Chemotherapeutic agents that are cytotoxic to tumors act by a variety of mechanisms. Those that are highly responsive to ionizing radiation and enhance the effectiveness of radiation treatment are termed radiation sensitizers. Radiation sensitizers act in a number of ways to make cancer cells more susceptible to death by radiation than the surrounding healthy cells. Several radiation sensitizers are now available for the treatment of solid tumors. This review will discuss the biology that underlies chemotherapy and radiation interactions for one radiosensitizer- gemcitabine(Drug information on gemcitabine) (Gemzar)-and will provide a brief assessment of how to modify treatment techniques by taking advantage of the natural history of the disease. In addition, the review will attempt to offer insight into trial design modifications for various cancers and will discuss the concept of molecular targeted therapy for improved efficacy of radiation sensitizers. The rationale for combining chemotherapy and radiation includes the notion that chemotherapy may eradicate sites of disease that have yet to appear or manifest themselves. If chemotherapy can, in fact, eradicate malignant cells, then the interaction between the chemotherapeutic agent and radiation is not really necessary. However, it is important to understand that the addition of another agent to radiation will only improve the therapeutic index if the tumor cell kill is increased more than the increase in toxicity to neighboring normal cells. Indeed, antitumor effects should be greater than the toxic side effects of the added therapy. Most cancer treatment now uses the combined approach of conventional chemotherapy with radiation. Based on cumulative evidence from numerous randomized clinical trials, this combined approach shows an in- creased survival benefit in the treatment of locally advanced cancers of the head and neck, lung, esophagus, stomach, pancreas, and rectum. Nonetheless, the underlying mechanism for how chemotherapeutic agents act as radiosensitizers remains largely unknown. We are only now beginning to understand how to use chemotherapy and radiation to optimize treatment, overall survival, and quality of life. What is known about one of the more widely used radiosensitizing agents, gemcitabine, is discussed below. Gemcitabine Gemcitabine (2'2'-difluoro-2'- deoxycytidine) is a nucleoside analog of cytosine arabinoside with clinical activity against solid tumors such as pancreas and non-small-cell lung cancers.[1,2] It is a prodrug that is metabolized by the enzyme deoxycytidine kinase, which phosphorylates the analog to the monophosphate and diphosphate and then to 2'2'- difluoro-2'-deoxycytidine triphosphate (dFdCTP) (Figure 1). The 5'-diphosphate and triphosphate forms are active metabolites, with dFdCTP as the toxic metabolite chiefly responsible for killing cells and producing cytotoxicity. As is common with many chemoradiation agents, the mechanism that produces cytotoxicity is often not the same mechanism that increases radiation sensitivity. Early in vitro studies showed that neither the increased pools of dFdCTP (the metabolite that causes cytotoxicity) nor the amount of gemcitabine in cells correlated well with the increase in radiation sensitivity. On the other hand, gemcitabine radiosensitization required that cells be depleted of dATP and exhibit a redistribution into S phase. In addition, gemcitabine did not affect either radiation-induced DNA damage or repair. Our working hypothesis is that sensitization occurs under S-phase progression on a damaged DNA template-one that has been damaged due to gemcitabine-induced depletion of dATP pools that lead to misincorporation and misrepair of incorrect bases after radiation and ultimately promotes cell death. In fact, gemcitabine is a very potent radiation sensitizer under cytotoxic conditions. In HT29 human colon cancer cells, exposure to 3 μmol/L gemcitabine for 2 hours resulted in approximately threefold increase in radiosensitivity. Even nontoxic doses of 10 nmol/L produced detectable sensitization at 24 hours. Plasma concentrations of 20 μmol/L are routinely achieved in patients receiving gemcitabine therapy. In the clinical setting, this information can be used to provide rational dosing regimens that take advantage of the dual properties of the drug: its cytotoxicity as well as its radiosensitivity. First of all, the findings show that radiosensitization can be achieved under noncytotoxic conditions and that doses of gemcitabine well below those required to achieve cytotoxicity may be used. Therefore, if the drug is given twice weekly at doses of 10 to 50 mg/m2, it would provide radiation sensitivity for the entire week and permit sensitization of all radiation fractions. Note that plasma level doses of ≥ 1 μmol/L (obtained with these lower