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The Oncologist, Vol. 10, No. 1, 34-51, January 2005; doi:10.1634/theoncologist.10-1-34
© 2005 AlphaMed Press

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Combined Modality Therapy of Gemcitabine and Radiation

Bea Pauwels, Annelies E.C. Korst, Filip Lardon, Jan B. Vermorken

Laboratory of Cancer Research and Clinical Oncology, Department of Medical Oncology, University of Antwerp, Antwerp, Belgium

Correspondence: Bea Pauwels, Ph.D., Laboratory of Cancer Research and Clinical Oncology, University of Antwerp, Campus Drie Eiken, Universiteitsplein 1, 2610 Wilrijk, Belgium. Telephone: 32-38202576; Fax: 32-38202248; e-mail: bea.pauwels{at}ua.ac.be


   LEARNING OBJECTIVES
Top
 Learning Objectives
Abstract
 Introduction
 Gemcitabine as a Single...
 Combination of Gemcitabine and...
 Conclusions
 References
 
After completing this course, the reader will be able to:

  1. List the advantages of combination chemotherapy and radiotherapy.
  2. Explain the rationale for doing in vitro research in the radiochemotherapy field.
  3. Describe the results of studies combining gemcitabine and radiotherapy.

Access and take the CME test online and receive 1 hour of AMA PRA category 1 credit at CME.TheOncologist.com


   ABSTRACT
Top
 Learning Objectives
 Abstract
Introduction
 Gemcitabine as a Single...
 Combination of Gemcitabine and...
 Conclusions
 References
 
The combination of gemcitabine and radiotherapy is a promising combined modality therapy. However, the clinical application of this combination has to be implemented carefully because of an increased toxicity to normal tissues. A body of experimental evidence shows that gemcitabine is a potent radiosensitizer in vitro and in vivo. The observations so far indicate that various mechanisms are responsible for the radiosensitizing effect. Although it is often difficult to transfer experimental data to the clinic, these studies offer the possibility to develop an improved schedule of administration for patient treatment, based on rational evidence in tumor biology. In the current review, the preclinical data that support the use of gemcitabine as a radiosensitizing agent and the clinical trials that have been conducted to date are summarized.

Key Words. Gemcitabine • Radiotherapy • Radiosensitization • In vitro • In vivo • Clinic


   INTRODUCTION
Top
 Learning Objectives
 Abstract
 Introduction
Gemcitabine as a Single...
 Combination of Gemcitabine and...
 Conclusions
 References
 
While the results of radiotherapy have substantially improved over the years, one-third of patients with solid tumors receiving curative treatment will suffer local recurrence due to residual tumor [1–3]. Treatment failures can be attributed to factors associated with the treatment delivery (e.g., suboptimal dose delivery or geographical miss) and with the biological response of the tumor cells to ionizing radiation. Attempts to improve the efficacy of radiotherapy have focused on improved methods to deliver the dose (e.g., conformal therapy and radiosurgery) or on studies to modulate the biological response to ionizing radiation [4]. Hyperfractionation and combinations of radiotherapy and chemotherapy have improved survival in patients with tumors known to be intrinsically radioresistant (e.g., non-small cell lung carcinomas [NSCLC], brain tumors, head and neck carcinomas) [3, 5]. Also new approaches targeting specific molecular pathways (e.g., p53 or the ataxia telangiectasia protein pathway) involved in radiation response are being tested in the laboratory [6].

The combination of radiotherapy and chemotherapy is advocated primarily because of the independent effects of each modality. Radiotherapy is aimed at controlling the primary tumor, while chemotherapy can also be used to eradicate distant metastases. Some chemotherapeutic drugs destroy tumor cells by their own cytotoxic action and additionally enhance the effects of radiotherapy. Chemotherapeutic drugs that have the potential to produce substantial sensitization of tumor cells to radiation treatment are defined as radiosensitizers, and the process is called radiosensitization. Because of this radiosensitization, the results of the combined use of radiation with cytotoxic drugs have been encouraging, both in terms of increased local tumor control and decreased distant failures.

Improved treatment results have been demonstrated in several in vitro and in vivo studies and in clinical trials in patients with locally advanced solid tumors during the past decade. Additive or synergistic effects against a tumor without substantial increase in toxicity to normal tissue may then lead to a therapeutic advantage.

