Annual Meeting of the GTA
May 11-12, 2017
John M. Clayton Hall Conference Center
University of Delaware
Mode of Action Approaches that Identify Genotoxic Mechanisms in Mammalian Cell Systems
Speakers Bios, Abstracts & Presentations
Curtis Harris, MD
National Cancer Institute; Editor-in-Chief for Carcinogenesi
Harris has received numerous honors throughout his distinguished career including the Alton Ochsner Award relating Smoking and Health (American College of Physicians), Deichmann Award (International Union of Toxicology), Charles Heidelberger Award (International Society of Gastroenterological Carcinogenesis), the Distinguished Service Medal (the highest honor of the U.S. Public Health Service), NCI Outstanding Mentor Award in 2007 and 2013, Ph.D. (Honorary) Nippon University School of Medicine, the AACR-Princess Takamatsu Award, and most recently in 2014 the ILCA Nelson Fausto Award and AACR-American Cancer Society Award for Research Excellence in Cancer Epidemiology and Prevention. He serves as an honorary member for the Japanese Cancer Association and is a Fellow at the American Society of Clinical Investigation and the AAAS.
Harris has published more than 500 journal articles, 100 book chapters, and edited 10 books, and holds more than 30 patents owned by the U.S. Government. He also serves as Editor-in-Chief for the journal, Carcinogenesis, and has held or currently holds elected offices in scholarly societies and non-profit foundations including the American Association of Cancer Research, the International Society of Differentiation, the Keystone Symposia on Molecular and Cellular Biology, International Liver Cancer Association and the Aspen Cancer Conference. Harris has a wide range of scientific interests and accomplishments spanning molecular genetics and epigenetics of human cancer to molecular epidemiology of human cancer risk and mechanistic biomarkers of cancer diagnosis, prognosis and therapeutic outcome.
Precision medicine: Lung biomarkers
More than half of all new lung cancer diagnoses are made in patients with locally advanced or metastatic disease, at which point therapeutic options are scarce. It is anticipated, however, that the widespread use of Low-Dose Computed Tomography (LDCT) screening, will lead to a greater proportion of lung cancers being diagnosed at an early, operable, stage. Still, the overall rate of recurrence for surgically treated Stage I lung cancer patients is up to 30% within 5 years of diagnosis. Thus, the identification and clinical application of biomarkers of early stage lung cancer are a pressing medical need. The integrative analysis of "omic," clinical and epidemiological data for single patients is a core principle of precision medicine. Through rigorous bioinformatics and statistical analyses we have identified biomarkers of early-stage lung cancer based on DNA methylation, expression of mRNA and miRNA, inflammatory cytokines, and urinary metabolites. Beyond a more comprehensive understanding of the molecular taxonomy of lung cancer, these biomarkers can have very practical implications in the context of unmet clinical needs of early stage lung cancer patients: First, current guidelines for LDCT screening broadly include individuals based on age and history of heavy smoking. Tumor-derived circulating biomarkers in the blood and urine associated with lung cancer risk could narrow and prioritize individuals for LDCT screening. Second, a high number of nodules are identified by LDCT, of which fewer than 5% are finally diagnosed as lung cancer. Biomarkers may help discriminate malignant nodules from benign or indolent lesions. Third, the expected rise in the numbers of lung cancer patients diagnosed at an early stage will necessitate new treatment options. Circulating, urinary and tissue-based biomarkers that molecularly categorize Stage I patients after tumor resection can help identify high-risk patients who may benefit from adjuvant chemotherapy or innovative immunotherapy regimens.
1. Vargas, A.J. and C.C. Harris, Biomarker development in the precision medicine era: lung cancer as a case study. Nat Rev Cancer, 2016. 16(8): p. 525-37.
2. Robles, A.I. and C.C. Harris, Integration of multiple "OMIC" biomarkers: A precision medicine strategy for lung cancer. Lung Cancer, 14 June 2016: p. [Epub ahead of print].
3. Robles, A.I., et al., An Integrated Prognostic Classifier for Stage I Lung Adenocarcinoma Based on mRNA, microRNA, and DNA Methylation Biomarkers. J Thorac Oncol, 2015. 10(7): p. 1037-48.
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Cristian Tomasetti, PhD
Johns Hopkins University
His expertise is in the mathematical modeling of cancer evolution (Tomasetti et al. PNAS 2010, Tomasetti et al. PNAS 2013, Tomasetti et al. PNAS 2015), cancer etiology (Tomasetti et al. Science 2015, Tomasetti et al. Science 2017), and the analysis of sequencing data from tumors and ctDNA (Tie et al. Sci Transl Med 2016, Anglesio et al. New Engl J Med 2017).
Dr. Tomasetti has given 40 invited talks across the world and has been recently the recipient (PI) of a 1.33 million 3-year grant from the John Templeton Foundation.
