On completion of the Phase I trial, the basic toxicity pro \201le of the agent in question should be known and an appropriate dose for further trials identi \201ed. Phase II and III Unless the drug has proved to be unacceptably toxic, it will then be subje ct to Phase II, and if successful, Phase III testing. The aim of Phase II trials is to asse ss the ef \201cacy of the drug. Each Phase II trial will be undertaken in patients with on e particular tumor type and they will all be treated with the same dose and schedule. Whist toxicity will continue to be monitored, the patients \200\231 disease will also be assesse d for response. The aim of Phase III trials is to compare the new agent with existing best treatment for the disease in question. If a new drug is proven to have an acceptable toxicity pro \201le, ef \201cacy in one or a number of disease types, and to compare favorably against existing standard treatments fo r the particular disease setting, then the compound may be submitted to regulatory authorit ies to register it as a product \200\224the culmination of a costly process which can take in the region of ten years to complete. Novel radiotherapeutic approaches Radiotherapy is an effective anti-cancer treatment modality. Increasing the dose delivered to the tumor while sparing the surrounding nor- mal tissue will commonly improve local tumo r control. Novel radio- therapeutic approaches aim to: Increase the dose delivered to tumors, by more effectively target- ing the tumor volume (bu t not at the expense of normal tissue damage). Increase the biological effectiveness of rad iation by affecting the radiosensitization of the tumor (but not of normal tissue). (cid:2) (cid:2) OHO-40(635-654) 11/18/02 9:34 AM Page 647 NOVEL RADIOTHERAPEUTIC APPROACHES 647 (cid:2) Use different forms of radiation which have improved biological effects. (cid:2) Use changed schedules of radiation delivery in order to increase the differential between tumor kill and normal tissue damage. Targeted radiotherapy The biological properties of the tumor itself provide the basis for s elective irradiation. In principle, this strategy should be capable of eradicating tumor ce lls anywhere in the body, but it is currently at an early stage of development for many sit es. Iodine (well differentiated thyroid carcinomas) (cid:2) (cid:2) Monoclonal antibodies to cancer cell surface antigens (B-cell lymphoma) (cid:2) Catecholamine precursor analog meta-iodo-benzl-guanine (MIBG) (neuroblastoma) (cid:2) Somatostatin (neuroendocrine tumors) Until recently, the nuclides used in targeted radiotherapy were selected because of their availability and low cost rather than on accoun t of their physical or radiobiological properties. Considerable experience has been gained with (cid:2) emitters such as iodine-131 and yttrium-90, but short lived (cid:3) emitters ( e.g. astatine-211 or bismuth-212) or Auger-emitting radionuclides (e.g. iodine-125) are mor e potent cell killers and may \201nd a clinical role. Targeting strategies to improve clinical results include: Improving the speci \201city of the existing targeting agents (cid:2) (cid:2) Regional targeting (e.g. intraperitoneal therapy) (cid:2) Multiple treatments (cid: 2) Decreasing side-effects (myelosuppression due to a radiation of bone marrow is the most important) by using peripheral blood stem cell support or haemopoietic growth factors (cid:2) Combination of targeted radiotherapy with other treatments The effectiveness of tar geted radiotherapy is strongly dependent on the extent to which a suf \201cient dose may b e delivered to critical clusters of tumor clonogens, and micodosimetric studies have shown that \202uc- tuations in dose uniformity can adversely affect treatment outcome. The opti mal strategy may be to use \200\230cocktails \200\231 of radionuclides, or to supplement t argeted radiotherapy with external-beam irradiation. Boron neutron capture therapy is a specialized approach to targeted therapy in which non-ra dioactive boron atoms are selectively incor- porated into tumor cells. When exposed to a be am of \200\230slow \200\231 neutrons the boron emits (cid:3) particles locally within the tumor, causing dense ionizations and DNA damage. So far, clinical experience in brain tumor s has produced controversial results. OHO-40(635-654) 11/18/02 9:34 AM Page 648 NOVEL THERAPEUTIC STRATEGIES 648 Improvements in external-beam radiotherapy delivery Improvements in focusing the external r adiotherapy beam on the tumor and avoiding normal tissue should allow a safe increase in th e dose of radiotherapy delivered. Conformal therapy This multi-step technique uses 3D image reconstruction and treatment plan ning, combined with sophisticated treatment veri \201cation, to con- form a high dose of r adiotherapy to the target volume (often irregu- larly shaped) but maintain a low