Radiotherapy by itself induces complex changes both in tumors and in the adjacent normal tissues. The response to chemotherapy is similarly complex; in addition to many of the factors that determine response to radiation treatment there are also problems of drug delivery, drug resistance, and metabolism. The combination of radiotherapy and chemotherapy involves the combined complexity of both modalities, plus the interactions between them. Before novel treatment strategies are attempted in the field of chemoradiation, there is need for adequate preclinical data that establish whether the addition of drugs to radiation is likely to be of clinical benefit. Basic knowledge that needs to be sorted out in a laboratory setting includes: A) is there an additive or synergistic effect between the drug and radiation; B) is the timing between the drug administration and radiation important in achieving synergism; C) can the in vitro effect be translated into an animal model; and D) is this interaction likely to be clinically relevant, making clinical trials warranted?

Gemcitabine with radiotherapy is a promising combination therapy. However, the clinical application of this combination has to be implemented carefully because of an increased toxicity to normal tissues [7]. In this review, preclinical studies of the radiosensitizing effect of gemcitabine are summarized, with emphasis on the importance of intensive in vitro research on the combination of chemotherapy and radiotherapy. In addition, the extrapolation of preclinical research to the clinical use of radiochemotherapy is discussed.


   GEMCITABINE AS A SINGLE AGENT
Top
 Learning Objectives
 Abstract
 Introduction
 Gemcitabine as a Single...
Combination of Gemcitabine and...
 Conclusions
 References
 
Gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdC) has shown activity in various solid tumors, including NSCLC, small cell lung cancer, head and neck squamous cell cancer, germ cell tumors, and tumors of the bladder, breast, ovary, cervix, pancreas, and biliary tract [8–17], as well as some hematologic malignancies [18, 19]. The compound was synthesized in the 1980s at Lilly Research Laboratories, Eli Lilly and Co.; Indianapolis, IN [20]. It was initially intended for development as an antiviral agent because of its ability to inhibit DNA and RNA viruses. However, the therapeutic index was insufficient because of cytotoxicity to the parental cells. As a result of this, it was further developed as an anticancer agent with, as shown below, a unique mechanism of action.

Gemcitabine is a deoxycytidine analogue with structural similarities to cytarabine (Ara-C) (Fig. 1Go). Gemcitabine differs from Ara-C through its different schedule dependency, greater membrane permeability, affinity for deoxycytidine kinase (dCK), longer intracellular retention, and its multiple self-potentiating mechanisms of action [21] (Fig. 2Go).



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  		Figure 1. Structural formulas of deoxycytidine (CdR), cytarabine (Ara-C), and gemcitabine (dFdC).

 


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  		Figure 2. Metabolism and mechanisms of action of gemcitabine. 1) facilitated diffusion human nucleoside transporters (hNT); 2) deoxycytidine kinase (dCK); 3) deoxycytidine deaminase (dCDA); 4) deoxycytidylate deaminase; 5) ribonucleotide reductase; 6) CTP-synthetase; and 7) DNA polymerase.

 
Mechanism of Action
Membrane transport of gemcitabine is mediated by the facilitated diffusion human equilibrative or concentrative nucleoside transporters hENT or hCNT [22, 23]. Gemcitabine uptake is apparently mediated by hENT1, hENT2, and hCNT1 but not by hCNT2 [23–25]. Gemcitabine also appears to be a substrate for hCNT3 [26]. It is activated intracellularly by deoxycytidine kinase, which adds a phosphate to the 5' position of the ribose leading to the monophosphate dFdCMP. Subsequent phosphorylation yields the active metabolites gemcitabine diphosphate (dFdCDP) and triphosphate (dFdCTP), which target DNA and RNA and are presumed responsible for the cytotoxic effect [27, 28].

The main action of gemcitabine is thought to be the competitive incorporation of dFdCTP and dCTP into DNA [22, 27, 28], after which DNA polymerase is able to add only one more nucleotide before there is DNA fragmentation and cell death. This so-called masked chain termination prevents exonuclease recognition and excision of gemcitabine. Gemcitabine cytotoxic activity has been correlated with dFdCTP formation, its incorporation into DNA, and its inhibition of DNA synthesis [27–30]. Gemcitabine induces an S-phase arrest [31, 32], and triggers apoptosis both in human leukemic cells [33, 34] and in solid tumors [31, 35]. However, it is still unclear by which mechanism(s) gemcitabine incorporation leads to cell death. Only recently, it was demonstrated that gemcitabine can enhance topoisomerase I (Top 1)-mediated DNA cleavage in vitro. Top 1-deficient cells were also found to be resistant to gemcitabine, suggesting that the induction of Top 1-mediated DNA breaks can play a role in the cytotoxicity of gemcitabine [36, 37].

dFdCDP interferes with the enzyme ribonucleotide reductase, causing depletion of deoxynucleotide triphosphates necessary for DNA synthesis, and this might play a role in apoptosis by influencing DNA repair. At the same time, several self-potentiating mechanisms have been described, including inhibition of dCMP-deaminase and CTP-synthetase [38, 39], which lead to an increase of intracellular levels of the active compound (Fig. 2Go) [7, 40, 41].