Stem cell divisions, somatic mutations, and cancer etiology
Cancers are caused by mutations that may be inherited (H), induced by environmental factors (E), or result from intrinsic DNA replication errors (R) during cell divisions. Recently we reported a striking correlation between the number of normal stem cell divisions and the risk of 25 cancer types (Tomasetti and Vogelstein, Science 2015). After reviewing those findings, we extend the analysis to 69 countries throughout the world. Finally, we provide further evidence for the major role of R in cancer etiology by an independent approach based solely on cancer genome sequencing and epidemiologic data that enable the estimation of the proportion of cancer mutations due to E, H, and R. The results are consistent with epidemiological estimates of the fraction of cancers that can be prevented by changes in the environment. Moreover, they accentuate the importance of early detection to reduce deaths from the many cancers arising from unavoidable R mutations.
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Song Wu received his PhD in Statistics from University of Florida. He is currently an Associate Professor in the Department of Applied Mathematics and Statistics at Stony Brook University. He also serves as a core member in the Cancer Center Cancer Genomics Group, an affiliated member in Institute for Advanced Computational Science (IACS), and the scientific director for Bioinformatics and Computational Core of Stem Cell Center at Stony Brook University. Before joining Stony Brook University, he worked as an Assistant Member in the Department of Biostatistics at St Jude Children’s Research Hospital and served in the Protocol Review Committee between 2008 and 2011.
Dr. Wu also had intensive training in biology. He received his BS in Biological Sciences from University of Science and Technology of China, and had been a PhD candidate in Molecular Cell Biology, particularly on cancer biology, in the University of Florida for three years. Given his mixed background, his primary research is on the development and application of novel statistical methodologies for studying and understanding genetic basis, or biological mechanisms, of complex traits in both model organisms and natural populations, such as linkage and linkage disequilibrium mapping, next-generation sequencing data analysis and pathway analysis. More recently, he is also interested in applying big data analytics to solve statistical genomics/genetics problems.
Dr. Wu has published more than 50 papers in his research areas. He is currently serving as an Editorial Board Member for the ‘Statistical and Computational Genetics’ section of BMC genetics, as Academic Editor/Editorial Board Member and Statistical Advisor for PLOS ONE, as an Editorial Board Member for Journal of Biometrics and Biostatistics, and as a Review Editor for Frontiers in Statistical Genetics and Methodology.
Evaluating Intrinsic and Extrinsic Cancer Risk
Determining the contributions of intrinsic and extrinsic risk factors to human cancers is critical to strategizing research investment and public health measures aimed at reducing cancer. If a majority of cancer risk is determined by unmodifiable intrinsic factors, cancer prevention focused on modifying the environment would be largely ineffective favoring resource allocation to treatment and secondary prevention. If, however, extrinsic cancer risk is a dominant component, as in smoking and lung cancer, significant efforts should be exerted on identifying and eradicating causal and contributory factors. To clarify these issues, here we review and discuss the existing evidence on the relative importance of intrinsic and extrinsic factors in cancer burden using evidence from three major areas of study: cancer epidemiology, cancer biology, and mathematical modelling. The utility and limitations of different approaches will also be discussed. Our main conclusion is that at the current stage, the proportion of extrinsic risk appears to be quite high for many cancers, including most common cancers. Hopefully our study can help guide research development aimed at determining the contribution of intrinsic and extrinsic factors, defining those factors, and devising approaches to mitigate exposure or consequences of these factors, thus developing a scientific basis to inform public policy on more productive and effective cancer research.
Dr Dutta received her BSc with Hons in Zoology (1995), MSc in Zoology with specialization in Genetics (1997) and a PhD in Zoology (Radiation and chemical induced DNA damage and repair) in 2004 from North Eastern Hill University in Shillong, India. Her Post-Doctoral training was done at Columbia University, New York, under the supervision of Dr. David J Brenner. During her post-doctoral training, Dr. Dutta worked on various DOE grants studying radiation induced chromosome damage and also on a NIAID grant building high throughput versions of cell culture, micronuclei and gamma-H2ax assays.
Dr. Dutta has co-authored several peer-reviewed publications and patents in the area of DNA damage. She is a member of the GTA and is currently the newsletter co-editor.
De-risking an MLA positive test result for a pharmaceutical agent
This is a case study to discuss some of the safety testing studies that were performed on a Pharmaceutical Agent that belongs to the class of nucleoside analogs. The compound tested was initially determined to be Ames negative. This was then followed up with a Mouse Lymphoma Assay that was found to be positive with a predominance of large colonies indicating potential mutagenicity. Upon consultation with the regulatory agency, a follow up Transgenic Rodent assay was initially recommended and the agency finally agreed to accept a 28-day Pig-a study in its place. At the end of the 28-day study, pig-a data indicated a positive response for mutant RBCs and reticulocytes with a dose-related trend. This is an exception in the group of compounds that this pharmaceutical agent belongs to in that it was found to be a strong mutagen in contrast to other compounds of this group investigated so far.