dose to th e non-target tissues of the patients. The commercial development of multi-leaf collimators for linear accelerators and megavoltage imaging systems has made this treatment available i n a number of large radiotherapy treatment centers. Early clinical results suggest that nor mal tissue side-effects can be reduced and dose escalation is possible e.g. in the treatmen t of localized prostate cancer. Intensity-modulated radiation therapy (IMRT) Here the intensity of the radiotherapy beam is varied across the treat- ment \201eld to ensure that a uniform dose can be achieved in a regular or irregular shape target while avoiding delivery of a high dose to the surroundin g structures. Essentially, this may add to conformal therapy by further reducing the dose t o sensitive structures. Currently, 14 institutions worldwide specialize in this technology, prioritizing treatment of intracranial tumors and cancer of the head and neck and prostate. The beam can be produc ed by a modulatory accelerator or with the addition of a special collimator, but the tech- nique is computationally complex. Intra-operative radiotherapy Radiotherapy can be delivered to a tumor volume under direct v isual localization during an open operation within a designated radio- therapy suite. A lar ge single dose is delivered at the one procedure. The potential advantage of a targeted boo st (given in addition to external- beam fractionated therapy) is balanced by the theoretica l limitations of the biological effects of the single large dose. A few centers in the world currently use this technique and some results suggest a survival bene \201t. There remains controversy over the dif \201cult logistics of delivery and fu rther larger clinical studies are needed to de \201ne and quantify any bene \201t. Improvements in radiotherapy fractionation In general, tumors and critical normal tissues ( those which limit the dose which may safely be delivered in a course of radiotherapy) are a ssociated with different fractionation sensitivities. Thus, reducing the OHO-40(635-654) 11/18/02 9:34 AM Page 649 NOVEL RADIOTHERAPEUTIC APPROACHES 649 daily dose per fraction will allow a greater total dose to be delivered to the tumor withou t exceeding the normal tissue tolerance and the therapeutic index may be favorably altered. Studies have demonstrated fast rates of growth in certain tumors (e.g. squamous cell carcin omas of the head and neck region) such that clonogens have potential doubling times of less than \201ve days. This implies that the time taken to deliver a radical course of conven - tionally fractionated radiotherapy should not be extended. Indeed, clinical trials have d emonstrated bene \201ts with treatment acceleration where a radical course of treatment ma y be completed in two weeks (with thrice daily fractions), rather than the more usual 4 \200\2236 weeks. Although the potential bene \201ts of acceleration and changed frac- tionation are clear, the impact of their adoption on overall survival is likely to be modest and only on subgrou ps of patients. Even amongst tumors of common histology, there are wide variations in the r adio- biological parameters such as potential doubling time. There is interest in the development of predictive assays, from which it may be possible to tailor a schedule of radiotherapy to the individual tumor. Particle beams Conventional radiotherapy uses uncharged electromagnetic radiation of suf \201cient energy to ionize molecules in tissues \200\224so-called \200\230ionizing radiati on \200\231. Particle radiation has a greater ionizing effect per unit dose and charged par ticles also have favorable dose-absorption pro \201les. Neutron therapy Neutrons are the commonest type of particle beam used for radio- therapy bu t the clinical results obtained are controversial. With a high LET (linear energy transfer) they produce more cell killing per dose than megavoltage X-rays. This may be particularly advantageous in hypoxic areas of tumor. Clinical trials have been carried out for sali- var y gland tumors, prostrate adenocarcinomas, soft tissue sarcomas, paranasal sinuses, and mel anomas. The main problems have been the severity of the normal tissue reactions and lack of a clear clinical advantage. The poor dose distribution and beam delivery systems of the early experimental neutron faci lities may have hampered the clinical results. Proton beam therapy These heavily charged particles are produced by a cyclotron and deliver their energy and therefore biological effects in a small-de \201ned area at the end of th eir range (often millimeters) \200\224the so-called \200\230Bragg peak \200\231. This redu ces the dose to underlying normal tissue. Complex multiple beams can be used to deliver a h igh dose to a small area. So OHO-40(635-654) 11/18/02 9:34 AM Page 650 NOVEL THERAPEUTIC STRATEGIES 650 far their