Molecular Genetics in Gynecological Oncology

Recent scientific developments have touched all areas of gynecological oncology including prevention, diagnostics, therapy modalities and post treatment care. All these lead to the ultimate goal of individualized care with improvement of the disease free and the overall survival by providing the best quality of life during and after treatment. One area supporting this attempt is the integration of molecular genetics.

In no era to date have there been as many advances in the scientific development, growth in theory and practice as in the last century. Initial key elements were the understanding of the cell and tissue as well as research in molecular structures and interactions. In 1938 Warren Weaver defined for the first time the term molecular biology as an “assortment of connected experimental systems for the physical, chemical and functional characterization of living organisms on the basis of relevant biological macro molecules”. His future view was further developed from different working groups and has led to new theories based upon actual molecular discoveries and an explosion of molecular understanding. Max Dellbrück postulated the theory of functionalism (1942), Maurice Wilkins and Linus Pauling the theory of structuralism (1948), Francois Jakobs and Elie Wollman the theory of regulation (1951) and James Watson and Francis Crick the information theory after the discovery of the double helical structure of DNA (1953). The revolutionary discovery of Watson and Crick started the era of the “omics-revolution”, e.g., genomics, proteomics, ribonomics, phenomics, epigenomics - the science of molecules and cellular phenomenons. In February 2001, the draft sequence of the human genome project (HUGO) was published in Nature and Science: 63 % or 2.21 billion base pairs were completed with the goal to achieve 100 % by spring 2003, the 50th year anniversary of Watson's and Crick's discovery. Following HUGO the next important step was the initiation of the human proteome project (HUPO) with the goal to functionally characterize gene products and their alterations. These two international collaborative projects have yielded a growth of knowledge, which connects the understanding of physiology and pathology of intra- and intercellular regulation cycles as well as the use of molecular techniques and targets for prevention, diagnostics and therapy.

Knowledge concerning molecular connections has always been a part of the historical and clinical development of gynecological oncology. Good examples are the discovery of steroid hormone receptors and the functionality of their ligands; the steroid hormones themselves and their resulting therapeutical options or the discovery of transmembrane receptors and the development of specific antibodies. Therefore, the selection and evaluation of relevant knowledge and ultimately the integration into the daily routine is an imminent challenge and often a hard to cover task next to the existing daily routine. This integration should be evaluated in view of technical, medical, sociological and economical terms.

The scientific advancement regarding the sequencing of the human genome was only possible through the continuous development of different molecular methods and their use for the analysis of molecular structures. Based on the molecular knowledge new approaches of molecular diagnostics and molecular therapies were developed.

Milestones in the development of diagnostic methods were the introduction of restriction fragment length polymorphisms (RFLP, 1980), the polymerase chain reaction (PCR, 1985) capillary electrophoresis (1990), thermo-stable polymerases (1995), the DNA chip analysis method (1996), and single nucleotide polymorphisms (SNPs) (1998). For molecular analysis solid or liquid, fixed or un-fixed, or native materials, e.g., serum, ascites, pleura exsudate, and solid tissue samples of different origins are used. The best choice for routine DNA analyses are classic and methodologically secure approaches like LOH, FISH and DNA sequencing. RNA analysis is directly dependent on the fixation of the sample and is therefore subject to problems. Nevertheless, RNA has especially become important in the comparative analysis of mRNA expression patterns of two different tissues using the “chip” technology, which bears an enormous scientific and clinical potential. However, the major part of molecular routine analysis is targeting proteins. The spectrum of analysis for qualitative and quantitative protein detection is especially useful for serum protein analysis, using RIA, ELISA (e.g., tumor markers, steroid hormone receptors), immuno-histochemical methods (e.g., steroid hormone receptors, growth factors and growth factor receptors, adhesion molecules, proteases) or the SALDI-method (e.g., unknown protein selection).

With the use of the first gene therapeutic treatment in the year 1990, there are great expectations in the possibility of molecular therapy. Currently, there are receptor mediated therapies like the classical hormone and anti-hormonal therapies and immuno-therapies (e.g., antibody therapies, like the Her-2/neu antibody ‘Herceptin’) used in practice. Receptor independent therapies were developed mostly for the treatment of carcinomas. Gene therapy strategies, which include the transfer of a specific gene using retroviruses (e.g., the MDR-1 gene in fibroblasts ex vivo, or the interleukin 4 gene in hematopoietic stem cells ex vivo for mamma carcinoma therapy, the wild type p53 gene in tumor cells ex vivo or the HSV-tk gene in tumor cells ex vivo for ovarian carcinoma therapy), have only experimental significance. Other experimental approaches test the targeted killing of carcinoma cells via the transfer of a toxin (e.g., the diphtheria A protein). These gene therapy strategies have not fulfilled expectations to date, therefore a routine protocol for receptor independent therapy strategies is not yet practical.

