction of dominant lethality following oral dosing of males with 200 mg/kg of cyclophosphamide was investigated using a factorial experimental design. Males from 3 genotypes, BALB/c, CBA/Ca and CBA/Ca X C57BL/6JF1 hybrid (CBB6F1) were mated to 6 females of the same genotype as the males over 3 weeks. Cyclophosphamide reduced the mating frequency of the BALB/c and CBA/Ca males. The total number of implants/female was reduced in all 3 genotypes with the greatest effect in the first 2 weeks after the males were treated. The proportion of early deaths/litter was significantly increased in CBA/Ca and CBB6F1 but the increase was smaller and non-significant with BALB/c. There was a high incidence (29.8%) of early deaths in the control BALB/c females. Statistical analysis of the ratio of early deaths to total implants in a litter using either the Freeman-Tukey binomial or the arc-sine transformation gave similar and satisfactory results. Analysis of early death data rather then the ratio of early deaths: total implants would have led to misleading conclusions. The implications of the use of a factorial design in dominant lethal assays for the detection of strain variation in mutagenic response without an increase in animal usage is discussed.

Thioguanine-resistant mutant frequency in T-lymphocytes from a healthy human population.

Resistance to 6-thioguanine in T-lymphocytes was used to study in vivo somatic mutations in normal healthy adults. Donor age had a significant effect on mutant frequency at the hprt locus, showing an increase of 0.09/10(6) cells per year of age. No significant increase was associated with sex of donor, smoking habits, alcohol or coffee/tea intake, or X-ray exposure. The lower mutant frequency seen with contraceptive pill usage was probably due to the age difference between the groups of users and non-users.

High resolution metrical analysis applied to the assessment of damage associated with induced mutations in the mouse.

Morphometric methods were used to investigate variation in the skeletons of 1030 offspring produced from matings of male DBA/2J by female C57BL/6J mice. 751 offspring originated from males that had received a single intraperitoneal injection of ethyl nitrosourea (EtNU) at a dose of 250 mg/kg. The remainder of the mice served as controls. The male parents of the controls were injected only with the buffer used as vehicle for the EtNU. Offspring were obtained for 3 weeks following injection. The treated males were then sterile for about 8 weeks. Immediately after the sterile period another sample of progeny was obtained. In the treated group, litter sizes at birth and weaning were reduced and survival to adulthood was lower. However, none of the differences were statistically significant. The skeletons were evaluated by two independent approaches. The first relied upon gross observation for unusual phenotypic variation, the second on a series of metrical measurement and coordinate data. A considerable amount of variation was recorded by both approaches. Some of the variants were severe but others were mild and perhaps of little or no importance to the health of the mice. The gross observation method produced no evidence for increased mild or severe variants in any group of offspring from the treated mice. The metrical methods also showed no evidence for treatment-related effects in offspring produced during the first 3 weeks of mating. However, in offspring produced after the sterile period, a pronounced, very highly statistically significant increase in all levels of metrical variation was observed. This treatment group revealed both increased variant measures and increased numbers of mice with variant measures. Much of this variation was so slight that it would have escaped notice were it not for the exacting measurements used in the analysis. Our morphometric approach is an analytically powerful tool, suitable for detecting variation in virtually any biological structure that can be measured. If the increased variation reported here is due to induced mutations, the effects would be consistent with that expected from slightly harmful mutations distributed throughout the mouse genome. It is appropriate to consider such effect in connection with genetic risk estimation.

Variation in sister-chromatid exchange among 106 members of the general U.K. population.

SCE scores of lymphocytes from 106 people revealed that the majority of background variation in SCE was between cells within individuals. Highly significant differences existed between individuals. Lesser, but still highly significant differences also existed between replicate cultures. Inter-individual variation was contributed to by each person's sex and their smoking habits. SCE frequency was not influenced by any of the other factors considered, age, drinking habits and diagnostic X-ray exposure of persons or lymphocyte number and proliferation rate in cultures.

Ethylene dibromide: negative results with the mouse dominant lethal assay and the electrophoretic specific-locus test.

Ethylene dibromide (1,2-dibromoethane; EDB) was tested for the induction of dominant lethal and electrophoretically-detectable specific-locus mutations in the germ cells of DBA/2J male mice. Males were treated with a single intraperitoneal injection of 100 mg/kg EDB and mated to two C57BL/6J females. In the dominant lethal assay, matings were carried out to measure the effect of EDB on meiotic and postmeiotic stages; germ cells representing spermatogonial stem cells were analyzed in the electrophoretic specific-locus test. Neither of these germ cell tests produced any evidence that EDB is a germ cell mutagen. It appears from these data and those reported in the literature that EDB, a genotoxic carcinogen that affects male fertility in some mammalian species, is not mutagenic in the germ cells of the male mouse.

Principal component analysis of Draize eye Irritation tissue scores from 72 samples of 55 chemicals in the ECETOC data bank.