use has been limited to super \201cial tumors such as uveal melanomas. Higher-en ergy beams now allow deeper-seated tumors to be treated e.g. chordoma of the clivus. Approximately 2000 \200\2233000 patients are treated worldwide each year and most of the tr eatment centers are still based in physics labora- tories. Improvements may be made by bett er tumor selection and technical advances in treatment delivery. Radiosensitization If tumor cells can be made more sensitive to the delivered radio- therap y, improvements in local control can be gained only if radio- sensitization is selective fo r the tumor. Hypoxic cell sensitizers Hypoxic tumor cells are resistant to radiation. The delivery of hy poxic cell sensitizers should theoretically improve tumor cell kill. Many international ran domized clinical trials have been negative, per- haps due to the inability to stratify and select for hypoxic tumors. More recently, trials with, for example tirapazamine, have demon - strated bene \201t, but these agents are still under evaluation. Synchronous chemotherapy Chemotherapy delivered with radiotherapy may provide bene \201t o ver and above the addition of more cell kill. This may be due to the inhi- bition of DNA re pair by the agent or some other mechanism of tumor radiosensitization. More clinical studie s are required to de \201ne the optimal combination of chemotherapy and radiotherapy and t o ensure this is a selective improvement for the tumor and does not just produce additive t oxicity. The approach of targeted delivery of radiosensitizing agents selectively to tumors (e.g. via liposomes) may provide therapeutic gain. Hyperthermia Hyperthermia enhances the effect of radiotherapy by direct cell killing and ra diosensitization. This interaction is time-dependent, maximum with simultanous treatment. I t is considered an experimental therapy, as clinical studies are ongoing, and one major lim itation is the necessary equipment needed to deliver heat above 41 C. Targeted radiotherapy Introduction Targeted radiotherapy means the selective irradiation of tumor cells by radion uclides that are conjugated to tumor-seeking molecules OHO-40(635-654) 11/18/02 9:34 AM Page 651 TARGETED RADIOTHERAPY 651 (targeting agents). Ideally, the targeting agent should be perfectly ef \201cient in \201nding every tumor cell in the body and should be absolutely discriminatory in \201ndi ng only tumor cells. Real agents fall well short of this ideal. In practice, a targeting ag ent has therapeutic potential if it can be stably conjugated to an appropriate radionuclide and allows signi \201cantly higher radiation doses to be delivered to tumor masses than t o critical tissues. This is a \200\230therapeutic ratio \200\231 criterion similar to that which applies to most forms of cancer treat- ment. However, targeted radiotherapy also pos sesses unique features. Targeting agents Tumor targeting depends on the existence of biological differences between normal and tumor cells. Several categories of targeting agent have been used, or are under development: (cid:2) Monoclonal antibodies \200\224limited discriminatory ability \200\224epitopes are heterogenous and unstable in tumor cell population \200\224poor penetration of tumor mass \200\224murine antibodies provoke host response \200\224used in cancer of ovary, colon, brain with modest response \200\224best response in B-cell lymphoma (cid:2) MIBG \200\224taken up by catecholamine-synthesizing cells of sympa- thetic nervous system \200\224taken up by neuroblastoma, phaeochromocytoma \200\224diagnostic and therapeutic (cid:2) Future \200\224melanoma \200\224glioma, squamous cell carcinoma: overexpressed cell receptor epidermal growth factor (EGF) Radionuclides for therapy Radionuclides which have potential for targeted therapy are the ( cid:3), (cid:2), and Auger particle emitters. Though particle-emitting radionuclides Table 40.1 Targeting agents for targeted radiotherapy Biological differential Targeting agent Target tumor Epitope Antibodies Various Noradrenaline transporter MIBG Neuroblastoma Melanin synthetic pathway Methylene blue Melanoma EGF receptor overexpression EGF Squamous carcinoma, glioma Proliferative differential IUDR Brain tumors Estrogen nuclear receptor Estrogen Breast cancer Genomic aberration Oligonucleotide ? OHO-40(635-654) 11/18/02 9:34 AM Page 652 NOVEL THERAPEUTIC STRATEGIES 652 usually produce some (cid:4)-ray photons as well, the photons make little contribution to t he therapeutic effect. The physical half-life of a targeting radionuclide must be long enough to allow radiochemic al conjugation and the homing of the conjugate to its target tumor cells. However, it is no t optimal to use radionuclides with half-lives longer than the time scale over which the co njugate will maintain its biochemical integrity and therefore target- ing speci \201city. In practice, clinical experience with targeted radiotherapy is largely con \201ned to (cid :2)-emitters, particularly 131I and, to a lesser extent, 90Y. The advantages of 131I are it s availability, ease of conjugation, and clinical familiarity. 