Inactivation of gemcitabine occurs through deamination of gemcitabine itself or of dFdCMP to the metabolites 2',2'-difluoro-2'-deoxyuridine (dFdU) or 2',2'-difluoro-2'-deoxyuridine monophosphate (dFdUMP). Deamination is caused by the enzymes deoxycytidine deaminase and deoxycytidylate deaminase, respectively (Fig. 2Go). dFdU is 1,000-fold less active than gemcitabine [42; Pauwels et al., unpublished results]. Collectively, the various effects of gemcitabine result in a unique mechanism of action.


   COMBINATION OF GEMCITABINE AND RADIOTHERAPY
Top
 Learning Objectives
 Abstract
 Introduction
 Gemcitabine as a Single...
 Combination of Gemcitabine and...
Conclusions
 References
 
In addition to its cytotoxic effect, gemcitabine is a potent radiosensitizer. Nucleoside analogues are considered to be attractive compounds to combine with ionizing radiation for several reasons. First, as DNA synthesis inhibitors, nucleoside analogues have the potential to inhibit the repair of genomic damage induced by ionizing radiation. Second, because they are preferentially cytotoxic to proliferative cells, these analogues may decrease the number of tumor clonogens and thus slow down cell repopulation during fractionated radiotherapy. Tumor shrinkage induced by these compounds may improve tumor oxygenation and thus counter the detrimental effect of tumor hypoxia on radiation response. Third, as DNA chain terminators, nucleoside analogues may, following incorporation into the DNA repair patch, trigger an apoptotic response similar to that observed during the replication phase. Since DNA damage is induced in all phases of the cell cycle by radiation, this mechanism offers the prospect of extending the cytotoxicity of these analogues to non-S-phase cells [43].

Several preclinical and clinical studies have been and are being performed to investigate the radiosensitizing effect of gemcitabine and optimize the clinical application of the combination gemcitabine and radiotherapy, but the mechanism of radiosensitization by gemcitabine is still not fully elucidated, and the optimal treatment schedule still has to be defined.

Radiosensitization In Vitro
Gemcitabine is a potent radiosensitizer of rodent and human tumor cells, including pancreatic, non-small cell lung, head and neck, colorectal, breast, and cervical cancer (Table 1Go). Depending on the cell line tested, the drug concentration used, the schedule of administration, and the cell proliferative status (e.g., plateau versus exponential growth), dose enhancement ratios (DER) in the range of 1.1–3.0 have been reported.


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  		Table 1. Summary of the available data on the radiosensitizing potential in vitro of gemcitabine
 
The advantage of gemcitabine is that it can induce radiosensitization at concentrations which are 1,000 times lower than typical plasma levels obtained with the drug [44]. The sensitizing effect of non- or hardly cytotoxic concentrations increases with increasing gemcitabine concentrations [45–54].

This radiosensitization induced by gemcitabine is clearly schedule dependent. It has been shown that radiosensitization can be induced by either a long (24-hour) exposure to low concentrations of gemcitabine or by a brief (2-hour) exposure to higher, clinically relevant concentrations [55]. In addition, the DER increased proportionally to exposure time when cells were treated with non- or slightly cytotoxic concentrations using different incubation times [32, 49, 51].

Radiosensitization of human solid tumor cell lines with gemcitabine was found optimal when drug exposure preceded radiation exposure [56], and the maximal DER was observed when cells were incubated for a total of 24 hours [46]. Several studies found that irradiation applied 12–72 hours after 24-hour drug exposure induced lower DERs than in experiments in which radiation followed gemcitabine exposure immediately [41, 48, 49]. Our studies also showed a decreasing radiosensitizing effect with a greater interval between gemcitabine treatment and radiation [32]. However, Lawrence et al. [55] quoted that gemcitabine is relatively resistant to excision from DNA and thus has an extended biological activity. As a result of this, radiosensitization was thought to be retained for some time after gemcitabine had been removed from the medium. In fact, they observed ongoing radiosensitization up to 2 days after drug removal in HT-29 cells.

