Regine Kollek

Transforming genes and target cells of murine spleen focus-forming viruses.

Publisher Summary This chapter outlines the factors that determine leukemogenesis induced by acutely transforming retroviruses by the interaction with either myeloid stem or progenitor cells of the mammalian system. The acutely transforming retroviruses described will be limited to the spleen focus-forming viruses, defined as those retroviruses that induce a proliferative hematopoietic disease upon the intravenous injection of adult animals. The chapter discusses the molecular features of the three known groups of acutely transforming murine viruses: (1) viruses with recombinant env genes, (2) viruses with the mos oncogene, and (3) viruses with the ras oncogene. Data pertaining to the relevant proto-oncogenes and their products are also presented. It outlines the critical factors of the viral genome or transforming gene, based on structure-function analysis, which determine the oncogenicity of the retroviruses and its target cell specificity. It also discusses retrovirus-target cell interaction and outlines the experiments that are still necessary to fully understand the oncogenesis induced by spleen focus-forming viruses in the myeloid system.

Prospects and Limits of Pharmacogenetics The Thiopurine Methyl Transferase (TPMT) Experience

Thiopurine drug metabolism is a quintessential case of pharmacogenetics. A wealth of experimental and clinical data on polymorphisms in the thiopurine metabolizing enzyme thiopurine methyl transferase (TPMT) has been generated in the past decade. Pharmacogenetic testing prior to thiopurine treatment is already being practiced to some extent in the clinical context, and it is likely that it will be among the first pharmacogenetic tests applied on a regular basis.We analyzed the published TPMT data and identified some lessons to be learned for the future implementation of pharmacogenetics for thiopurines as well as in other fields. These include the need for comprehensive and unbiased data on allele frequencies relevant to a broad range of populations worldwide. The nature and frequency of TPMT gene polymorphisms in some ethnic groups is still a matter of speculation, as the vast majority of studies on TPMT allele distribution are limited to only a small subset of alleles and populations. Secondly, an appreciation of the limits of pharmacogenetics is warranted, as pharmacogenetic testing can help in avoiding some, but by far not all adverse effects of drug therapy. An analysis of six clinical studies correlating adverse thiopurine effects and TPMT genotype revealed that an average of 78% of adverse drug reactions were not associated with TPMT polymorphisms. Pharmacogenetic testing will thus not eliminate the need for careful clinical monitoring of adverse drug reactions. Finally, a careful approach toward dose increases for patients with high enzyme activity is necessary, as TPMT-mediated methylation of thiopurines generates a possibly hepatotoxic byproduct.

Disclosure of individual research results in clinico-genomic trials: challenges, classification and criteria for decision-making

While an ethical obligation to report findings of clinical research to trial participants is increasingly recognised, the academic debate is often vague about what kinds of data should be fed back and how such a process should be organised. In this article, we present a classification of different actors, processes and data involved in the feedback of research results pertaining to an individual. In a second step, we reflect on circumstances requiring further ethical consideration. In regard to a concrete research setting—the one of clinico-genomic research—we discuss what kinds of difficulties have to be faced when returning individual research results to trial participants. In a last step, we elaborate on a stepwise model to trigger the individual feedback process. Hence, this paper gives guidance on how to feedback individual research results in a specific research setting and responds at the same time to new challenges in the debate on the duty to return individual research findings.

Individualized Pharmacogenetic Therapy: A Critical Analysis

Objective: Individualized, or personalized, therapy is highlighted as the declared goal of pharmacogenetics. In this paper, the content and significance of the individualization concept are analyzed. Method: Our analysis is based on a systematic reading of the current literature pertinent to pharmacogenetics. Results: This analysis reveals that the pharmacogenetic understanding of individualization is based on a biomechanistic paradigm. In contrast to a notion of individualized therapy based on a biopsychosocial paradigm, this biomechanistic concept does not provide for individualization in psychosocial terms, but instead leads to the stratification and classification of patient populations. This finding does not necessarily cast doubt on the efficacy of pharmacogenetics, but does call its underlying ideology into question. Conclusion: The term ‘individualization of therapy’ does not reflect the real potential of pharmacogenetics, but instead represents a widely used and theoretically unjustified publicity slogan.