Presentation not available
Stephanie Kellum received her BS degree from Towson University, Towson, MD in Biology. She has more than 8 years of experience in tissue culture and genetic toxicology. She is currently the study director for the in vitro chromosomal aberration and micronucleus assays in the Genetic Toxicology group at DuPont Haskell Global Centers for Health and Environmental Sciences, in Newark, DE. She is also experienced in flow analyses, and was recently the co-lead for the development of an in vitro method for the evaluation of proliferation and CAR/PXR-mediated gene expression in primary mouse and human hepatocytes.
Trend or no trend, that is the question
Recent updates to OECD test guidelines have placed an increased emphasis on statistical dose-response analysis. A statistical trend test is in most cases required in order to prove a clear negative result, as in comparison to the previous adaptations of the guidelines where a trend test was only required to support a positive finding. Although the importance of the statistical analysis has been put in the forefront for evaluation of the test results there is no firm guidance or communicated consensus on what statistical test to use. Although expert judgment is also called for, our experience is that statistical significance on the 5% level tends to trump any other factor, such as data being inside the 95% confidence limits of the negative control range or of very low magnitude. There is also little guidance on how to follow-up an inconclusive or equivocal result. A reoccurring issue that has become troublesome is dose-response analysis. What conclusion should be drawn for a study that has a statistically significant trend test with no other indication of test chemical associated mutagenicity? Is an apparent (by the eye) or statistically significant trend reason to invalidate or change the overall outcome of a previously conducted study where there was no requirement to analyze for dose-responsiveness unless the results where positive? In light of the increased relevance of statistics we have identified a need for more harmonious and standardized methods of statistical evaluation to insure consistency between laboratories and to eliminate surprise rejections of the data. There also needs to be a practical approach at following up an equivocal result, especially when pertaining to a preexisting study, that will minimize the use of additional animals. In this presentation we will present in vitro and in vivo case studies where the question of the relevance of the dose-responsiveness trend became an issue, and how we proceeded to find resolutions.
Presentation in PDF
Worker safety limbo: how low should I go?
In preparation for commercial synthesis of a highly potent genotoxic drug, the worker safety assessment required an understanding of where in the synthetic route the molecule became an active genotoxicant. The active pharmaceutical ingredient (API) was thoroughly characterized as to its mechanism of action and toxicity in vivo and required strict environmental controls during preparation and handling. However, no toxicity information was available for synthesis intermediates. Since, it was not possible to isolate the entire synthesis process; the synthesis intermediates were screened in a high throughput/high content in vitro micronucleus assay with DNA damage and cellular toxicity biomarkers. The API produced a unique biomarker signature, which allowed a comparison between the intermediates and the API in both potency and cellular response. Benchmark dose modeling was used to determine which intermediates required strict control like the API and which could be controlled like any other reactive intermediates.
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Liz is a graduate of the University of Connecticut with a degree in Cytogenetics (now called Diagnostic Genetic Sciences). She is secretary of the Society of Biomolecular Imaging and Informatics (SBI2).
Mechanistic biomarkers using imaging technologies improve the risk assessment of drug development candidates
The screening of chemical drug development candidates for genotoxicity is usually accomplished through the miniaturization of regulatory accepted in vitro assays. While this approach can result in highly predictive results for the outcome of the regulatory testing battery, it provides little mechanistic information and the molecular target(s) often remain undefined. The lack of mode of action (MoA) information often leads to the unnecessary attrition of potentially valuable drug development candidates and drives the need for approaches that allow for a better mechanistic understanding and the risk to exposed humans. In this presentation we will show data from a screening flow micronucleus assay and the use of mechanistic biomarkers to discriminate between clastogens, aneugens and cytotoxicants. Using real life screening candidates, we will present how high content imaging and other technologies can be used to further improve the mechanistic understanding of aneugenic and clastogenic compounds. Finally, we will integrate the results from these assays to develop a simple model to identify the sequence of events starting with identifying the molecular targets and subsequent cellular consequences leading to the positive result in the in vitro micronucleus assay. The value of this approach and its impact on the risk assessment of pharmaceutical compounds will be discussed.
Presentation in PDF
Andrew Richard Collins
Oslo University (Retired)
Professor Collins received his first degree (1968) and PhD (1972) from Cambridge University, UK. After a year working with marine biologists in the Sudan, he returned to Cambridge as a researcher in the Department of Zoology, spent a year in Denver, Colorado (1979-80), moved to Aberdeen, Scotland, and worked for 10 years at the Rowett Research Institute before moving to Norway in 2002. He received his ScD degree from Cambridge University in 2006.