Recently, more information has become available on the radiosensitivity of gemcitabine-resistant cells [57]. In a new gemcitabine-resistant human lung cancer cell line (SWg), as well as in a gemcitabine-resistant human ovarian carcinoma cell line (AG6000), the sensitivity to ionizing radiation was not altered and resistance to gemcitabine did not eliminate the ability of gemcitabine to sensitize cells to radiation. Remarkably, greater sensitization effects have been observed also in radioresistant cell lines [51, 58].

During the past decade, extensive research has been conducted to clarify the mechanism of gemcitabine-induced radiosensitization. These studies focused on the effect on nucleotide pools, changes in the cell cycle distribution, on apoptosis, and on the characterization of intracellular reactions (DNA, RNA).

Nucleotide Pools
It has been shown that gemcitabine-induced dATP depletion may play a role in enhancing radiosensitivity. Gemcitabine was able to deplete the endogenous dATP in the cells by at least 80% as a result of the inhibition of ribonucleotide reductase. No significant differences in the other dNTP pools were observed after gemcitabine treatment under radiosensitizing conditions [46, 47, 52, 54].

Cell Cycle Distribution
Treatment with gemcitabine causes an accumulation of cells in the S phase of the cell cycle. This effect of gemcitabine seems important for the radiosensitizing effect. In our studies, we observed a clear correlation between the radiosensitizing effect and the amount of early S-phase cells [32]. Latz et al. [41] also indicated that cells must be in S phase to be radiosensitized by gemcitabine. They concluded that treatment of cells with gemcitabine immediately before irradiation eliminates, or at least greatly reduces, the variation in radiosensitivity during the cell cycle. The importance of S-phase accumulation for radiosensitization by gemcitabine was also stressed by the Michigan group [52, 54]. Using two human glioblastoma cell lines, Ostruszka et al. [54] demonstrated that U251 cells were radiosensitized at both IC10 and IC50 concentrations of gemcitabine. However, a variety of gemcitabine doses failed to radiosensitize D54 cells. An explanation for the difference in radiosensitization with gemcitabine was the observed difference in cell cycle distribution of the two cell lines at the time of irradiation. More than 70% of the U251 cells were in S phase, whereas <37% of the D54 cells were in S phase. Mose et al. [59] demonstrated that before progressing through the S phase, cells were blocked and partially synchronized at the G1/S border. Furthermore, they hypothesized that cells progressing following the block might accumulate proapoptotic signals caused by both radiation and gemcitabine, which will also result in cell death.

Chen et al. [53] and Robinson et al. [52] investigated the role of p53 in gemcitabine-mediated cytotoxicity and radiosensitization. Chen et al. reported that p53 status may influence gemcitabine-mediated apoptosis, cytotoxicity, and cell cycle progression, but a significant role of p53 in the radiosensitization could not be demonstrated [53]. The same was observed by Robinson et al. [52]. The use of isogenic cells differing in p53 status gave us the opportunity to investigate the effect of p53 status and transfection on the radiosensitizing and cell cycle effect of gemcitabine. Since both the radiosensitizing effect at equitoxic conditions and the effect on the cell cycle were independent of p53 expression, we also concluded that p53 protein does not seem to play a crucial role in the radiosensitizing mechanism of gemcitabine (Pauwels et al., unpublished results).

Apoptosis
It seems logical to investigate the role of apoptosis in gemcitabine-mediated radiosensitization, since this mechanism of cell death was shown to be the pathway by which the drug exerts its cytotoxic action [31, 33, 34, 60]. Lawrence et al. [61] found that apoptosis played an important role in the gemcitabine-mediated radiosensitization of apoptotic-prone HT-29 colon cancer cells, but not in that of the UMSCC-6 head and neck cancer cells and A549 lung cancer cells, which differ substantially in the ability to undergo radiation-induced apoptosis. Nevertheless, the latter two cell lines were also, though modestly, radiosensitized. These findings suggested that, although apoptosis can contribute to gemcitabine-mediated radiosensitization, the role of apoptosis depends on the cell line rather than representing a general property of the drug.

