International Conference on
Technology in Cancer Research and
Treatment in the New Millennium
Adenine Press
2066 Central Avenue
Schenectady, NY 12304
Phone: 518 456-0784
Fax: 518 452-4955
stone@adeninepress.com
http://www.cancerwatch.org
2066 Central Avenue
Schenectady, NY 12304
Phone: 518 456-0784
Fax: 518 452-4955
stone@adeninepress.com
http://www.cancerwatch.org
Pattern Recognition in Drug Discovery
W. Graham Richards, Ph. D.
Department of Chemistry, University of Oxford, New Chemistry Laboratory, South Parks Road, Oxford OX1 3QT, UK
Techniques developed in the area of artificial intelligence such as computer vision and object recognition can be adapted for use in computational chemistry and drug discovery. The alignment of molecules is often the first step in many design projects and this can be problematic if the structures are diverse, particularly with respect to size. It is possible to handle the problem however, using methods from computer vision. Similarly the multiscale approach used in medical imaging can be adapted to find binding sites in proteins.
Once available, knowledge of a binding site in atomic detail permits the screening of libraries of small molecules as potential drugs or inhibitors. We are undertaking a massive screening exercise of this nature using screen saver technology in the search for potential anti-cancer drugs.
Clinical Application of Proton MR Spectroscopy in the In Vivo Evaluation of Tumors: Diagnosis, Treatment Status, and Treatment Planning
Lester Kwock, Ph.D.
Department of Radiology, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7515
Proton magnetic resonance spectroscopy (1H-MRS) by itself or in conjunction with functional magnetic resonance imaging (MRI) techniques (diffusion and perfusion MRI) has been increasingly receiving more attention from radiologists, neurosurgeons, radiation and medical oncologists in the clinical evaluation of human tumors. In the past, most human MR spectroscopy studies were performed at major medical centers but the availability of commercial software spectroscopy packages has increased its use in daily clinical situations at other institutions worldwide. Good quality 1H -MRS can be performed on most clinical 1.5 Testla MR imaging units fitted with commercially available automated software. Single volume or multivolume 1H -MR spectra with volume elements as small as 0.5 cc can be obtained in time periods as short as 10 minutes. Thus, at many institutions, clinical 1H -MRS studies have been integrated into the routine clinical MR evaluation of not only tumor processes but also other disease processes (1-3).
1. Castillo, M. and Kwock, L., Clinical applications of proton magnetic resonance spectroscopy in the evaluation of common intracranial tumors, Topics in Mag. res. Imaging 10(2), 104-113, 1999.
2. Taylor, J.S., Langston, J.W., Reddick, W.E., Kingsley, P.B., et al., Clinical value of proton magnetic resonance spectroscopy for differentiating recurrent or residual brain tumor from delayed cerebral necrosis, Int. J. Radiation Oncology Biol. Phys. 36(5), 1251-1261, 1996.
3. Castillo, M., (ed.), Proton MR Spectroscopy of the Brain, Neuroimaging Clinics of North America, Vol. 8(4), W.B. Saunders Co., Philadelphia, 1998, pp. 713-926.
4. Kwock, L., Localized MR spectroscopy: Basic Principles, Neuroimaging Clinics of North America 8(4), 713-732, 1998.
5. Fulham, M.J., Bizzi, A., Dietz, M.J., Shih, H.H-L, et al., Mapping of brain tumor metabolites with proton MR spectroscopic imaging, Clinical Relevance, Radiology 185, 675-686, 1992
6. Vigneron, D.B., Nelson, S.J., and Kurhanewicz, J., Proton Chemical Shift Imaging of Cancer, in Magnetic Resonance Imaging of the Body, Chapter 12, 3rd edition, C.B. Higgins, H. Hricak, and C.A. Helms (eds.), Lippincott-Raven Press, New York, 1997, pp. 205-220.
After the Human Genome Project
Charles R. Cantor, Ph.D.
Sequenom Inc, San Diego CA and Sequenom GmbH, Hamburg, DE
Now that we have a nearly complete DNA sequence for the human genome and a number of model organism genomes, the course of future biomedical research is likely to be altered substantially. For example, discovery and mapping of DNA sequence polymorphisms in many species is already easier in silico than experimentally. High throughput analytical tools like automated mass spectrometry or expression profiling using DNA chips are now available but these require extensive computation both for experimental design and for analysis of the resulting deluge of data. In this presentation I will first look back and evaluate the success of the Human Genome Project, and then I will look forward to try to predict its impact in a number of aspects of future biomedical research.