Professor Collins has published around 300 scientific papers and has an h-index of 58. He was president of the UK EMS (2002-4), has served on editorial boards of Mutagenesis, Mutation Research Reviews, Free Radical Research, DNA Repair, British Journal of Nutrition, Cell Biochemistry and Function and is currently an editor of the European Journal of Nutrition. He has edited several special issues of journals. He was awarded the Jan Jessenius Gold Medal of the Slovak Academy of Science (2000), the first James Parry Award of the UK EMS (2011), and was elected a life fellow of UK EMS in 2013.
Elucidating mechanisms of DNA damage and repair with the comet assay
The alkaline comet assay is routinely used in genotoxicity testing to assess DNA strand breakage, both in vivo and in vitro. It is simple, sensitive, gives quick results, and can be applied to virtually any eukaryotic cell type and any tissue that can be disaggregated into single cells. In its original form it has limitations: a scientific one, that strand breaks and alkali-labile sites represent only some of the lesions that occur in DNA; and a technical one, that the number of samples that can be analyzed in one experiment is restricted by the size of electrophoresis tank. The simple change from one or two gels per slide to 12 mini-gels on a slide or 96 on a GelBond film has greatly increased the throughput of the assay. To deal with the issue of limited lesion detection, many years ago we introduced an extra step in the assay, incubating the nucleoids after cell lysis with lesion-specific enzymes – particularly formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III to recognize oxidized purines and pyrimidines respectively. Fpg, it turns out, detects not just 8-oxoguanine but other kinds of base damage, and so it increases the sensitivity of the assay for general detection of genotoxic agents. The high throughput assay, with Fpg, has recently been applied in a study of nanoparticle toxicity.
Efficient removal of DNA lesions is crucial for cell survival, and the comet assay can be applied to most of the various repair pathways. Fpg or T4 endonuclease V is used to follow the kinetics of base excision repair (BER) of oxidation damage and nucleotide excision repair (NER) of UV-induced lesions, respectively. NER can also be studied by incubating UV-irradiated cells in the presence of aphidicolin, which causes incomplete repair sites to accumulate. We have also developed in vitro repair assays in which a cellular extract is incubated with a substrate of nucleoids with specific lesions. Differential repair of damage in particular DNA domains has been investigated by combining fluorescent in situ hybridization of gene-specific probes with the comet assay.
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The 46 members of the in vivo Workgroup and data analysis team are mid-career and senior genetic toxicologist with experience designing, conducting and/or reviewing genetic toxicology data. The committee is chaired by Dr. David J. Kirkland (Genetox Consulting, UK) and co-chaired by Dr. Dan D. Levy (US Food and Drug Administration) and Matthew J LeBaron (The Dow Chemical Company) with program management assistance from Dr. Jennifer Tanir (HESI).
Each member of the committee is actively working in the field of genetic toxicology and has written numerous test reports, regulatory reviews and/or peer reviewed publications in the field.
HESI Genetic Toxicology Technical Committee evaluation of compounds tested in both comet and transgenic rodent mutation assays
Data on tissue-specific mutations and DNA damage are beginning to be requested in various regulatory guidelines now that OECD test guidelines for comet and the transgenic rodent mutations (TGR) assays have been published. Testing strategies (e.g. when to conduct a test) and critical details of test conduct (e.g. which tissues and routes of administration to use) are generally beyond the scope of OECD test guidelines. Analysis of newly available data from the two assays will provide support and justification for the development of testing strategies. A workgroup of the HESI Genetic Toxicology Technical Committee has begun to organize the available published data to support such analyses. We found data for around 90 compounds which had been tested in both assays. Some of the data was published using early and unreliable test procedures. Reliable analyses require reliable data, so two dozen experienced genetic toxicologists compiled key test parameters and results, including from carcinogenicity studies, into a standardized collection form and evaluated the methods and data reliability from over 500 studies. Preliminary data from the analyses will be presented to illustrate the challenges encountered as we finalize comparison criteria and conduct the full analyses. Ultimately data from the short term in vivo tests will be combined and compared to the results of in vitro testing and cancer bioassays.
Presentation in PDF
Mirjam Luijten, PhD
Centre for Health Protection, National Institute for Public Health and the Environment (RIVM)
Dr. Luijten received her MSc in Biomedical Sciences from Utrecht University in 1996, and her Ph.D. in Human Genetics from University of Amsterdam/Academic Medical Center in 2001. Her post-doctoral research included phytoestrogens and breast cancer (Dr. Coen van Kreijl), transgenic mouse models for carcinogenicity testing (Prof.dr. Harry van Steeg) and alternative test methods for reproductive toxicity (Prof.dr. Aldert Piersma), all at the National Institute for Public Health and the Environment (RIVM).