Intracellular Reactions
Evidence from Weiss et al. [62] suggests that a significant increase in residual DNA damage is a possible mechanism of radiosensitization by gemcitabine. They noted that when gemcitabine was added to radiotherapy, a considerable and dose-dependent increase of DNA double strand breaks (DSBs) remains 24 hours after radiotherapy (5.4-fold to 12.2-fold). However, Gregoire et al. [63] concluded that neither the induction nor the repair of DNA DSBs after ionizing radiation was affected by gemcitabine. In a later study [64], they confirmed this in cells with different intrinsic radiosensitivity (SCC61 and SQD9). They did show, however, that in experimental conditions in which substantial radioenhancement was present, gemcitabine decreased the repair of genomic lesions, which led to secondary chromosome breaks. Wachters et al. [65] addressed the possibility of whether gemcitabine might affect homologous recombination (HR)-mediated DSB repair or base excision repair (BER). They did this by comparing the gemcitabine-mediated radiosensitization in cell lines that were deficient in BER and in HR with that in their BER-proficient and HR-proficient parental counterparts. Gemcitabine did induce radiosensitization in BER-deficient cells, but did not induce this in the mutant cells deficient in HR. Therefore, they concluded that gemcitabine can sensitize cells to radiation by specific interference with the HR pathway.

Gregoire et al. [66] found in various cell lines an excellent correlation between the specific enzymatic activity of dCK, the enzyme necessary to activate gemcitabine intracellularly, and the radiosensitization of gemcitabine. Cell lines that expressed a high enzymatic activity of dCK were the most radiosensitizable. In their experiment, dFdCTP, the triphosphate form of gemcitabine, which is responsible for the cytotoxic effect of gemcitabine, did not seem to play an important role in this radiosensitization process. This was also found by the Michigan group [46, 56]. They showed that radiosensitization by gemcitabine in human tumor cells not only occurred at concentrations that are 1,000 times lower than the typical plasma levels found with the drug, but that this effect was independent of the intracellular concentration of dFdCTP as well as the relative dFdCTP/dCTP levels [46, 56].

As mentioned earlier, dFdU, the main metabolite of gemcitabine, is 1,000-fold less active than gemcitabine [42] (Pauwels et al., unpublished results). Nevertheless, we found that slightly cytotoxic concentrations of dFdU can cause radiosensitization (Pauwels et al., unpublished results). Because plasma concentrations of dFdU are maintained for a prolonged time (>24 hours) at levels known to cause growth inhibition, dFdU might be partly responsible for the radiosensitizing effect of gemcitabine.

To the best of our knowledge there is only one published study using transitional cell carcinoma (TCC) cell lines that did not demonstrate a radiosensitizing effect of gemcitabine, even though the drug itself had a dose-dependent cytotoxic and apoptosis-inducing effect on all TCC cell lines tested [67]. Another recent study observed an unusual radiation response of gemcitabine-treated HeLa cells [68]. Experiments with HeLa cells demonstrated the ability of gemcitabine to both enhance and reduce radiation cell kill under different conditions. The observed increase in survival rate was limited to noncytotoxic exposures and was highly dependent on cell kinetic changes. Shortening exposure time avoided cell cycle effect and eliminated the increased radiation survival rate [68].

The in vitro studies so far have demonstrated that gemcitabine, in the great majority of experiments, clearly enhances the radiation cell killing effect. For most cell lines, radiosensitization was evident at noncytotoxic concentrations. The extent of radiosensitization generally increases with both increasing gemcitabine concentrations and duration of exposure. However, no optimal schedule can be deduced from these data and the mechanism of radiosensitization still has not been fully elucidated. The first in vivo experiments to investigate the combination of gemcitabine and radiation have built on the work done in cell culture.

Radiosensitization In Vivo
Data on the combination of gemcitabine and radiation in vivo are more limited. However, available studies in vivo have confirmed the radiosensitization potential of gemcitabine. Furthermore, they provided insight into the timing of gemcitabine administration, relative to radiation exposure, which may have led to further optimization of the therapeutic index.

In various mouse models, the combination of gemcitabine and radiation resulted in a radiosensitizing effect with DERs ranging from 0.9–3.3 using different gemcitabine doses and treatment schedules (Table 2Go).