The genome project was essentially a brute force approach that used newly developed technology to achieve great economies of scale. However, as we go forward, the increasing power of available technology once again will allow hypothesis testing to drive many aspects of human genetic research. For example, Sequenom's genotyping technology uses an array of nano-liter samples to allow automated input and MS analysis. Up to 3840 samples can be analyzed in a single run, in about one hour. Genotypes are measured by using a primer extension reaction to convert DNA single nucleotide polymorphisms (SNPs) into length changes. The development of new SNP assays is totally automated. This allows whole genome SNP scans to be carried out at reasonable costs and in reasonable time frames. We are in the process of assaying all coding SNPs against several different population pools. Perhaps the most interesting of these are pools of healthy people stratified by age. Using these pools SNPs responsible for significant human morbidity or mortality can be discovered because their allele frequency will decrease as a function of age. A number of interesting genes with major health impact have already been discovered this way in a remarkably short time period.
Applications of Dynamic Contrast Enhanced MRI in Oncology
Orhan Nalcioglu, Ph.D., FIEEE, FAAPM, FISMRM
Center for Functional Onco-Imaging, University of California – Irvine, CA 92697-5020
Formation of new blood vessels within tumors is essential for the growth and spread of cancer. This process, termed angiogenesis, depends on the recruitment of pre-existing blood vessels from normal surrounding tissue. Tumors release specific proteins, such as vascular endothelial growth factor (VEGF) to stimulate blood vessel formation. New vessel formation results in a higher vascularity and a leakier structure, which can be detected by dynamic contrast enhanced MRI. This technique has been shown as a non-invasive means to measure the vascular characteristics in tumors. In the past several years we have been working on development of the technique in conjunction with pharmacokinetic modeling analysis to derive the parameters characterizing vascular volume and vascular permeability in tumors. The former is related to the blood flow supplied to the tumor, and the latter is related to the leakage status (or, maturity) of vessels, which are two important physiologic properties of a tumor. We have shown in animal models that vascular volume can be used to predict the outcome of chemotherapy, and vascular permeability may serve as an indicator for aggressiveness of tumor’s growth. We found that faster growing tumors have leakier vessels (with higher vascular permeability), and tumors with higher grade also have leakier vessels. In this presentation we will describe the technique and present several applications to demonstrate how the technique may be used for diagnosis and to assess response to treatment.
Alphavirus Vectors as Tools in Cancer Gene Therapy
Kenneth Lundstrom, Ph. D.
F. Hoffmann-La Roche, Research Laboratories, Basel, Switzerland
Introduction
Viral vectors have shown great potential of gene delivery both in vitro and in vivo and should therefore be suitable for cancer gene therapy applications. Expression vector systems have been developed for three common alphaviruses, Semliki Forest virus (SFV) (1), Sindbis virus (2) and Venezuelan equine encephalitis virus (VEE) (3). Different types of expression vectors have been constructed (Figure 1): 1). Replication-competent vectors, containing the full-length viral genome and an additional subgenomic promoter and the foreign gene to be expressed. 2). Replication-deficient vectors, containing the viral nonstructural (replicase) genes and the foreign gene of interest. These vectors require a helper vector virus packaging. 3). Layered DNA vectors, where an RNA polymerase II expression cassette drives the transcription of self-replicating RNA. Mainly the replication-deficient vectors and the layered DNA vectors have been used for cancer gene therapy applications. Features that have made alphavirus attractive are rapid high-titer (109-1010 infectious particles / ml) recombinant particle production, broad host range, efficient RNA replication in the cytoplasm of host cells, extreme expression levels of heterologous proteins and induction of apoptosis of host cells.