90Y has some physical advant ages but is unfamiliar and the conjugates are less stable. (cid:3)-emitters have high radiobiological effectiveness and short-range emissions but are dif \201cult to obtain and have inconveniently short half-lives. Experience with (cid:3)-e mitters is so far con \201ned to the labora- tory, but encouraging clinical potential has been demonstrated. Auger electron emitters have been little used for targeted therapy because the short range of the Auger electron requires a DNA- targeting agent. Radiobiological considerations Targeted radiotherapy differs from conventional irradiation in its lower dose rate, dependence on the biodistribution and pharmaco- kinetics of the rad ioconjugate, particle range effects, and the role of tumor heterogeneity. The effects of pa rticle range have been studied by mathematical modeling and in experimental tumor models an d have produced therapeutic results of potential clinical importance. Combined modalities Targeted radiotherapy using (cid:2)-emitters inevitably results in a wh ole- body radiation dose because of limited targeting speci \201cities (cross- Table 40.2 Radionuclides for targeted radiotherapy Radionuclide Half-life Emitted particles Particle range (cid:2) 90Y 2.7 days 5mm (cid:2) 131I 8 days 0.8 mm (cid:2) 67Cu 2.5 days 0.6 mm (cid:2) 199Au 3.1 days 0.3 mm (cid:3) 211At 7 hours 0.05 mm (cid:3) 212Bi 1 hour 0.05 mm 1 (cid:5)m 1 (cid:5)m 125I 60 days Auger electrons 123I 15 hours Auger electrons OHO-40(635-654) 11/18/02 9:34 AM Page 653 TARGETED RADIOTHERAPY 653 targeting to normal tissues) and because of radionuclide in the gener- al circulation. Radi obiological modeling suggested that systemic tar- geted radiotherapy might best be regarded as a kind of non-uniform TBI \200\224with higher doses being given to tumor cells than to normal tissues. Therefore, systemic targeted radiotherapy might be most appropriate for pat ients for whom conventional TBI, with marrow rescue (or other forms of hematological suppor t), was already an option. These considerations suggest the possible advantages of a combined modality regime incorpor ating targeted radiotherapy, TBI, and mar- row rescue. Computer simulation studies of the e ffect of combined modality treatment have suggested that disseminated tumors of differing s ize may be optimally treated by different components of the combination. For example, distr ibuted single cells and very small micrometastases are effectively treated by TBI (and by c hemo- therapy), targeted 131I is ideal for treatment of larger micrometastases, whilst macr oscopic tumors are best dealed with by local modalities (radiotherapy, surgery). These concepts are now being applied in the targeted radiotherapy of neuroblastoma using 13 1I-MIBG in combination with TBI or with high-dose chemotherapy. Combined modality treatment of B-cell lymphoma using radio-labeled antibodies, TBI, or systemic chemo- therapy and hae mopoetic rescue may be an appropriate next step. Regional treatment (cid:2) Higher concentration of targeting agent (cid:2) Higher radiation dose to tumor depo sit (cid:2) Cancer of the ovary \200\224intraperitoneal 131I or 90Y conjugated anti- bodies (cid:2) Cancer of the bladder \200\224intravesical radio-labeled antibody (cid:2) Brain tum ors \200\224intrathecal or intraventricular administration; CSF Injection of radiolabelled agent directly into macroscopic tumor e.g. cystic glioma (cid:2) OHO-40(635-654) 11/18/02 9:34 AM Page 654 This page intentionally left blank OHO-41(655-664) 11/18/02 9:39 AM Page 655 Chapter 41 Gene therapy for cancer Gene delivery 656 Tumor-suppressor and drug-resistance genes 657 Genetic pro-drug activatio n therapy 659 Genetic immunotherapy 660 Anti-sense DNA oligomer treatment: blocking oncogen e expression 662 Genetic tagging 663 Future directions 663 Further reading 663 OHO-41(655-664) 11/18/02 9:39 AM Page 656 GENE THERAPY FOR CANCER 656 Gene therapy is the transfer and expression of genetic material into human cells for a ther apeutic purpose. New insights into the control and growth of human cells and their deregula tion in cancer have co- incided with advances in recombinant DNA technology. Novel methods of introducing \200\230therapeutic \200\231 DNA into cancer cells have allowed gene therap y to progress to clinical trials. New anti-cancer strategies are urgently needed as most cancers are still ultimately resista nt to conventional treatment modalities. Germ- line gene therapy has been deemed unethical, so current develop- ments involve introducing foreign genes into somatic (non-sex) cells o nly. In the UK, clinical gene therapy