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  		Table 2. Summary of the available in vivo studies on the radiosensitizing potential of gemcitabine
 
The effect of radiosensitization of gemcitabine has also been studied in normal tissues. In mouse jejunum, a small radiosensitizing effect (DER 1.3) was observed for a 3-hour time interval between drug treatment and single-dose irradiation, whereas a slight radioprotection was observed for a 48-hour time interval (DER 0.9). Classen et al. [69] found that the timing of single-dose gemcitabine relative to single-fraction radiotherapy had no major influence on the acute skin reactions or on the fibrotic changes both in skin and underlying soft tissue in a nude mouse model. Also, the mouse lung tolerance was not greatly affected by gemcitabine with single-dose radiation, no matter which interval (long or short) was used between radiation and drug administration [70]. On the other hand, normal tissue toxicity was found increased when gemcitabine was administered more than once [71]. Joschko et al. [72] compared daily gemcitabine administrations with weekly and twice-weekly administrations, each time given in combination with radiotherapy and each time using the maximum tolerated dose (MTD) of gemcitabine. They found the highest DER with the weekly and twice-weekly regimens. Significant increases in toxicity occurred with radiation plus twice-weekly administration of gemcitabine, but radioenhancement was already seen using gemcitabine doses well below the MTD. From the above, it can be deduced that both gemcitabine toxicity and efficacy are strongly dependent on the schedule used. The drug alone is well tolerated, even in a fractionated prolonged treatment setting. In all schedules, treatment with high drug doses have no surplus value; on the contrary, there is a threshold dose beyond which efficacy no longer increases, but toxicity does [73].

Most in vivo research has focused on the schedule dependency of the radiosensitizing effect of gemcitabine (Table 2Go). While radiosensitization was typically observed for all time intervals tested before or after irradiation, the maximal effect was observed when gemcitabine was administered between 24 and 60 hours prior to irradiation for both local tumor cure and tumor regrowth delay. Mason et al. [71] tested a single (25 mg/kg) dose of gemcitabine versus repeated (two times or five times) doses (12.5 mg/kg) of gemcitabine, each time given 24 hours before fractionated radiotherapy (3, 5, or 7 Gy daily for 5 days, Table 2Go). Because toxicity to normal tissue was increased with the repeated gemcitabine administrations, the highest therapeutic gain (1.4) was achieved with the single dose of gemcitabine 24 hours before the start of the fractionated radiotherapy. Fields et al. [74] compared radiotherapy with or without gemcitabine delivered either once (800 mg/kg) or twice weekly (150 mg/kg), 6 hours prior to irradiation. In these circumstances they observed a better therapeutic index with the twice-weekly regimen. Cividalli et al. [73], comparing different gemcitabine doses at different intervals prior to radiation (20 minutes, 4 hours, and 24 hours), observed more impact of the radiation dose than the gemcitabine dose, but the 24-hour interval seemed crucial. Interestingly, in one study the gemcitabine/radiotherapy combination also decreased the rate of developing lung metastases [75]. This observation was confirmed in a second study with a larger number of mice [71]. This observation supports the principle of combined modality therapy in that gemcitabine helped in minimizing the primary tumor burden, translating into a decreased systemic spread of tumor cells.

Unfortunately, only limited data are available on factors that can be associated with the radiosensitization in vivo of gemcitabine. The study of Grégoire et al. [76] in crypt cells tended to support the hypothesis that inhibition of DNA synthesis and cell cycle redistribution are surrogates for radiosensitization. Mason et al. [71] indicated in their study that apoptotic death of S-phase cells exposed to gemcitabine and reoxygenation of the resistant hypoxic fraction of tumor cells were major mechanisms for tumor radioenhancement by gemcitabine. Therefore, elimination of the S-phase tumor cells may aid the radiation response by not only causing cell cycle synchronization but also by leading to reoxygenation of hypoxic cells.

The question remains how results of these in vivo experiments could help to design clinical trials with a better treatment schedule for the combination gemcitabine and radiotherapy. The earlier-mentioned data of Joschko et al. [72] indicated that significant enhancement was seen in particular with the weekly and twice-weekly dosing of gemcitabine, but that the weekly dosing seemed less damaging to normal tissues. In contrast to these findings, Fields et al. [74] found a higher therapeutic index with the twice-weekly dosing when compared with the once-weekly dosing of gemcitabine in both cases combined with daily radiotherapy. Also, Cividalli et al. [73] concluded that administration of low gemcitabine doses twice weekly, starting 24 hours before the first x-ray fraction of a conventional x-ray treatment or a hypofractionated treatment, might be an interesting protocol for clinical study.

Clinical Application of Gemcitabine as a Radiosensitizer
Preclinical data, from both in vitro and in vivo studies, predicted that gemcitabine would be a very potent and useful complementary treatment to ionizing radiation. However, clinical trial design depends not only on preclinical data, but also on clinical estimates of normal tissue toxicity. The latter, however, is difficult to predict from preclinical models.