Several human prostate tumor cell lines as well as ex vivo infected prostatic duct epithelia showed high levels of SFV-mediated b-galactosidase expression and induction of apoptosis (4). Stereotactic injections of SFV-LacZ virus into the striatum and amygdala of rat brain resulted in high local reporter gene expression (Figure 2) (5). Therapeutic effect of intratumoral injection of SFV vectors expressing the p40 and p35 subunits of interleukin-12 has been tested in a mouse B16 melanoma model resulting in significant tumor regression and inhibition of tumor blood vessel formation (6). Repeated injections increased the anti-tumor response and most encouragingly no immune reaction against SFV was detected. In another study, nude mice with implanted human lung carcinomas were injected with SFV-LacZ, SFV-GFP and empty SFV vectors, which all induced a p53-independent apotosis (7). Repeated injections on 3 consecutive days followed of 3 more injections a week later resulted in significant tumor regression, but no immune response against SFV.
For safer and more precise gene delivery with alphavirus vectors attempts have been made to target the virus infection. The introduction of IgG binding domains of protein A in the E2 envelope protein of Sindbis virus generated chimeric vectors with a 105-fold lower infection rate of normal host cells (8). However, cells treated with monoclonal antibodies to cell surface proteins resulted in efficient infection through the protein A domains. Furthermore, introduction of a- and bhCG gene sequences into the Sindbis virus envelope allowed specific infection of choriocarcinoma cells (9).
Production of Retrovirus-Like Particles
SFV vectors have been used for production of retrovirus-like particles. Co-transfection of BHK cells with SFV vectors expressing the gag-pol, env and LTR-y+-neo-LTR from Moloney murine leukemia virus (MMLV) led to packaging of retrovirus-like particles possessing reverse transcriptase activity (10). In another approach, retrovirus virion RNA transcribed by the SFV vector was introduced into retrovirus packaging cell lines, which resulted in fully functional retrovirus particles (11). Moreover, replacement of the SFV envelope protein genes with the env gene from murine leukemia virus (MuLV) generated minimal virus particles with a targeted infection of cells carrying MuLV receptors (12).
Vaccine Strategies
Alphavirus vectors have been used in the form of replication-deficient virus particles, naked RNA or layered DNA vectors for immunization of animals to obtain cytotoxic T cell (CTL) responses and protection against viral or tumor challenges (13). A single intramuscular injection of of SFV-LacZ RNA prolonged the survival of mice with established tumors and protected mice from tumor challenge (14). In another study it was demonstrated that immunization with 5 ¥ 106 SFV particles expressing human papillomavirus early genes E6 and E7 protected animals from cervical cancer challenge (15). The use of alphavirus-based self-replicating DNA vectors is tempting because 1,000-fold lower DNA concentrations compared to conventional DNA vectors induce antigene-specific responses (16).
Vector Development
The strong cytotoxicity and short-term expression have limited the use of alphavirus vectors. A novel noncytopathogenic Sindbis virus vector with a point mutation in the nsP2 gene was obtained (17). Recently, novel SFV vectors showed less inhibition of host cell protein synthesis and substantially prolonged survival of host cells (18). Moreover, alphavirus vectors allowing persistent replication have been developed (19). These vectors should permit long-term expression of recombinant proteins. In conclusion, all these novel vectors should contribute to potentially new gene therapy applications of alphavirus vectors, including antisense, ribozyme and RNA interference technologies, to increase efficacy in cancer therapy.
References
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3. Davis et al., Virology 171, 189-204 (1989).
4. Hardy et al., Int. J. Mol. Med. 5, 241-245 (2000).
5. Lundstrom et al., Gene Ther. Mol. Biol. 3, 15-23 (1999).
6. Asselin-Paturel et al., Gene Ther. 6, 606-615 (1999).
7. Murphy et al., Gene Ther. 7, 1466-1482 (2000).
8. Ohno et al., Nat. Biotech. 15, 763-767 (1997).
9. Sawai & Meruelo, Biochem. Biophys. Res. Comm. 248, 315-323 (1998).
10. Li & Garoff, Proc. Natl. Acad. Sci. USA 95, 11658-11663 (1996).
11. Wahlfors et al., Hum. Gene Ther. 8, 2031-2041 (1997).
12. Lebedeva et al., J. Virol. 71, 7061-7067 (1997).
13. Lundstrom, Intervirology, in press (2001).
14. Ying et al., Nat. Med. 5, 823-827 (1999).
15. Daemen et al., Gene Ther. 7, 1859-1866 (2000).
16. Berglund et al., Nat. Biotech. 16, 562-565 (1998).
17. Agapov et al., Proc. Natl. Acad. Sci. USA 95, 12989-12994 (1998).
18. Lundstrom et al., Gene Ther. 4, 23-31 (1999).
19. Perri et al., J. Virol. 74, 9802-9807 (2000).
Transimmunization: Large scale Dendritic Cell loading with Apoptotic Tumor Cell Antigens
Carole Berger, Ph.D., Michael Girardi, M.D., Richard Edelson, M.D.