Pharmacogenetics, Adverse Drug Reactions and Public Health

Adverse drug reactions (ADRs) are a major public health problem. Pharmacogenetic testing prior to drug treatment is supposed to considerably alleviate this problem. The state of pharmacogenetic development was assessed by a systematic literature review, supplemented by expert interviews. Analysis of three case examples revealed that – with the exception of thiopurine methyltransferase (TPMT) – studies are lacking which unambiguously prove the clinical value of pharmacogenetic testing. Testing can prevent some, but by far not all ADRs. Since it does not compensate for clinical monitoring, pharmacogenetics can be regarded as add-on technology, applied in addition to established methods. A non-representative, explorative survey conducted amongst members of the German Society of Laboratory Medicine revealed that the demand for testing is limited and has not increased much, although a certain increase is expected in the future.

Viral transfer, transcription, and rescue of a selectable myeloproliferative sarcoma virus in embryonal cell lines: expression of the mos oncogene.

A derivative of the myeloproliferative sarcoma virus (Neor-MPSV) carrying the mos oncogene and dominant selection marker for neomycin resistance (Neor) was introduced into embryonal carcinoma and embryo-derived cell lines by transfection and infection using pseudotypes with Friend helper virus (Friend murine leukemia virus [F-MuLV]). Cells resistant to G418 (a neomycin analog) were cloned and expanded. Transductants retained an undifferentiated phenotype as judged by morphology, tumorigenicity, and cell-surface antigen analyses. Nucleic acid analysis of infectants revealed both Neor-MPSV and F-MuLV proviruses, although no virus was released. G418-resistant transductants remained nonpermissive for the expression of other proviruses and for subsequent superinfection. Northern analysis showed expression of full-length Neor-MPSV, as well as mos-specific subgenomic RNA. mos sequences were deleted from Neor-MPSV (Neor mos-1), and pseudotypes were used to infect embryonal carcinoma cells. No morphological differences were observed in either mos+ or mos- transductants as compared with parental cell lines. However, mos+ transductants showed an enhanced anchorage-independent growth compared with that of mos- transductants in agar cloning. PCC4 transductants were induced to differentiate with retinoic acid and superinfected with F-MuLV. Infection with viral supernatant in fibroblasts and in mice confirmed the rescue of biologically active Neor-MPSV.

The myeloproliferative sarcoma virus retains transforming functions after introduction of a dominant selectable marker gene

The dominant neomycin resistance gene (neoR) was introduced into the genome of the myeloproliferative sarcoma virus (MPSV), a replication-defective retrovirus carrying the mos oncogene. The resulting selectable neoR-MPSV virus did not lose its acute transforming property, unlike the results of attempts by other groups to insert marker genes into oncogenic viruses. NeoR-MPSV DNA was used to generate infectious virus by transfection followed by rescue with Friend or Moloney murine leukaemia virus. Infection of fibroblasts with this virus resulted in morphologically transformed cells which were resistant to the neomycin analogue G418. Segregation of the two functions (transformation and G418 resistance) was not observed in more than 500 independent viral transfers to fibroblasts. Furthermore, neoR-MPSV retained the leukaemogenesis-inducing properties of the wild-type virus. Myeloproliferation and G418-resistance transfer did not segregate after passage in mice.