gemcitabine doses) can be tolerated under a twice-weekly schedule compared with a weekly schedule. In addition, these lower doses are sufficient to provide continued radiosensitization. If the goal is to produce cytotoxicity with radiosensitization, a chemotherapeutic dose (1,000 mg/m2) can be administered, which should radio- sensitize radiosensitize for at least part of the week. To determine whether gemcitabine is being administered to a patient under conditions that produce radiosensitization, it may be of interest to assess simultaneous intracellular dATP pools and to determine the cell cycle distribution in tumor biopsies. Gemcitabine as a Radiosensitizer In vivo studies were conducted to gain further insight into how the dosing schedule of gemcitabine could affect the therapeutic index. One study included an intraperitoneal dose of gemcitabine at 50 mg/kg given either before or after a single 25-Gy fraction.[ 8] Regrowth delays were longest when gemcitabine was administered 24 to 60 hours before irradiation, and were in agreement with earlier preclinical studies. In addition, a single 5-mg/kg dose, which was found to have a minimal effect on tumor regrowth, was able to enhance the response to radiation, confirming that minimally cytotoxic doses of gemcitabine are radiosensitizing as predicted by earlier preclinical results. The same approach has recently been taken for the treatment of head and neck cancers with the use of gemcitabine as a radiosensitizer. Initial clinical studies used relatively low doses of gemcitabine given once weekly for patients with untreated stage IV squamous cell cancer of the head and neck. Treatment with weekly gemcitabine (at escalating doses beginning at 300 mg/m2) and standard external beam radiation (70 Gy in 2-Gy fractions using the shrinkingfield technique) was used. In this trial, severe acute and late mucosal and pharyngeal-related dose-limiting toxicity required de-escalation of gemcitabine in successive patient cohorts. Even 50 mg/m2 gemcitabine given once weekly was not tolerable. Subsequent preclinical studies using a mouse model for oral toxicity revealed that equitoxic combinations of twice-weekly gemcitabine with radiation were significantly more effective against flank tumors than once-weekly gemcitabine. A clinical trial was designed using this concept and is cur- rently accruing patients. Is it also possible to take advantage of the cytotoxic properties of gemcitabine as well as its radiosensitizing properties? Based on studies performed in cell culture, it is clear that higher doses of gemcitabine are both radiosensitizing and highly cytotoxic. In fact, the drug could produce profound radiosensitizing effects at doses of 1,000 mg/m2. Therefore, a full dose of gemcitabine along with a full dose of large-field radiation should be used with caution because the combination is extremely powerful and may lead to unacceptable toxicity. Combined Cytotoxicity and Radiosensitivity of Gemcitabine A different approach has been taken for the treatment of pancreatic cancer in a phase I dose-escalation trial. Here, a standard dose of gemcitabine was combined with a lower radiation dose in order to radiate the primary tumor alone without the inclusion of normal-appearing regional lymph nodes in patients with unresectable or incompletely resected advanced pancreatic cancer. The reduced radiation dose was used for safety purposes in order to reduce the risk of side effects. In addition, the treatment volume was reduced in order to target the primary tumor, and chemotherapy was used to control occult disease. Therefore, the study attempted to maximize the use of both therapies in order to obtain an optimal response by limiting the radiation portals. Gemcitabine was delivered at 1,000 mg/m2 weekly every 28 days, and the radiation dose was escalated with a starting dose of 24 Gy in 1.6- Gy fractions. The fractions were escalated by increasing the fraction size by 0.2 Gy and keeping the duration constant at 3 weeks. An example of the treatment volume used in the doseescalation trial is shown in Figures 2 and 3 (right panels) as compared with the volume generally used for prophylactic radiation shown on the left. Results of this phase I study showed that hematologic toxicities were no different than those reported in other studies using gemcitabine alone. Therefore, with the use of small radiation volumes, the toxicity was quite tolerable. The mean weight loss was only about 1%, and the recommended dose was 36 Gy in fifteen 2.4-Gy fractions. With a median follow-up of 22 months for 37 patients, the overall median survival was 11.6 months (range: 9.9-19.2 months). Moreover, three patients have survived more than 20 months, including one with biopsy-proven liver metastases. Another study evaluated weekly gemcitabine at doses of 350 to 500 