trials are currently regulated by the Gene Therapy Ad visory Committee (GTAC). Table 41.1 Strategies for cancer gene therapy Somatic correction of gene defect Antisense to mutant oncogene Expression of tumor suppress or gene Genetic pro-drug activation Genetic immunomodulation Polynucleotide vaccination Cancer vaccines Vectoring of biotherape utic genes to tumors and T cells Dendritic cell immunity Gene marking Increased normal tissue tolerance Gene delivery The addition, substitution, or ablation of DNA sequences may be achieved by any of the foll owing: Injection of naked DNA into skeletal muscle by simple needle and syringe. (cid:2) (cid:2) DNA transfer by liposomes (delivered by the intravascular, intra- tracheal, intraperitoneal, or intracolonic routes). (cid:2) DNA coated on the surface of gold pellets that are air-propelled into the epidermis (the \200\230gene gun \200\231). (cid:2) Biological vehicles (vectors) such as viruses and bacteria. Viruses are genetically engineered not to replicate once inside the host. They are currently the most ef \201cien t means of gene transfer. OHO-41(655-664) 11/18/02 9:39 AM Page 657 tumor-SUPPRESSOR AND DRUG-RESISTANCE GENES 657 Other techniques involve fusion of whole cells or viral envelopes, elec- troporation, micro -injection, or chemical precipitation of DNA into cells. The ef \201ciency of transfer of therapeutic DNA required (dictated by the nature of the g enetic defect) in \202uences the choice of vector e.g. for gene replacement, high-ef \201 ciency viral vectors are desirable, whereas short-term gene expression to prime an immune r esponse or sensitize cells to radiotherapy may be achieved by liposomal delivery. Some of these strategies can be achieved ex vivo by transfer of a therapeutic gene into iso lated cancer or non-cancer cells that are then reimplanted into the host. Others require de livery and expression of genes to target cancer cells in vivo (at much lower ef \201ciency than ex vivo transfer) by exploiting transcriptional differences of speci \201c genes bet ween cancer and normal cells. The ef \201ciency of gene transfer also varies greatly accor ding to cell type targeted (low in neural and haemopoietic cells; high in myocytes, \201b roblasts, hepatocytes; and variable among different tumors). Tumor-suppressor and drug-resistance genes The inactivation of tumor suppressor genes such as retinoblastoma (Rb1), p53, Nm23, p16, or E-cadherin may result in the initiation or the progression of a cancer. Replacement with n ormal copies of these genes using viral vectors has resulted in suppression and/or reversal of the malignant phenotype in in vivo tumor models. Furthermore, combining successful rest oration of genes such as wild type p53 and sequential administration chemotherapy (such as cisplatin) appears to be synergistic in reducing the malignant expression in these cell lin es. The clinical ef \201cacy of intra-tumoural adenoviral delivery of wild type p53 into p53-m utant squamous cell head and neck tumors is currently being evaluated. The MDRI multiple drug-resistance gene has been transduced ex vivo into normal marrow and b lood-derived stem cells to produce a selectable population of cells resistant to high doses of chemotherapy, which could then be re-infused into the patient. This has allowed higher doses of taxol to be administered in breast and ovarian cancer patients. O H 6 5 8 O 4 1 ( 6 5 Table 41.2 Viral vectors for gene therapy 5 6 Retrovirus Adenovirus Adeno-associated virus Herpes virus 6 4 ) Advantages High viral titres Small genome High viral titres Small genome 1 1 Carries 8 \200\22330 kb insert Carries 10 kb insert Carries up to 15 kb insert Carries 4 \200\2235 kb insert / 1 Stable co-linear integration 8 / 0 Highly ef \201cient gene transfer Ef \201cient gene transfer Ef \201cient gene transfer Can infect non-dividing cells Highly ef \201cient gene transfer Can infect non-dividing cells 2 9 Naturally replication-incompetent Neural tropism : 3 9 Disadvantages A M Immunogenicity Immunogenicity Immunogenicity Immunogenicity Requires actively dividing cells Transient expression Large genome Not well studied P a Small DNA sequences only carried Small DNA sequences only carried Small DNA sequences only carried Lytic virus g e Low titre, transient expression 6 5 Random integration 8 Insertional mutagenesis OHO-41(655-664) 11/18/02 9:39 AM Page 659 GENETIC PRO-DRUG ACTIVATION THERAPY 659 Genetic pro-drug activation therapy The fundamental problem with current chemotherapy is its lack of selectivity. If drug-activ ating genes could be inserted that would be expressed only in cancer cells, then giving an appropriate pro-drug could be highly selective. There are many examples of genes preferen- tially expressed in tumors. In some cases their promotors have been isolated and coupled to drug-activating enzymes (suicide