Yale University School of Medicine
An ideal anti-cancer immunotherapy would be tumor-specific, capable of being adapted to the changing tumor population, simple to rapidly construct, and safe to administer. It would allow for the immunization against a spectrum of the tumor's antigens, without the need for their identification. We are now aware that extracorporeal photopheresis (ECP) - in which a patient’s leukocytes are isolated, passed though an ultrathin clear plastic plate, and exposed to 8-methoxypsoralen and ultraviolet A light prior to reinfusion - is a relatively efficient dendritic cell therapy and the first FDA approved selective immunotherapy for cancer. ECP has demonstrated efficacy in the treatment of cutaneous T cell lymphoma (CTCL), as well as oligoclonal T cell mediated diseases such as graft-versus-host-disease (GVHD). ECP involves the simultaneous induction of monocyte-to-dendritic cell (DC) differentiation and malignant/pathogenic T cell apoptosis. In CTCL patients, the transition of monocytes to immature DCs was observed by the expression of cytoplasmic CD83 and membrane CD36 in the absence of membrane CD14 staining, as well as induction of membrane CD83 expression. Differentiating DCs were highly phagocytic and readily engulfed apoptotic malignant T cells. Differentiating DCs demonstrated functional activity of antigen presenting cells (APCs) by stimulating marked proliferation of normal alloreactive lymphocyte responders, indicating increased expression of membrane MHC class II. This approach provides a clinically practical means of developing tumor-loaded cells maturing DCs without the requirement of exogenous cytokines, identification of tumor antigens, or excessive cellular manipulation or isolation. Development of anti-tumor vaccines via this method of large scale dendritic cell loading with apoptotic tumor cell antigens is potentially useful in the immunotherapy of other cancers.
Effective Delivery of Therapeutic Agents by in vivo Electroporation
Richard Heller Ph. D., Loree Heller Ph. D., M. Lee Lucas M.S., Richard Gilbert Ph.D. and Mark J. Jaroszeski Ph. D. Department of Surgery, College of Medicine; College of Engineering; Center for Molecular Delivery University of South Florida, Tampa, FL.
The efficient delivery of therapeutic molecules to tissues is an important tool for the treatment of a variety of diseases. The uptake of molecules through the cell membrane can be facilitated by use of electroporation, a physical phenomena that temporarily permeabilizes cell membranes. When membranes are in a permeabilized state it is possible for molecules that do not normally pass through the membrane to gain intracellular access. In vivo electroporation can be used for drug or plasmid DNA delivery either alone or in combination. With respect to drug delivery, previous studies by our group in both animal and human trials have obtained response rates of >80% for several types of malignancies. Clinical studies were very successful in the treatment of skin malignancies achieving objective rates of 99% and complete response rates of 91% in patients with melanoma and basal cell carcinoma. Previous studies have also demonstrated that ECT could be used to effectively treat human rhabdomyosarcomas in athymic rats. It is believed that this procedure can be utilized as a limb sparing procedure in the treatment of this disease.
A key issue in the development of effective gene therapy protocols is the development of appropriate delivery methods. A common problem of gene therapy treatments is inefficient gene delivery and insufficient expression. Typically, the goal is to target gene delivery to a particular type of cell or to cells within a specific tissue. The delivery of genes that code for biologically active compounds is envisioned as a treatment for many diseases including cancers and metabolic disorders. With respect to gene delivery, our group has initiated several studies to investigate the use of in vivo electroporation for plasmid DNA delivery in a variety of tissues. Protocols were developed to allow delivery of the plasmid directly to either tumor, normal skin, normal liver or normal muscle. Initial work was performed using plasmids coding for either the reporter gene luciferase or -galactosidase. Different electric pulse conditions were needed to obtain peak expression at each of the various sites. However, in each case expression levels were significantly increased when compared to injection alone. To test the therapeutic potential, several cytokine genes were delivered to tumors either alone or in combination. Although the work is in its initial stages, encouraging results have been obtained. Long term complete regressions have been obtained with various treatment protocols of a murine melanoma. This work is being continued and expanded to optimize the procedure and confirm these initial results. (Supported by research grants from the NIH - R21 DK055588 and RO1 CA76181 also supported by the Center for Molecular Delivery, Univ. of South Florida).