mg/m2 (for 7 weeks) with largefield external-beam radiation at doses of 30 to 33 Gy in 10 to 11 fractions delivered over the first 2 weeks. In this trial, the combination produced significant nonhematologic side effects in over half of the 18 patients (nausea/ vomiting and dehydration; 44% required hospitalization). The difference, however, between this weekly dose trial and the previously described dose-escalation trial includes the fact that radiation volumes were different, as was the dose per fraction. Therefore, it is possible that the use of largevolume radiation therapy with a strongly cytotoxic drug such as gemcitabine may not be tolerable for prolonged dosing schedules. On the other hand, radiation therapy using targeted volumes that identify the gross tumor volume with a modest margin allows cytotoxic chemotherapeutic agents to be tolerated at much higher doses. As such, decreasing the radiation volume is a way to intensify both therapies when used in combination. Intensifying Cytotoxicity With Radiosensitivity One may wonder if it is possible then to intensify systemic therapy. One preclinical study suggests that treatment effects may be intensified by using a doublet combination plus radiation. Using two human pancreatic cancer cell lines, cells were exposed to three different treatment schedules to mimic clinical treatment regimens prior to radiation therapy: (1) sequential dosing of gemcitabine for 2 hours followed by cisplatin(Drug information on cisplatin) for 2 hours; (2) gemcitabine for 2 hours followed by a washout, a replenishment of medium for 24 hours, and then cisplatin for 2 hours; or (3) gemcitabine for 24 hours with concurrent cisplatin for the last 2 hours. With these regimens, it was found that cisplatin did not produce radiosensitization. However, the drug was synergistically cytotoxic with gemcitabine without compromising gemcitabine-mediated radiosensitization. As a result, a phase I trial of cisplatin and gemcitabine plus radiation in patients with locally advanced unresectable cancer of the pancreas is being developed. Molecular Targeted Therapies Are there other potential radiosensitizers that have not yet been explored? Studies of how normal cells integrate signals to affect a response would suggest that there are. For example, a quick glance at the growth factor receptors and their intracellular activation pathways illustrates that many receptors are involved in signal integration, including the erbB2 family of receptors, the insulin growth factor receptor, fibroblast growth factor receptor, and the integrins (Figure 4). With such careful integration, under normal circumstances no single pathway appears to dominate. With the onset of abnormal growth, however, many cells will overexpress a single growth factor family, such as the epidermal growth factor receptor (EGFR), during carcinogenesis (Figure 5). When this occurs, cell signaling is no longer integrated but driven by one single growth factor receptor pathway. In some cases, overexpression is the activating lesion. While some cancerous cells overexpress one or more growth factor receptors, overexpression does not always indicate an activating lesion. Therefore, performing routine immunohistochemical procedures following cell inhibition will not consistently indicate which tumors are actually being driven by one or more aberrant pathways. Instead, it appears that one of the better ways is to perform a biopsy in advance of administering a drug and then another following drug administration to determine if a specific pathway has been suppressed. Nonetheless, biopsies, when possible, are not a fair measure of change; biopsies represent only a single point in time and do not adequately monitor the change in a tumor or in the normal tissue during the course of treatment. Therefore, clinical performance should involve a number of different sources of data from clinical specimens and from new tests, such as magnetic resonance spectroscopy, to permit the measurement of tissue changes (eg, metabolite changes) over time. Molecularly targeted therapy can be advantageous if it produces a selective cytostatic effect. This could block tumor cell growth during a protracted course of radiation, thereby increasing the effectiveness of treatment. In addition, even modest radiosensitization, as long as it is selective, could be beneficial. Several groups have taken advantage of this concept by introducing a farnesyltransferase inhibitor that inactivates the Ras pathways. While only modest radiosensitization in culture has been observed with farnesyltransferase inhibitors- enhancements of 1.2 to 1.3 as compared with values of 1.8 to 3 with chemotherapeutic agents-it is conceivable that this extent of selectivity could produce benefit when repeated 30 to 40 times during a course of radiation. More importantly, if the cytostatic effect of a molecular targeted therapy is further combined with the cytotoxic effect of a chemotherapeutic agent, an extremely potent and favorable outcome may arise. Some of the recently pursued targeted therapies include CI-1033, gefitinib(Drug information on gefitinib) (Iressa), and erolitnib (Tarceva). These are all 4-anilinoquinazolines with the ability to inhibit tyrosine kinase receptors such as EGFR and HER2 receptor kinases.