Molecular genetic methods have found entry into routine diagnostics. Next to technical details, like reproducibility, quality assurance, sensitivity, specificity, or quality control, particularly the technical potential from the analysis of a single gene or a combination analysis of multiple genes has to be discussed critically. These new technical possibilities of molecular diagnostics and their conclusive therapies have a deep influence in the relationship of medical doctors and patients who seek advice at different times in their lives. In this regard, medical aspects of predictive genetic diagnostics of late manifesting diseases and actual genetic diagnostics influence all aspects of gynecological oncology.

Predictive genetic diagnostics of late manifesting diseases is per definition the postnatal investigation and advising of individuals with genetic alterations, but who are pre-symptomatic and/or healthy. The goal is to gain information about the presenting genetic alteration. The probability of the potential disease risk is based upon the generalization of actual scientific knowledge as well as calculations using known risk models (for risk of disease or presence of mutations). With the cloning of genes in regards to disease disposition for carcinoma diseases (e.g., hereditary colorectal carcinoma syndromes [HNPCC; 2 p15 MHS2, 3 p21 MLH1, 2 q32 PMS1, 7 p22 PMS2, 5 q11 MSH3, 2 p22 MSH6]; hereditary breast-/Ovarian-Syndrome [HBOC; 13 q12 BRCA2, 17 q21 BRCA1]; Li-Fraumeni-Syndrome [LFS; 17 p13 TP53]) has increased the questioning of so-called “gene tests” in the frame of predictive postnatal diagnostics. Because predictive genetic diagnostics deals with the information risk of healthy individuals who have not yet developed the disease, the information acquired about a genetic modification or an increased health risk has to be dealt with special care. The use of genetic tests for late manifesting diseases is connected with a “new state of mind”. In the case of predictive genetic diagnostics a decision for or against genetic diagnostics as well as for the resulting options (e.g., early detection; prevention by medicaments or surgery, specific forms of therapies) has to be made by the individual. The above stated problems represent a new dimension of the relationship between the patient and medical doctor. In order to meet the expectations, equal and individual advising and possibly help with an interdisciplinary concept is necessary. Presently, for this reason, basic rules for clinical, human genetic and psychotherapeutic advice and help as well as for implementing genetic diagnostics, an interdisciplinary approach is used to evaluate the new knowledge in the best way for the patients (e.g., guidelines for the diagnostics of the genetic disposition for cancer diseases 1998). These individual requirements speak against the introduction of general routine genetic screening rather than for targeted genetic tests of susceptibility genes in the frame of predictive genetic diagnostics. Although genetic alterations, which are tested by using predictive genetic diagnostics, mostly exhibit a high penetrance and therefore a high chance to cause a disease, the majority of single diseases, however are not explainable due to the presence of one single high penetrance gene mutation. In the last years it was shown that predisposition can also be due to the presence of low penetrance genes as well as the accumulation of polymorphism patterns in a single gene resulting in an elevation of risk or modulation of disease. Only after additional involvement of cofactors, which are still partly unknown, the predisposition will become relevant. Therefore, it will not only be important in the future to detect mutations with high penetrance genes, but also to detect mutations of low penetrance genes as well as the pattern of polymorphisms.

Actual genetic diagnostics comprises the detection of genetic alterations in abnormal tissues. Genetic analyses are performed, however, the direct transfer of gained knowledge into the clinical work at present is only feasible with single diseases. Mostly, discrepancies can be seen between the scientific results from the basic and clinical diagnostic research as well as with the therapy. For example, an almost exponential growth of scientifically detected multiple genetic alterations in mamma carcinoma has been published. However, the transfer of this knowledge into the clinical work focuses only on a few modifications in the steroid hormone receptor, and the Her-2/neu oncogene and protein. Other factors for prognosis and prediction (e.g. p53, PAI-1, uPA, MIB1, CD95) are in discussion, but are to date not relevant for the clinical routine. Therefore, the routes of the carcinoma therapy focusing with the anti-hormone and the antibody therapy only relate to two cellular pathways. In the last year a new diagnostic approach, the analysis of expression profiles using chip technology, opened the possibility to detect genetic alterations or differences in the gene expression pattern with clinical therapeutic consequences. An individual RNA expression profile for the discrimination of BRCA1 and BRCA2 induced tumors or for the selection of therapies of sporadic tumors using estrogen receptor dependent genes, or expression of genes involved in metastasis show future possibilities in the clinical application of molecular diagnostics.