Dr. Luijten has authored or co-authored over 50 peer-reviewed publications. Dr. Luijten is an editorial board member of Mutation Research Reviews, Mutation Research Genome instability and disease, and Frontiers in Medicine. She is member of the OECD Expert Group on Non-Genotoxic Carcinogens and of the OECD Expert Advisory Group on Molecular Screening and Toxicogenomics, in which she chairs the AOP training committee. She is the chair of the Clean Sheet Workgroup of the Genetic Toxicology Technical Committee (GTTC) of ILSI-HESI and member of the ILSI Europe Expert Group ‘Threshold of Toxicological Concern’. She is also board member of the Dutch Environmental Mutagen Society (DEMS).
A mechanism-based testing strategy to identify non-genotoxic carcinogens
Assessment of genotoxic and carcinogenic potential is considered one of the basic requirements when evaluating the potential hazards and risks of chemicals for human health. Chemical substances that are carcinogenic may cause cancer via either genotoxic or non-genotoxic mechanisms. Test strategies currently in place focus primarily on identifying genotoxic potential due to the strong association between the accumulation of genetic damage and cancer. However, 10-20% of the substances classified by IARC as probably, possibly or proven carcinogenic to humans are assumed to lack genotoxic potential. Using only genotoxicity assays to predict carcinogenic potential has the significant drawback that risks from non-genotoxic carcinogens remain largely undetected in the absence of a carcinogenicity study. Furthermore, test systems and methods already developed to reduce or replace animal use are not easily accepted and implemented by either industries or regulators. Consequently, novel scientific knowledge and emerging technologies are incorporated in regulatory toxicology at a slow pace. Using both test methods for cancer hazard identification that have been adopted by the regulatory authorities and promising alternative methods, we proposed a generally applicable tiered test strategy that can be considered capable of detecting both genotoxic as well as non-genotoxic carcinogens. Moreover, it will improve understanding of the underlying mode of action, which is fully in line with the ongoing transformation of regulatory toxicology. In this strategy, the prediction of carcinogenic potential (or lack thereof) of non-genotoxic chemicals largely relies on data from sub-chronic toxicity studies. A case study performed to evaluate the usefulness of this strategy showed that this approach needs further refinement by adding in extra parameters to better recognize the mode(s) of action involved and assess the human relevance. This is essential for all stakeholders, including industries and regulatory bodies. Several options how to achieve this refinement will be presented and discussed.
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Dr. Schuler received his Ph.D. from the University of Kaiserslautern in Germany where he worked on the detection of chromosomal damage induced by environmental and synthetic estrogens. During a postdoctoral fellowship with Dr. David Eastmond at the University of California, he developed fluorescence in situ hybridization methods for the detection of aneugenic and clastogenic damage in vitro and in rodent target tissues in vivo. Dr. Schuler joined Pfizer Global Research and Development in 2000 and is responsible for the risk management of early drug development candidates and development of novel risk management tools. He is involved in the International Working Groups on Genetic Toxicology (IWGT), the OECD in vitro micronucleus testing guideline working group, and the ILSI/HESI efforts developing follow-up strategies for in vitro genetic toxicology positive findings.
Mode of Action Approaches that Identify Genotoxic Mechanisms in Mammalian Cell Systems
Interpretation of Genetic Toxicity Benchmark Dose (BMD) Values - Comparisons Across Covariates, Determination of Endpoint-specific BMRs (Benchmark Response), and Estimation of Human Exposure Limits
The results of genetic toxicity tests have traditionally been evaluated using dichotomous binning (i.e., yes or no) that merely identifies genotoxic hazard. However, there is increasing interest in quantitative analyses of dose-response data; moreover, determination of point-of-departure (PoD) metrics for regulatory interpretation of assay results. The benchmark dose (BMD) approach determines the dose that elicits a small, pre-specified effect size (i.e., the BMR or Benchmark Response); the recently developed combined-covariate approach permits robust comparisons of BMDs across covariates such as compound, tissue, sex, exposure regime, or cell line. The approach was applied to the analysis of in vitro micronucleus (MN) frequency dose-response data for a series of aneugens, two mutagenic azo compounds, and a topoisomerase II inhibitor. The aneugen analysis yielded compound groupings based on BMD confidence intervals; analyses of the azo compound data confirmed the influence of endogenous metabolism on genotoxic potency. Similarly, analyses of in vivo MN dose-response data permitted robust ranking for a series of clastogens. Additionally, empirical comparisons of Pig-a mutant phenotype frequency data permitted effective investigations regarding the effects of sex and post-exposure sampling time on the mutagenicity of N-ethyl-N-nitrosourea. BMD-covariate analysis of Muta™Mouse lacZ mutant frequency dose-response data revealed robust tissue differences in benzo[a]pyrene potency across tissues, with ranking permitting the formulation of mechanistic hypotheses regarding the manifestation of tissue-specific effects. Additional analyses employed BMD values and their confidence intervals to examine variations in the in vivo mutagenicity of EMS (ethyl methanesulfonate) across multiple variants of the Transgenic Rodent gene mutation assay (e.g., Muta™Mouse, gpt delta mouse, BigBlue® mouse). Despite published observations to the contrary, the results failed to show any evidence of potency differences attributable to assay variant. Although most published studies employing the BMD approach employ uniform BMRs across multiple endpoints (e.g., 10% or 1 standard deviation above control), recent work noted that comparisons across endpoints are confounded by variations in maximum response. Using the mathematical framework publish developed by Slob, dose-response data for 9 data-rich compounds were used to determine effective BMR values for in vitro and in vivo genetic toxicity endpoints (e.g., transgene mutant frequency, Pig-a mutant phenotype frequency, MN frequency). The results suggest that the BMR employed for MN induction should be far lower than that employed for gene mutation or DNA adduct endpoints. Finally, BMD values derived from in vivo dose-response data were used to estimate human exposure limit values for risk assessment. Analyses of Muta™Mouse BaP data provided a human exposure limit value approximately 50-fold greater than the geometric mean oral exposure level in North America. Similarly, the analyses revealed MOE (Margin of Exposure) values ranging from a low of 7200 to a high of over 500,000.