The Radiation Oncologist’s Perspective on Stereotactic Radiosurgery
William F. Regine, M.D.
Department of Radiation Medicine, University of Kentucky, Lexington, KY 40536-0084
Unfavorable Gliomas
The basis for single-fraction Stereotactic Radiosurgery (SRS) is largely historical in nature and rooted in conventional thinking. This is derived from the original use of SRS in the treatment of arteriovenous malformations (AVMs), where the benefit of single-fraction high-dose radiation is clearly optimal in terms of addressing AVM obliteration kinetics. However, tumor cell kinetics are not the same as AVM obliteration kinetics and therefore may not be optimally addressed by single fraction SRS. In addition, fractionated (F) SRS, as compared to single-fraction SRS, should allow for sparing of normal tissue damage. The relatively noninvasive nature of SRS allows for the potential of exploiting the use of FSRS and also allows for consideration of delivering FSRS in a split-course fashion. This provides an additional advantage over what can normally be achieved by use of stereotactic brachytherapy, in that sterotactic brachytherapy is likely to be performed only once in the course of a patient's primary treatment. This feasibility, along with the previous discussion, serves as the rationale for our current study design combining FSRS boost in a split-course fashion pre- and post-conventional external beam radiation therapy (CEBRT). This strategy exploits tumor and/or normal tissue cell kinetics, inclusive of attempting to counteract the initial accelerated tumor growth phase pre-CEBRT, thereby decreasing the rate of clinical tumor progression during CEBRT. This split-course design should also help to counteract the effect of accelerated tumor repopulation post-CEBRT. Our unique experience with this approach in patients with unfavorable gliomas will be presented.
Brain Metastases
While whole brain radiation therapy (WBRT) remains a standard of care in patients with brain metastases, it's potential neurocognitive morbidity remains a poorly understood concern. Despite this, and with an increasing role of surgery and/or SRS in the primary management of patients with brain metastasis, recently reported experiences withholding WBRT as part of primary therapy for brain metastases have not analyzed the potential effects on neurological functional status and/or neurocognition associated with the increased risk of brain tumor recurrence seen with such a strategy. We recently evaluated the risk of symptomatic brain tumor recurrence and associated neurologic deficit in 36 patients treated for newly diagnosed unresected brain metastases treated by Gamma Knife SRS alone followed by planned observation. Among the 17 patients (47%) developing brain tumor recurrence, 71% (12/17) were symptomatic and 59% (10/17) had an associated neurologic deficit. Also of interest, the author (WFR) performed a secondary analysis of Radiation Therapy Oncology Group (RTOG) study 91-04, a randomized phase III study of accelerated hyperfractionation (AH) versus standard accelerated fractionation (AF) in over 300 patients with unresected brain metastases. Use of AH as compared to AF-WBRT was unassociated with a significant difference in neurocognitive function as measured by Mini-Mental Status Exam (MMSE) in this patient population with unresectable brain metastases and limited survival. However, control of brain metastases had a significant impact on MMSE. It is only among patients with "uncontrolled" brain metastases that a "meaningful" change/drop in MMSE score (1-point) is seen and becomes statistically significant at 3 months. Details of these studies will be presented and implications with regards to the complementary role of WBRT in patients undergoing SRS for brain metastases will be discussed.
Targeting Biological Pathways in Breast Cancer
Debu Tripathy, M.D.