Despite the positive aspects for gynecological oncology due to the progress of molecular knowledge, the chances and risks of a “geneticalization of medicine” have to be discussed controversially. The progress of developed approaches and connected increase of new discoveries raises questions which target social and socioeconomic aspects. For example, the question of the transfer, examination and free access of knowledge, the social examination of results, individual anxieties as well as the historical and social urges are connected to the progress and knowledge of molecular associations. Among many, three individual aspects have to be defined: 1) the fear of loosing privacy as well as total exposure, 2) general and free access to scientific information without limitations through patents and 3) financing the integration of molecular knowledge into the health care system.

The growth of knowledge through predictive genetic examinations, so-called “gene tests” can have different advantages and disadvantages for the individual person by loss of privacy and total exposure. Regarding the above, individuals should be prospectively informed with consideration to the individual psyche. The possibilities for a primary, secondary or even tertiary prevention becomes possible through a definition of the risk burden. Similarly, this also bears the risk of discriminations against positive individuals in professional and social levels in the frame of social economical aspects like retirement-healthcare- and social security insurances. Thus, for some diseases special considerations were implemented to prevent disadvantages for clients by insurance companies through the definition of genetic risks. An open question for patients is, if gene tests in contrast to a thorough family anamnesis, or specific laboratory technical examinations, will yield better information remains to be shown. Because only approximately 360 diseases are known to date - with an estimate of 34 000 identified genes thus far - which could be possibly diagnosed using gene tests, the fear for loss of privacy and total exposure is not justified. Next to proven genetic alterations is also the modulation of penetrance through other genes and their modifications, or even possibly through environmental or developmental factors not to be defined. Therefore, in most cases the form, time point or risk of the disease cannot be determined. Despite these biases more than 60 % of the German population accepts genetic testing. A disadvantage of genetic examinations like abortions, misuse of data, eugenics and discriminations however, are viewed critically.

Today, financial limitations in the German healthcare system already exist. With the introduction of diagnosis related groups (DRG), disease management programs (DMP) as well as quality assurance programs (QAP) the compatibility of the health care system should be maintained with limited resources. Molecular gynecological oncology with a continuous growth of knowledge, which is transferred step by step into the healthcare system (translational genetics [from the bench to the bedside]) bears under the above mentioned programs new financial problems. Especially the access to scientific information and its uses is problematic with the patenting of genes and gene functions. Additionally, the introduction of diagnostic tests and newly discovered medicaments, which were elucidated from the knowledge of gene functions, are covered by monopole positions of patenting companies. For example, the analysis of BRCA1 and BRCA2 is patented worldwide by the Bio-Tech company Myriad Genetics (USA). Therefore, commercial genetic testing performed world wide, outside from scientific studies, is only feasible with permission of this company. Governments and companies need to discuss the application and the costs of these analyses in the frame of public health care. The implications for molecular diagnostics are that samples either have to be sent worldwide or a price has to be paid ($ 600.00 for BRCA1/2 analysis). The use of specific antibodies i.e. Trastuzumab (Herceptin) for the treatment of patients with metastasizing breast carcinoma (150 mg sample, dosage 2 mg/kg/once/week costs 750 EUR) reveals the same financial burden. The members of the healthcare system - government, insurance plans, and physician unions - all have to discuss with companies regarding the introduction of their products into the present financially limited healthcare system. The entire healthcare spectrum is therefore not only based upon the general, local or national criteria of the system, but also under the international, monopolistic and commercial aspects. The new dimensions for the healthcare system nowadays are hard to predict.

The future vision of molecular gynecological oncology participates at the accelerating development of gene technology and their future visions. In five years, all main genes of importance will be known and it is possible that the transfer of genetic and functional information to the clinical setting occurs; the chip technology and bioinformatics will be more miniaturized and therefore cheaper. In ten years a more detailed biological knowledge about functions in physiology and pathophysiology of cellular pathways will exist. The development and a slow initiation of gene therapy will progress; the chip technology using different cellular aspects (DNA, RNA, protein) will be a routine daily medical work. In fifteen years pharmacogenetics will be the area of individualization for preventive, diagnostic and therapeutic treatments. Genetic disease modifiers will gain relevance. In twenty years the routine genetic testing will be overall integrated, a life risk profile will be created which will lead to targeted preventative measurements and early interventions. With that the individual life quality will be improved. In twenty-five years genes and their testing will be history. The use of gene therapy or targeted therapy strategies based upon functional cellular knowledge will be routine. Molecular genetics stands for an unforeseen new and exciting future of gynecological oncology.

Matthias W. Beckmann