genes). Genetic pro-drug activation therapy (GPAT, also k nown as GDEPT and VDEPT) exploits differences in gene expression between cancer cells in th is way to increase the speci \201city of cell destruction. Two targeting strategies may be used independently or in combina- tion to keep suicide gene expression limited to malignant tissue: (cid:2) Transcriptional t argeting exploits regulatory sequences of genes overexpressed in cancer cells to drive the expression of a suicide gene selectively within tumor cells e.g. ERBB2 in breast cancer, ty rosinase in melanoma. (cid:2) Transduction targeting relies on preferential delivery of vectors constitutively ex pressing a suicide gene into actively dividing cells only e.g. glioma cells and not into no rmal neighboring central nervous system cells. Improvements in the ef \201cacy of GPAT have resulted from successfully modifying both ret roviral and adenoviral tropism, and the use of chi- maeric transcriptional elements combini ng strong tissue-speci \201c pro- moters with tissue-speci \201c enhancers to improve tar geting further e.g. combining the ERBB2/MUC-1 transcriptional elements to drive the HSV-tk gene in breast cancer cells increases their sensitivity to pro- drug activation compared wi th the use of single elements to drive the same suicide gene expression. An important additional feature of GPAT systems is the \200\230bystander effect \200\231. This is the process whereby non-transduced cells in a mixed pop- ulation die in the presenc e of a given pro-drug due to diffusion, active transportation, or recruitment of a local im mune response. This effect varies in magnitude with the GPAT system and tumor cell line stu died, but as little as 2% transduction of a colorectal tumor cell line results in almost co mplete cell kill upon exposure to a pro-drug. This may indicate that sustained high ef \201ciency gene transfer may not necessarily be required. transductional targeting of gliom as and mesotheliomas, transcriptional targeting of ovarian and breast carcinomas, and melan omas using adenoviral or retroviral transduction or direct intralesional injection of plasm id DNA. Current clinical trials consist of OHO-41(655-664) 11/18/02 9:39 AM Page 660 GENE THERAPY FOR CANCER 660 Table 41.3 Prototype systems used for genetic pro-drug activation therapy Enzyme Pro-drug Active drug Viral thymidine kinase triphosphate Ganciclovir Ganciclovir Cytosinedeaminase 5- \202uorocytosine 5- \202ourouracil Linamarase Amygdalin Cyanide Nitroreductase CB1954 Nitrobenzamidine Cyp1A2 Paracetamol Toxic metabolites Superoxide dismutase Radiation DNA damage Table 41.4 Cloned genes and their promoters that may be coupled to drug-activating enzymes for tumor-selective expression Selective gene (cid:2)-fetoprotein Prostate-speci \201c antigen Tumor(s) Hepatoma, germ cell tumors Prostate cancer Thyroglobulin Thyroid carcinoma Tyrosinase Melanoma Carcinoembryonic antigen Gastrointestinal and other cancers Polymorphic epithelial mucin Breast cancer Neuron speci \201c enolase Small cell lung cancer c-erbB2 Breast and gastrointestinal cancers Genetic immunotherapy Attempts at enhancing the naturally weak immunogenicity to tumors have resulted from a clea rer understanding of antigen recog- nition, processing and presentation at the molecular le vel, and in particular, the nature of effector (T cell) responses to antigenic stimu- latio n. A number of approaches are currently being evaluated: (cid:2) Systemic immunotherapy for cancer with recombinant cytokine therapy is associated w ith low response rates at the expense of high systemic toxicity. Small doses of cytokines a re delivered at the tumor site by inserting cytokine genes into cultured tumor- OHO-41(655-664) 11/18/02 9:39 AM Page 661 GENETIC IMMUNOTHERAPY 661 in \201ltrating lymphocytes (TILs) ex-vivo, then re-infusing the cells. Anti-tumor ef \201cacy of this subset of T cells has been shown to improve with the transfer of the genes for tumor necrosis factor and interleukin-2 (IL-2). (cid:2) Transducing tumor cells ex-vivo with the same cytokine, allo- geneic HLA (human leu cocyte antigen), or genes encoding co- stimulatory molecules, such as the B7 family of gene s, prior to re-infusion (after irradiation of eliminate malignant activity) so that T-cell recognition of tumor antigens is enhanced. CTLs recognize tumor-speci \201c antigens prese nted on the surface of these cells. They are induced by the local secretion of the trans- f erred cytokine gene product to expand, target, and destroy cancer cells. (cid:2) Polynucleotide (naked DNA) vaccinations (as opposed to previous vaccinations consis ting of peptides, whole tumor cells, or tumor cell lysates) have great therapeutic potentia l in that delivery of genes that express unique oncoproteins (such as KRAS or lym- phoma id iotypic protein) endogenously within a cell, may then