UCSF Carol Franc Buck Breast Care Center
University of California at San Francisco
1600 Divisadero St, 6th floor, Box 1714
San Francisco CA 94115-1714
Our expanding knowledge of genetic and biochemical abnormalities responsible for the development of cancer has led to the identification of new targets for therapy that may be more effective and less toxic. The identification of the HER2/neu oncogene 20 years ago led to further studies that linked amplification and overexpression of this gene more a more aggressive phenotype in early stage breast cancer. The gene encodes a transmembrane tyrosine kinase growth factor receptor whose signaling pathway can lead to several end results such as cell proliferation and alterations in responsiveness to hormonal and chemotherapeutic agents. Monoclonal antibodies to this receptor can inhibit cancer cell growth and also modulate responsiveness to certain chemotherapy drugs. Phase I, II and III clinical trials have now shown that a humanized monoclonal antibody to HER2/neu, trastuzumab, can lead to transient but clinically significant remissions in some patients with advanced metastatic breast cancer whose tumors overexpress HER2/neu. A randomized trial comparing chemotherapy alone to chemotherapy plus trastuzumab showed an improvement in the likelihood of response, duration of remission and survival with the addition of trastuzumab. While this therapy represents a large step forward in targeted cancer therapy, the infrequent side effect of cardiomyopathy serves to remind us that even cancer-related pathways are important in normal physiology. Furthermore, the fact that many patients are resistant to this treatment signifies that many carcinogenic signals are redundant, and that other modulating influences must be identified in order to continually improve clinical results. Currently, numerous strategies against other growth factor receptors and other components of the signaling pathways are being tested in the laboratory and the clinic. Screening for small molecules or natural compounds with properties that modulate these pathways may lead to more candidate cancer drugs with greater specificity and clinical efficacy.
Enhancing the Effectiveness of Gene and Drug-based Cancer Therapy by Electropermeabilization
Dietmar P. Rabussay, Ph.D.
Genetronics, Inc., San Diego, CA
The effectiveness of potentially powerful cancer therapeutics is often limited by their inability to efficiently penetrate the cell membrane. This is true for certain chemotherapeutic drugs, as well as DNA for cancer gene therapy. Electropermeabilization (EP, also known as "electroporation") of the cell membrane renders the membrane temporarily permeable by inducing "pores" across the lipid bilayer. EP is achieved by applying electrical pulses of short duration (micro to milliseconds) and high field strength (100-1500 V/cm) directly to the tissue to be treated, using invasive needle electrodes. This treatment increases intracellular delivery of DNA several hundred-fold and the delivery of certain anticancer drugs up to 5000-fold.
Anticancer drugs may be injected intratumorally or intravenously, followed by EP of the tumor to be treated. EP is administered by inserting needle-array electrodes into the tumor tissue and applying the electrical pulse. This treatment has been shown to be highly effective in eliminating various types of tumors in mice, using different anticancer drugs. Bleomycin was found to be the most effective drug in combination with EP. Upon treatment with bleomycin/EP, tumor cells are rapidly destroyed within 24-48 hours after treatment. Histological and other analyses show that most of that cell death is caused by apoptosis and that certain pro-apoptotic gene expression is enhanced while antiapoptotic gene expression is decreased. In mice, treated tumors generally shrink within 10 days, dry out, and the resulting eschar falls off within 3 to 4 weeks after treatment, exposing healthy tissue at the site of the former tumor. While bleomycin/EP treatment is highly effective against tumor tissue, normal tissue is only mildly and transiently affected.
Phase I/II clinical studies have shown bleomycin/EP to be effective in treating squamous cell carcinoma of the head and neck, basal cell carcinoma and melanoma. The MedPulser® system consisting of pulse generator and disposable needle-array electrodes has been approved for drug delivery in Europe and will be commercially available from Genetronics in the near future.
For cancer gene therapy studies, a system similar to the MedPulser has been developed for delivering plasmid DNA into tumor cells or cells of healthy tissue. Using this system, retardation of tumor growth or complete tumor regression in mice has been obtained with several gene constructs. EP has also proven highly effective for enhancing the immune response to DNA vaccines, both the humoral as well as the cellular response. This enhanced response was not only shown in mice but also in larger animals, including monkeys.
In conclusion, EP appears to be an attractive methodology to enhance the potency of anticancer drugs, cancer gene therapy and cancer DNA vaccines, by increasing the effectiveness of transmembrane delivery of drugs and DNA. While drugs and DNA may be delivered directly into cancer cells resulting in local treatment of tumors, DNA may also be delivered into healthy cells which then produce anticancer agents, thus facilitating systemic treatment of cancer.