In the past few years, several clinical trials of the combined use of gemcitabine and radiotherapy have been conducted to explore its effect on cancers where either the role of radiotherapy is well established (e.g., head and neck cancer), and/or where gemcitabine itself has shown clinical activity (e.g., NSCLC) (Table 3Go).


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  		Table 3. Phase I and II trials of concurrent gemcitabine and radiation
 

Phase I Studies
Blackstock et al. [77] and Pipas et al. [79] determined the MTD, dose-limiting toxicities (DLTs), and potential antitumor activity of twice-weekly administrations of gemcitabine and concurrent radiation in patients with pancreatic cancer. Both concluded that radiation with twice-weekly administration of low doses of gemcitabine was well tolerated and showed promising activity in patients with advanced pancreatic cancer. There was a remarkable consistency in outcome of both studies. The MTD was 40 mg/m2 in the study reported by Blackstock et al. [77] and 50 mg/m2 in the study reported by Pipas et al. [78]. Doses investigated in these studies ranged from 10 mg/m2 to 60 mg/m2. In two of six patients treated by Pipas et al., severe upper gastrointestinal (GI) complications (ulceration, bleeding) were noted only at the highest dose level, occurring approximately 1 month from completion of therapy [78].

Phase I studies in patients with pancreatic cancer using a once-weekly gemcitabine dose schedule suggested that with conventional radiotherapy regimens the MTD is in the range of 250–350 mg/m2 [79, 80]. Similar dose ranges of gemcitabine have been proposed for chemoradiation in lung cancer patients based on data obtained in phase I studies using the once-weekly dose schedule [81, 82]. Van Putten et al. [82] indicated that 300 mg/m2/week of gemcitabine was feasible for 6 weeks together with radiotherapy as long as the radiation treatment volume did not exceed 2,000 cm2. In addition, in patients with advanced unresectable NSCLC who achieved clinical downstaging so as to re-enter resectability, surgery proved to be possible without any increase in morbidity or mortality [81].

Worrisome late GI toxicities, including ulceration, bleeding, strictures, and fistula, have been observed by other investigators who also studied the once-weekly gemcitabine regimens, but either at higher doses or with larger radiation dose fractions [83, 84]. In the Amsterdam study, using a dose of 300 mg/m2 of gemcitabine concurrent with radiotherapy (three fractions of 8 Gy on days 1, 8, and 15), ulcerations in the stomach and duodenum occurred in 37.5%, 20.8% had ulceration with bleeding, and 1 of 24 patients developed a fistula between aorta and duodenum [84].

The doses of gemcitabine that patients with head and neck cancer tolerate on a weekly basis during radiotherapy are generally lower. Eisbruch et al. [85] reported that due to severe acute and late mucosal- and pharyngeal-related DLTs, gemcitabine doses had to be de-escalated from 300 mg/m2/week to 150 mg/m2/week and even 50 mg/m2/week in successive patient cohorts in their phase I study. However, even at this lowest level, severe late toxicities were observed. No late DLT was observed at 10 mg/m2/week. It is not completely clear why the duodenal mucosa has a higher tolerance to the radiosensitizing effects of gemcitabine compared with oral/pharyngeal mucosa.

In most studies reported in Table 3Go, gemcitabine was administered as a 30-minute infusion. When gemcitabine was administered as a 24-hour continuous intravenous infusion given weekly with concurrent radiotherapy, doses of 50 and 100 mg/m2 were well tolerated, while higher doses resulted in severe toxicity. It appears that the MTD for weekly 24-hour infusion of gemcitabine combined with daily radiation therapy (and an accumulation dose of 40 Gy) is 100 mg/m2, at least when it concerns GI malignancies [86]. In this study, in which 13 patients with unresectable colorectal cancer and 12 patients with advanced unresectable pancreaticobiliary cancer were included, an overall response rate of 75% (50% clinically complete) was found.

Phase II Studies
In a phase II study, Van Laethem et al. [87] evaluated the feasibility and tolerability of postoperative administration of gemcitabine alone followed by concurrent gemcitabine and upper abdominal radiotherapy after curative resection of pancreatic head adenocarcinoma. Before the concurrent administration of gemcitabine and radiotherapy, patients were given three cycles of gemcitabine alone (1,000 mg/m2 on days 1 and 8 every 3 weeks). This was followed by gemcitabine 300 mg/m2 weekly plus 40 Gy in a split course. This regimen was well tolerated and was completed by all patients except two: one received only 20 Gy because of World Health Organization grade 4 vomiting and thrombocytopenia and the other stopped radiotherapy after 32 Gy because of early disease progression. No reduction in gemcitabine dose during radiotherapy was necessary; no toxic death was noted and no late toxicity developed. In a study of gemcitabine combined with radiation in patients with localized, unresectable pancreatic cancer, weekly gemcitabine at a dose of 1,000 mg/m2 for 7 weeks was given as an induction phase. Patients who showed clinical benefit and in whom reduced or stabilization of tumor size was confirmed by computed tomography entered the chemoradiotherapy phase of the treatment. This treatment schedule was well tolerated and can provide prolonged clinical benefit and disease stabilization with localized, unresectable pancreatic cancer [88].