[ 15] Their antitumor activities generally revolve around their ability to reversibly or irreversibly inhibit one or more tyrosine kinase receptors and thus limit or inhibit tumor cell growth. While gefitinib and erlotinib are reversible inhibitors that exhibit receptor selectivity, CI-1033 appears to bind all tyrosine kinase receptors irreversibly and thus may have a larger spectrum of activity. An example of the synergistic relationship between CI-1033 and radiation therapy has been demonstrated in an animal model in which LoVo colon carcinoma tumors were implanted into the flanks of nude mice (TS Lawrence, unpublished data, 2002). Following implantation, the mice were treated with either radiation therapy (2 Gy for 15 fractions), CI-1033 (20 mg/kg/d for 5 days a week, for a total of 15 treatments), radiation plus CI-1033, or no treatment. Figure 6 shows the relative tumor volume for all treatment groups and illustrates the synergistic effect found with combination therapy. Whether the results are due to the cytostatic or cytotoxic effects of CI-1033 remain unclear; however, analysis of tissue sections showed the greatest cell death occurred with combination treatment. Other targeted therapies include inhibitors of other specific growth factor tyrosine kinases. In addition, gene therapy is being used for the treatment of solid tumors. With gene therapy, specific genes, such as those expressing cytokines or suicide genes, are in- troduced into tumor cells. In some cases, a radiation-sensitive promoter is placed upstream of the gene and, thus, activated with the delivery of ionizing radiation. In other cases, the gene delivery is used to generate high local concentrations of chemotherapy or a cytokinelike tumor necrosis factor. While these models are still in the early stages of testing, they show promise. The question remains whether targeted molecular therapy can increase the therapeutic index of standard therapy. While data remain inconclusive, it is clear that these targeted therapies do not rely on pure sensitization and, thus, are not being used for such purposes. Instead, it is more important that these therapies kill the primary tumor while sparing the normal tissues. The therapeutic index should affect the tumor more than it affects normal tissue. Conclusions It is becoming increasingly clear that to improve patient quality of life, delivery of all therapies should focus on a more targeted approach that limits toxicity and preserves normal tissue function. Future research should focus on methods of individualizing cancer therapy. These may be based on rapid assays that evaluate mechanisms of resistance or the use of selective inhibitors of one or more tumor populations for inclusion in combined- modality protocols. Thus, the future of clinical trial design with radiotherapy must include chemotherapeutic and/or biologic therapies and ways to both analyze the drug and evaluate the therapeutic index, not just the radiosensitizing properties of the added agent. Techniques such as magnetic resonance spectroscopy will assist in the measurement of intracellu- lar metabolites over time in order to monitor changes in the disease. Some chemotherapies, such as gemcitabine, fluorodeoxyuridine, and cisplatin, all active in the micromolar range, will require positron emission tomography imaging of the drug. Their activity will require the analysis of key enzymes such as ribonucleotide reductase in the case of gemcitabine or thymidylate synthase in the case of fluorodeoxyuridine. To appropriately use many of these potentially potent newer agents, the natural history of the disease should be taken into consideration. This will help identify how each drug can be administered. In the case of gemcitabine, the delivery can be modulated to emphasize its cytotoxic nature (a delivery that is appropriate for cancers with a high likelihood of metastases, such as pancreatic cancer), or emphasize its radiosensitizing property (such as with the delivery of verylow- dose gemcitabine). With radiotherapy, targeted treatment can be accomplished by threedimensional conformal therapy that produces a truly targeted dose of radiation to a more site-specific location. The present and the future of truly targeted therapy require that important improvements be incorporated into the treatment regimens with a cautious but systematic approach.