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Dr. Marchetti’s academic training includes a Doctoral Degree, magna cum laude, from the University of Rome, La Sapienza in Italy (1990). Following his doctoral studies, Dr. Marchetti performed postdoctoral research in reproductive biology at the Louisiana State University Medical Center in Shreveport, Louisiana (1991-1994) and in molecular cytogenetics at the Lawrence Livermore National Laboratory in Livermore, California (1994-1997).
Dr. Marchetti has authored or co-authored over 90 peer-reviewed publications. He is an Adjunct Research Professor at Carleton University and is the chair of the Germ Cell Workgroup of the Health and Environmental Science Institute’s (HESI) Genetic Toxicology Technical Committee (GTTC) and a member of the steering committee of the GTTC. He is also a member of the Organization for the Economic Co-Operation and Development Expert Group on Genotoxicity Testing. Dr. Marchetti is a long-standing member of the Environmental Mutagenesis and Genomic Society having served on the council, executive board, as co-chair of several special interest groups, and just completed a 5-year term as the Editor-In-Chief of Environmental and Molecular Mutagenesis (EMM). He serves on the editorial boards of EMM, Mutation Research Genetic Toxicology and Environmental Mutagenesis, and Scientific Reports. In 2015, he received the Health Canada Deputy Minister’s Award for Excellence in Science.
Advances in assessing genetic hazards in germ cells
Genetic damage contributes to a wide range of human diseases and there is renewed interest in identifying environmental agents and lifestyle factors that increase the risk of heritable genetic hazard. The publication of OECD test guideline 488 for the detection of mutations in rodent germ cells and the inclusion of germ cell mutagenicity as a health hazard criterion in the Globally Harmonized System of classification and labelling have led to an increased demand for germ cell testing for regulatory submission purposes. It is also increasingly recognized that diverse genomic changes can be induced in germ cells and impact the health of the offspring. Therefore, assays that assess different mutational mechanisms in germ cells are required for comprehensive characterization of the genotoxic effects of chemicals. The goal of our research is to develop, validate, and implement novel methods for identifying environmental factors that cause heritable genetic damage using the MutaMouse transgenic rodent model. We have developed an integrated approach where different genotoxic endpoints (DNA damage, point mutations and tandem repeat mutations) can be investigated. Our results show that: 1) stem cell spermatogonia are susceptible to mutation induction and that peak responses occur in dividing spermatogonia; 2) tandem repeat mutations are induced in mouse spermatogonia and appear to occur at doses that do not produce increases in point mutations; 3) male germ cells have a spectrum of spontaneous and induced mutations that is different from somatic tissues suggesting that germ cells may have unique mutational mechanisms; 4) assays for detecting DNA damage in sperm can be readily integrated into standard regulatory designs investigating genotoxicity in somatic cells; and, 5) benchmark dose values for germ cell genotoxic effects are similar to those observed with the micronucleus assay in bone marrow. These findings illustrate the utility of these methods to study germ cell effects and advance our understanding of the mechanisms of induction of heritable genetic damage and the potential consequences of exposures to environmental chemicals on abnormal reproductive outcomes.
Presentation in PDF
Pfizer Worldwide Research and Development
Michelle received her undergraduate degree in Biology from Quinnipiac University in New Haven, CT (1993) and her graduate degree in Biology (MA.) from Brown University in Providence, RI (2000).
Michelle has authored or co-authored several peer-reviewed publications in the area of mutagenicity (in vitro and in vivo) and the use of in silico tools for assessing mutagenicity of impurities. Michelle has also contributed to the ICH M7 Addendum. She is a long-time member of the GTA, was secretary in 2015 and is currently chair-elect for the GTA
Use of expert knowledge in review of (Q)SAR mutagenicity predictions for impurities: case examples from the industry
ICH M7 notes that Ames testing is not needed for impurities when there are no mutagenicity alerts in two complementary (Q)SAR methods. It is also recommended that expert knowledge can be provided to assess relevance of positive, negative, conflicting and inconclusive predictions to justify a conclusion about mutagenicity. This presentation will discuss case examples where expert knowledge was provided to regulators to support a mutagenicity assessment particularly for conflicting predictions. When available, regulatory feedback on the assessment will be provided. Additionally, a case example will be presented where additional support beyond a two system negative prediction was requested by a regulatory authority.