The multivariate statistical method Principal Component Analysis (PCA) has been applied to a set of data from the ECETOC reference chemical data bank. PCA is a multivariate method that can be used to explore a complex data set. The results of the analysis show that most of the variability in the values for tissue damage scores for the 55 chemicals can be described by a single principal component which explains nearly 80% of the variability. This component is derived by giving approximately equal weight to each of the 18 individual measures made on the tissues over the 24-, 48- and 72-hr observation period. The principal component scores on the first component (PC I) are very highly correlated with the maximum individual weighted Draize scores or total Draize scores (TDS) derived using the Draize scoring method. A second principal component, describing about 7% of the variability, contrasts damage measured on the iris and cornea with that measured on the conjunctiva. Plots of principal component scores show the overall pattern of responses. In general, low measures of the TDS and a positive (PC I) score are associated with iris and conjunctival damage (damage to the iris was never recorded in the absence of damage to the conjunctiva). High TDS and negative PC I scores are associated with corneal and/or iris and conjunctiva damage. Plots of the principal component scores identify some chemicals that appear to cause unusual patterns of damage and identify some individual animals as having outlying or idiosyncratic responses. However, the analysis suggests that (i) there is only limited evidence for differential responses of different tissues and (ii) that attempts to identify alternative tests which predict specific types of tissue damage based on the results collected in a Draize test are likely to be unsuccessful. It indicates that further refinement of the results of the in vivo Draize test will not arise from more detailed analysis of the tissue scores but by refinement in the understanding of the mechanisms associated with the test. PCA was shown to be a powerful statistical tool for the investigation of complex data sets and provides a succinct description of such data sets, allowing patterns to be identified and the potential to develop further hypotheses for investigation.

Principal Component Analysis of Tissue Scores from Substances used in the COLIPA Eye Irritation Validation Study.

Principal component analyses (PCA) have been carried out on the tissue scores from Draize eye irritation tests on the 55 formulations and chemical ingredients included in the COLIPA Eye Irritation Validation Study. A PCA was carried out on the tissue scores 24, 48 and 72 hours after instillation of the substances. The first Principal Component (PC I) explained 77% of the total variation in the tissues scores and showed a high negative correlation (r=-0.971) with the scores used to derive the Modified Maximum Average Score (MMAS). The second component (PC II) explained 7% of the total variability and contrasted corneal and iris damage with conjunctival damage as in a similar analysis carried out previously on the ECETOC databank. The third component (PCIII), while only explaining about 3% of the variability, identified individuals treated with formulations that were observed to have low corneal opacity but large corneal area scores. This may represent some particular manner of scoring at the laboratory administering the Draize test or a specific effect of some formulations. A further PCA was carried out on tissue scores from observations at 1hr to 21 days. PC I in this analysis explained 62% of the variability and there was a high negative correlation with the sum of all the tissue scores, while PC II explained 14% of the variability and contrasted damage up to 72 hours with damage after 72 hours. A number of formulations were identified with relatively low MMAS scores but tissue damage that persisted. PCA analysis is thus shown to be a powerful method for exploring complex datasets and for identification of outliers and subgroups. It has shown that the MMAS score captures most of the information on tissue scores in the first 72 hours following exposure, and it is unlikely to be of any advantage in using individual tissue scores for comparisons with alternative tests. The relationship of the classifications schemes used by three alternative methods in the COLIPA study with the results of the PCA were investigated and the implications of the effect of persistence of tissue damage for various classifications schemes visualized.

A summary report of the COLIPA international validation study on alternatives to the draize rabbit eye irritation test.

The principal goal of this study was to determine whether the results from a set of selected currently available alternative methods as used by cosmetics companies are valid for predicting the eye irritation potential of cosmetics formulations and ingredients and, as a consequence, could be valid replacements for the Draize eye irritation test. For the first time in a validation study, prediction models (PMs) that convert the in vitro data from an assay to a prediction of eye irritation were developed for each alternative method before the study began. The PM is an unequivocal description of the relationship between the in vitro and the in vivo data and allows an objective assessment of the reliability and relevance of the alternative methods. In this study, 10 alternative methods were evaluated using 55 test substances selected as representative of substances commonly used in the cosmetics industry (23 ingredients and 32 formulations). Twenty of the single ingredients were common to the European Commission/British Home Office (EC/HO) eye irritation validation study (Balls et al., 1995b). The test substances were coded and supplied to the participating laboratories. The results were collected centrally and analysed independently, using statistical methods that had been agreed before the testing phase began. Each alternative method was then evaluated for reliability and relevance in assessing eye irritation potential. Using the criteria of both reliability and relevance as defined in the study, the preliminary results indicate that none of the alternative methods evaluated could be confirmed as a valid replacement for the Draize eye irritation test across the full irritation scale. However, three alternative methods-the fluorescein leakage test, the red blood cell assay (classification model) and the tissue equivalent assay-each satisfied one criterion of reliability or relevance. Further investigation of the decoded data from this study to explore more fully the relationship between the in vitro data and the in vivo data is recommended. Such a review may allow the development of new prediction models to be tested in a subsequent validation study.