Point mutations in the U3 region of the long terminal repeat of moloney murine leukemia virus determine disease specificity of the myeloproliferative sarcoma virus

The myeloproliferative sarcoma virus (MPSV) is made up entirely of sequences derived from the Moloney murine leukemia virus (Mo-MuLV) and the cellular mos oncogene. As other members of the Moloney murine sarcoma virus (Mo-MuSV) family, MPSV transforms fibroblasts in vitro and causes sarcomas in vivo. In addition, however, MPSV also causes an acute myeloproliferative disease in adult mice. The mos oncogene is essential for its transforming capacity, but sequences specific to the long terminal repeat (LTR) U3 region of MPSV account for its expanded target specificity as compared to Mo-MuSV (C. Stocking, R. Kollek, U. Bergholz, and W. Ostertag, Proc. Natl. Acad. Sci. USA 82, 5746-5750 (1985)). The U3 region of the LTR of MPSV is, however, closely related to that of the Mo-MuLV, and it appeared likely that the difference between MPSV and Mo-MuSV was caused by a divergent evolution of Mo-MuSV LTRs. In this paper, we show that this is not the case. The few nucleotide differences in the LTR between Mo-MuLV and MPSV are crucial for the expanded host range of MPSV. Moreover, Mo-MuLV-related gag sequences retained in MPSV are not essential for the distinctive biological properties of MPSV.

Ethical and legal requirements for transnational genetic research

List of abbreviations 1. Introduction 1.1. From clinical to clinico-genomic research: New ethical and legal challenges 1.2. The ACGT project: Developing an ICT infrastructure 1.3. Aim and structure of the book 2. Ethical requirements 2.1. Introduction 2.2. Informed consent 2.3. The right to know, the duty to inform, and the quality of feedback 2.4. Summary of consolidated ethical requirements 2.5. Outlook: Ethical challenges in the european context 3. Legal requirements 3.1. Introduction 3.2. Theoretical analysis 3.3 Data protection within a trans-european research project - using the example of ACGT 3.4. Data protection framework within genetic research networks 4. Legal conclusion 5. References 6. Appendix 1- legal terminology 7. Appendix 2 - relevant regulation

Post-genomic clinical trials: the perspective of ACGT

During the last few years, the ‘omics’ revolution has dramatically increased the amount of data available for characterizing intracellular events. As a result, a lot of patterns of gene expression were found that could be used to classify molecular subtypes of tumours and predict the outcome and response to treatment. Currently, the main focus is on interlinking the various data sources generated by high-throughput array technologies. Various groups have applied network analysis to gene data sets associated with cancer. ACGT, a project funded by the European Commission in the Sixth Framework Programme, goes far beyond these networks by the integration of clinical data. The ultimate objective of the ACGT project is the provision of a unified technological infrastructure, which will facilitate the seamless and secure access and analysis of multi-level clinical and genomic data enriched with high-performing knowledge discovery operations and services. By doing so, it is expected that the influence of genetic variation in oncogenesis will be revealed, the molecular classification of cancer and the development of individualized therapies will be promoted, and finally, the in silico tumour growth and therapy response will be realistically and reliably modelled. Achieving these goals, ACGT will not only secure the advancement of clinico-genomic trials, but will also achieve an expandable environment to other studies’ technologies and tools. Today, it is recognized that the key to individualizing treatment for cancer lies in finding a way to quickly ‘translate’ the discoveries about human genetics made by laboratory scientists into tools that physicians can use in making decisions about the best way to treat patients. This area of medicine that links basic laboratory study to clinical data, including the treatment of patients, is called translational research and is promoted by clinico-genomic trials running in ACGT. These clinico-genomic trials are scenario based and driven by clinicians. Today, two main clinico-genomic trials and an in silico experiment are interconnected within the ACGT project. The realization of these trials will act as benchmark references for the development and assessment of the ACGT technology. All ethical and legal requirements for clinico-genomic trials will be respected. A data protection framework will be set up for ACGT, which consists of an ACGT Data Protection Board, a Trusted Third Party responsible for the pseudonymization of the patient’s data and contracts between all participating hospitals research units or other users of genetic data. Patients who take part in clinico-genomic trials may be helped personally by the treatment(s) they receive. They get up-to-date care from cancer experts, and they receive either a new treatment being tested or the best available standard treatment for their cancer. Of course, there is no guarantee that a new treatment being tested or a standard treatment will cure the patient. New treatments also may have unknown risks, but if a new treatment proves effective or more effective than standard treatment trial patients who receive it may be among the first to benefit.