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(Q)SAR Evaluation of potentially mutagenic impurities: regulatory experience with out of domain results
ICH M7 recommends using expert rule-based and statistically-based (Q)SAR methodologies to predict Ames outcome for low level impurities. Predictions from the 2 methodology approach, supported by expert knowledge as necessary, are considered adequate for evaluating mutagenic potential. However, regulatory decision making becomes more complex when an out of domain result occurs (i.e., impurity structure falls outside of the chemical space adequately represented in a model training set). Based on regulatory experience, out of domain results are relatively common. Although ICH M7 does not specifically discuss this scenario, there are options to address the lack of a second valid prediction. These options include using a 3rd (Q)SAR model and/or applying expert knowledge.
This presentation will summarize recent FDA/OND experience with (Q)SAR submissions. In addition to providing recommendations regarding out of domain results, a brief summary of approaches used in regulatory submissions as well as case studies will be described.
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Dr. Goodwin received his BS in Biology from the College of William and Mary, Williamsburg, VA (2005) and a PhD in Cellular and Molecular Medicine from the Johns Hopkins University School of Medicine, Baltimore, MD (2010). He continued his research career at Johns Hopkins, first as a postdoctoral fellow at the Sidney Kimmel Comprehensive Cancer Center and then held a research faculty appointment in the Department of Medicine. Dr. Goodwin’s research interests included inflammation-associated tumorigenesis and pharmacological approaches targeting polyamine metabolism.
Dr. Goodwin has authored 11 peer-reviewed journal articles and book chapters. He has made scientific and educational presentations on regulatory topics to internal (FDA) and external audiences, including the 2016 Annual Meeting of the American College of Toxicology.
Safety assessment of potentially genotoxic impurities: case study from regulatory perspective
This case study will provide an example of the regulatory review process for the safety evaluation of potentially genotoxic impurities for a late stage small molecule drug. In silico and in vitro genetic toxicology assessment of a library of structures based on the principles of the ICH M7 Guidance will be discussed. A number of different strategies were utilized to qualify groups of structures, including in silico results, Ames assay data, structural analogy to non-genotoxic compounds, and inherent reactivity or purging during the manufacturing process. Particular focus will be placed on the review process within FDA, as well as a discussion of the acceptability of non-standard (miniaturized) Ames assay data.
Presentation in PDF
Stefan Pfuhler, PhD
Procter and Gamble
Role and status of 3D tissue models in genetic toxicology: skin and beyond
Regulatory restrictions on animal use have increased the reliance of risk assessors and regulators on in vitro test systems. In vitro assays are usually based on mammalian cell culture systems using ‘immortal’ cells with compromised cell functions. Such 2-dimensional static cell culture systems are artificial and far removed from the in vivo state, while 3D tissue constructs allow for more natural cell-cell and cell-matrix interactions and show ‘in vivo like’ behavior. Ideally, tissue-based assays could replace the animal studies as follow-up tools to verify results from standard in vitro assays. 3D tissues have developed over time as a standard platform for toxicological testing and efficacy testing and are increasingly being utilized for assessment of genotoxicity potential. For example, 3D human skin model-based genotoxicity assays have been developed and optimized to investigate dermal exposure scenarios which account for the vast majority of consumer exposures to chemicals used in cosmetic products. The two assays that have recently undergone validation, the Reconstructed Skin Micronucleus assay (RSMN) and the 3D skin Comet assay, have both demonstrated good transferability and inter- and intra-laboratory reproducibility and have achieved an excellent overall predictive capacity (about 90%) based on testing of >50 coded chemicals. In addition, initial studies with silica nanoparticles showed favorable results in an RSMN assay (and a lack of exposure) while producing unfavorable results in standard ‘2D’ cell cultures, thereby indicating the usefulness of these tissue-based methods for particulates. Recognizing the importance of exposure routes other than skin, the data generated to date using 3D liver and 3D lung models is more limited and their current status will be briefly reviewed. The next logical step towards modeling in vivo situations more completely would be combining more than one organ in an ‘organ on a chip’ approach. Initial data from such an approach, connecting human 3D skin models with HepaRG based liver mini-reactors via an artificial circulatory system will be shown. Such a system, once optimized, should allow for assessing route of exposure impact on toxic responses. Even more complete modelling of in-vivo-like distribution, metabolism and excretion of a test chemical may be achievable with the hen’s egg test for micronucleus induction (HET-MN). This assay uses fertilized hen´s eggs and its validation is in progress, thus far showing very good alignment with expected results including pro-mutagens that require activation via liver enzymes.