result in an MHC class I CD8+ respons e and proliferate activation of CTLs, rather than a less effective class II CD4+ response i nduced by exogenous peptides. This may be a further means of breaking down immunotolerance to tumors and lead to the generation of tumor-speci \201c responses. Targets for DNA thera py are shown in the table. The limitation to this approach is the paucity of truly tumor- speci \201c antigens which may be exploited as molecular targets. Most target antigens are tumor-associated (i.e. norm al cellular genes inappropriately expressed in cancer) and lack epitopes to activate T cell s. However, delivery of genes encoding the E6 and E7 proteins of HPV 16 and 18, in patients with advanced cervical cancer, resulted in antibody and HPV-speci \201c T-cell responses. (cid:2) Dendritic cells (DCs) are potent antigen-processing and antigen- presenting cells w hich are critical to the development of primary MHC-restricted T-cell immunity to infectiou s agents, in auto- immune diseases and anti-tumor immunity. Recent technological advances h ave allowed their expansion in vitro from peripheral blood precursors and marrow using cyto kines. Cultured DCs are able to take up exogenous antigen (as tumor protein, peptide, or RN A), or may be transduced with genes encoding tumor antigen using physical or viral methods of gene transfer, and then present it to T cells to induce a measurable anti-tumor effect. Early clinical trials have involved DCs pulsed with idiotypic pro- tein for relapsed B-cell lymphoma or whole tumor lysates for melanoma; in both cases antigen-speci \201c immunity has been demonstrated. OHO-41(655-664) 11/18/02 9:39 AM Page 662 GENE THERAPY FOR CANCER 662 Table 41.5 Targets for DNA vaccination strategies Target Class of antigen Associated cancer(s) p53 Mutated tumor suppressor >50% all human cancers RAS Mutated oncogene product >10% all human cancers; >80% pancreatic, colorectal ERBB2 Growth factor receptor Breast, ovary, stomach, pancreas Igidiotypes Idiotypicepitope B-cell lymphoma HPVE6, E7 Viral gene products Cervical cancer P210BCR/ABL Mutated oncogene product Chronic myeloid leukemia MAGE-1 Embryonic gene product Melanoma, breast Tyrosinase Normal differentiation antigen >50% melanomas Anti-sense DNA oligomer treatment: blocking oncogene expression (cid:2) Short (10 \200\22350 base), synthetic, nucleotide sequence (cid:2) Complementary to speci \201c DNA or RNA sequences (cid:2) Enter all cells easily (cid:2) Speci \201c to t arget individual oncogenes (cid:2) May produce downregulation (cid:2) Can inhibit transcrip tion/translation of single gene Inhibition can be caused by triggering RNase H \200\224deg radation of target RNA \200\224interferes with processing of pre-mRNA (cid:2) (cid:2) Examples include \200\224pancreatic carcinoma (HRAS) \200\224prostate cancer cell lines (Block bcl-2 expression) (cid:2) BUT \200\224poor ef \201ciency \200\224poor speci \201city \200\224may be incre ased cell invasiveness Initial trial results encouraging in AML and lymphoma patients (cid:2) OHO-41(655-664) 11/18/02 9:39 AM Page 663 FURTHER READING 663 Genetic tagging The insertion of a foreign marker gene into cells during a tumor biopsy, and replacing the marked cells prior to treatment can provide a sensitive new indicator of minimal residual d isease after chemo- therapy. Neomycin phosphotransferase (NeoR) \200\224an enzyme that meta bolizes the aminoglycoside, G418 \200\224has been retrovirally trans- duced ex vivo into pu rged marrow from AML and neuroblastoma patients prior to re-infusion. In those individuals with relapsed disease, as few as one in 106 cells expressing the NeoR gene has been detecte d by PCR, indicating failure of the purging process. Future directions Initial clinical studies indicate that gene therapy for cancer is safe and feasible, but ha mpered by inef \201ciency of current gene transfer tech- nology. The precise role for gene therapy in the treatment of cancer may be limited to an adjuvant or palliative role rather than attempting to eliminate bulk disease. It may also serve as an ef \201cient genetic m ark- er or enhance tumor sensitivity to chemotherapy or radiotherapy. Rapid progress in this \201eld has to be tempered by appreciation of potential new toxici ties. Transfer of DNA may initiate anti-DNA anti- body responses similar to those associate d with autoimmune diseases. The use of viral vectors may be associated with insertional mut a- genesis and activation of oncogenes leading to neoplasia, antiviral immunity, and propag ation of replication-competent viruses. Further reading Crystal, R.G. (2001) Adenoviral vectors in the gene therapy of cancer. Clin Can Res 7, W 836A Suppl 5. Huber, B., Austin, E., Davis, S., Richards, B. and Good, S. (1994) Metabolism of 5- \202uorocytosine to 5- \202uorouracil in human colorectal tumor cells transduced with the cytosine deaminase gene: signi \201cant antitumour effects when only a small percentag e of tumor cells express cytosine deaminase. Proc Nat Acad Sci USA 91, 8302 \200\2236. Xu, G., McLeod, H.L. (2001) Strategies for enzyme/prodrug cancer therapy. Clin Can Res 7, 3314 \200\22324. OHO-41(655-664) 11/18/02 9:39 AM Page 664 This page intentionally left blank OHO-App(665-682) 11/18/02 9:41 AM Page 665 Part 7 Appendices 1 NCIC common toxicity criteria (CTC) grading system (May 1991, revised) 667 2 Nomogram for determination of body surface area 681 OHO-App(665-682) 11/18/02 9:41 AM Page 666 This page intentionally left blank OHO-App(665-682) 11/18/02 9:41 AM Page 667 Appendix 1: NCIC common toxicity criteria (CTC) grading system (May 1991, revised) O H 6 6 8 O A p Grade 0 Grade 2 (Moderate) Grade 3 (Severe) Grade 1 (Mild) Grade 4 (Life-threatening) p ( 6 6 5 Allergy None Transient rash, fever <38 C, 100.4 F Urticaria, fever = 38 C, 100.4 F, mild bronchospasm Serum sickness, bronchospasm, req parenteral meds Anaphylaxis 6 8 2 ) BLOOD/BONE MARROW 1 \211 4.0 WNL 1 White blood cells (109/1) 2.0 \200\2232.9 1.0 \200\2231.9 3.0 \200\2233.9 <1.0 / 1 Platelets (109/l) 75.0 \200\223normal 50.0 \200\22374.9 25.0 \200\22349.9 <25.0 8 / Hemoglobin (g/dl) 10.0 \200\223normal 8.0 \200\2239.9 6.5 \200\2237.9 WNL \211 2.0 <6.5 0 2 Granulocytes (109/l) (i.e. neuts.+baso.+eosin) Lymphocytes (109/l) 1.0 \200\2231.4 0.5 \200\2230.9 <0.5 1.5 \200\2231.9 9 : \211 2.0 None 1.0 \200\2231.4 0.5 \200\2230.9 <0.5 1.5 \200\2231.9 4 1 Hemorrhage (clinical) (includes nosebleeds, menorrhagia, etc.) Mild, no transfusion (includes bruise/ haematoma, petachiae) Gross, 3 \200\2234 units transfusion per episode Gross, 1 \200\2232 units transfusion per episode Massive, >4 units transfusion per episode A M P a CARDIOVASCULAR g e Arterial Venous \200\223 Deep vein thrombosis not requiring anticoagulant therapy None None Transient events Deep vein thrombosis req anticoagulant therapy \200\223 Super \201cial (excludes IV site reaction \200\224code under skin Permanent event Pulmonary embolism 6 6 8 WNL = within normal limits O H O A p p ( Grade 0 Grade 2 (Moderate) Grade 3 (Severe) Grade 1 (Mild) Grade 4 (Life-threatening) 6 6 5 Dysrhythmias Recurrent or persistent, no therapy req None Req monitoring, or hypotension, or ventricular tachycardia, or \201brillation Asymptomatic, transient, req no therapy Req trt 6 8 2 ) 1 1 / Oedema None 3+ 2+ or dependent throughout day 1+ or dependent in evening only 4+, generalized anasarca 1 8 / 0 Function None Asymptomatic, decline of resting ejection fraction by <20% of baseline value Asymptomatic, decline of Mild CHF, responsive resting ejection fraction by to therapy >20% of baseline value Severe or refractory CHF 2 9 : 4 1 Hypertension None or no change Asymptomatic, transient increase by >20 mm Hg or to >150/100 if previously WNL. No trt req Recurrent or persistent increase by >20 mm Hg or to >150/100 if previously WNL. No trt req Hypertensive crisis Req therapy A M P a g e Hypotension None or no change Changes req no therapy (incl transient orthostatic hypotension) Req therapy & hospitalization; resolves within 48 hrs of stopping agent Req \202uid replacement or other therapy but not hospitalization Req therapy & hospitalization for 48 hrs after stopping agent 6 6 9 6 6 9 O H 6 7 0 O A p Grade 0 Grade 2 (Moderate) Grade 3 (Severe) Grade 1 (Mild) Grade 4 (Life-threatening) p ( 6 6 Ischaemia None or no change \202attening Non-speci \201c T wave Asymptomatic, ST & T wave changes suggesting ischaemia Acute myocardial Angina without evidence for infarction infarction 5 6 8 2 ) Pain (chest) Moderate None Severe Mild Life-threatening Pericardial None Asymptomatic, effusion, no intervention req Pericarditis (rub, chest pain, Symptomatic effusion, Tamponade, drainage ECG changes) 1 1 drainage req urgently req, or constrictive pericarditis req surgery / 1 8 / 0 2 9 COAGULATION : 4 \211 0.24 \227 N >2.00 \227 N >3.00 \227 N Fibrinogin 0.49 \200\2230.25 \227 N 1.51 \200\2232.00 \227 N 2.34 \200\2233.00 \227 N WNL 0.74 \200\2230.5 \227 N 1.26 \200\2231.5 \227 N 1.67 \200\2232.33 \227 N 0.99 \200\2230.75 \227 N 1.01 \200\2231.25 \227 N 1.01 \200\2231.66 \227 N 1 Prothrombin time WNL A M Partial thromboplastin time WNL P ENDOCRINE a g Yes \211 3 months Mild e Amenorrhoea \200\223 No \200\223 \200\223 6 Cushingoid Pronounced Moderate & \211 1/day Normal \200\223 \200\223 7 0 Hot- \202ushes None Frequent & interferes with normal function Mild or <1/day \200\223 O H O A p p ( Grade 0 Grade 2 (Moderate) Grade 3 (Severe) Grade 1 (Mild) Grade 4 (Life-threatening) 6 6 5 Gynaecomastia Pronounced or painful Normal \200\223 Mild \200\223 6 8 Impotence/libido Normal Decrease in normal function Absence of function \200\223 \200\223 2 ) 1 FLU-LIKE SYMPTOMS 1 / Arthralgia (joint pain) Moderate Severe \200\223 None Mild 1 8 /