Presentation not available
Application of New Cell Culture Systems: The Role for 3D Organ Models in Genetic Toxicology
Conventional in vitro toxicology models (i.e., Tox21 assays) have employed proliferating cells, typically transformed/immortalized, on rigid substrata to study chemical-induced toxicity potential. While these approaches have been successfully applied to identify chemical interaction potential with individual molecular pathways (i.e., nuclear receptors via transcriptional reporter), they lack the capability to model human tissue function and integrated pathway responsiveness to xenobiotic exposure. In Phase III of the Tox21 program, the National Toxicology Program is assessing the utility of in vitro models with improved physiological-relevance and xenobiotic metabolism competence for chemical toxicity research & screening. Data-rich high dimensional assays (i.e., transcriptomics, high content imaging) are being integrated with these models to probe tissue modeling and biological response to chemical exposures. In this presentation we will highlight our recent progress in this area including the development of highly differentiated 2D & 3D models (384-well format) with HepaRG cells with tissue-like drug metabolism competence, and their response to compound exposures (i.e., metabolically-activated) using our new transcriptomic platform to discriminate chemical-biological pathway perturbations in a potency-driven framework.
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MIMETAS’ Organ-on-a-Chip platform for predictive toxicology
Drug toxicity remains a major issue in drug discovery and stresses the need for better predictive models. Here, we describe the development of a perfused renal proximal tubule cell (RPTC) model in Mimetas’ OrganoPlates® to predict kidney toxicity. The OrganoPlate® is a microfluidic platform, which enables high-throughput culture of microtissues in miniaturized organ models. In OrganoPlates®, extracellular matrix (ECM) gels can be freely patterned in microchambers through the use of PhaseGuide technology. PhaseGuides (capillary pressure barriers) define channels within microchambers that can be used for ECM deposition or medium perfusion. The microfluidic channel dimensions not only allow solid tissue and barrier formation, but also perfused tubular epithelial vessel structures can be grown. We have developed several multi-cellular models for toxicity study including liver, brain, gut, and kidney. The presentation will focus on our kidney renal proximal tubular model which reconstructs viable and leak-tight boundaries for performing cytotoxicity, as well as transport and efficacy studies. Human RPTC (SA7K clone, Sigma) were grown against an ECM in a 3channel OrganoPlate®, yielding access to both the apical and basal side. Confocal imaging revealed that the cells formed a tubular structure. Staining showed tight junction formations (ZO-1), cilia pointing into the lumen (acetylated tubulin) and correct polarization with microvilli on the apical side of the tubule (ezrin). Tightness of the boundary over several days was shown by diffusion of a dextran dye added to the lumen of the tubule. Addition of toxic compounds, such as cisplatin, resulted in disruption of the barrier which could be monitored in time and correlated with DNA damage by dH2AX. The time point of loss of integrity corresponds with the concentration and the toxic effect of the compound. Furthermore, fluorescent transport assays showed functional transport activity of in- and efflux transporters. The 3D proximal tubules cultured in the OrganoPlate® are suitable for high-throughput toxicity screening, trans-epithelial transport studies, and complex co-culture models to recreate an in vivo-like microenvironment.
 S. J. Trietsch, G. D. Israëls, J. Joore, T. Hankemeier, and P. Vulto, “Microfluidic titer plate for stratified 3D cell culture.,” Lab Chip, vol. 13, no. 18, pp. 3548–54, Sep. 2013
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Standard genetic toxicology assays are intended for hazard identification only and provide very little mechanistic information. Without proper mode of action information, it is often very difficult to establish clear structure activity relationships and to design appropriate in vivo follow-up studies. This often leads to the discontinuation of valuable compounds that pose little to no risk to exposed humans. Recent developments in new assay platforms allow for the collection of mode of action information concurrently or as follow-up to the assays to positive responses in the standard genetic toxicology battery.
However, most of these technologies have not been evaluated in the context of their use to establish a cellular genotoxic mode of action.
The first part of the workshop aims to discuss technologies like toxicogenomics, flow cytometric biomarker assays and other mechanistic platforms that could help to establish a genotoxic mode of action. For each of these technologies, the basic platforms will be described and specific examples of each application will be shown. In addition, technologies will be evaluated for favorable and unfavorable properties using a SWOT (Strengths, Weaknesses, Opportunities, Threats) analysis.
The second part of the workshop is intended to establish key cellular and genetic events for defined molecular initiating events like tubulin poisons, alkylating agents and topoisomerase inhibitors. Similar to adverse outcome pathways (AOPs), the sequence of events will be summarized in a pathway map and possible ways to detect key cellular events will be proposed and discussed. The outcome of the workshop will be presented on the 2nd day of the GTA meeting.
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