Other phase II studies with concurrent gemcitabine and radiotherapy in locally advanced stage IIIB cervical carcinoma [89] and locally advanced squamous cell carcinoma of the head and neck [90] showed that the combination was well tolerated and effective. Dosages used in these studies were 300 mg/m2 and 50–100 mg/m2, respectively. Data indicating that the use of gemcitabine and radiotherapy might have superior effects come from randomized studies in which the control arm consisted of the currently used standard approach. As an example, 5-fluorouracil concurrent with radiotherapy so far has been considered the standard treatment for locally advanced pancreatic cancer. Recently, Li et al. [91] showed in a small randomized study that gemcitabine concurrent with radiotherapy was superior over the standard in terms of response, quality-adjusted life month survival, progression-free survival, and overall survival and was comparable to the standard in terms of tolerability. However provocative, the study was limited to 36 patients.

Notable case reports in the literature of normal tissue toxicities include those that either are felt to be related to the drug itself (e.g., acute interstitial pneumonitis) [92, 93] or to its relationship with radiation therapy (e.g., radiation recall dermatitis) [94, 95]. There is strong evidence from the clinical literature that tolerance to the combination of gemcitabine and radiation depends on the dose and schedule of gemcitabine, the fraction size of radiation, the volume of normal tissues irradiated, and the type of normal tissues irradiated. The lower MTDs with biweekly gemcitabine administration schedules in clinical studies are consistent with the preclinical data reviewed earlier that showed normal tissue toxicity occurring at lower gemcitabine doses with biweekly [71, 74] and daily dosing [71]. The phase I dose-escalation studies in patients with pancreatic cancer that have used radiation fields to confine the tumor alone [83, 96] were able to administer weekly doses two to three times higher than those that have used regional fields [88, 97, 98]. The gemcitabine infusion rate issue not withstanding, weekly doses in the range of 200–400 mg/m2 [88, 97, 98] were tolerated with fields that included the primary tumor and regional lymphatics, while doses in the range of 700–1,000 mg/m2 could be given with smaller fields [83, 96]. Overall, different studies have indicated that a large volume of normal tissue within the field of irradiation may lead to an increased risk of radiation injury with this combination [82, 83]. Moreover, there also appears to be an inverse linear relationship between the MTD of gemcitabine and the radiation dose used [99].

Gemcitabine Combinations with Radiotherapy
In an attempt to improve locoregional activity, potentially there may be a role for combining various radiosensitizers with radiotherapy. Several institutions and study groups have begun to investigate the addition of other cytotoxic agents to gemcitabine-based chemoradiation. Phase I studies of the combined modality therapy of gemcitabine and radiotherapy with cisplatin [100–108], paclitaxel [109], and mitomycin C [110] are already published. The results of these studies suggested that incorporation of more active drugs into the combined modality regimen might be beneficial. A striking example of this can be found in the observations made by Duenas Gonzales et al. [111] in patients with stage IB2-IIB cervical cancer. In this latter study patients were randomized to receive either cisplatin (40 mg/m2) or cisplatin/gemcitabine (40 mg/m2 and 125 mg/m2, respectively) weekly for 6 weeks concurrent with external-beam radiotherapy (50 Gy), followed by radical hysterectomy 4 weeks later. High-risk patients (based on the pathologic specimen) in both arms received adjuvant brachytherapy. In an interim analysis the investigators observed significantly more complete or near-complete pathological complete responses (56.5% versus 87%; p = 0.0273) and significantly fewer patients needed brachytherapy in the cisplatin/gemcitabine arm (p = 0.0273). As mentioned in this interim report, a phase III randomized trial is ongoing to confirm these data and to study any positive impact in survival.

In addition, as has been observed in studies in patients with NSCLC, through complimentary mechanisms, both sequential and concomitant combined modality therapy strategies can lead to improved survival. Therefore, a further improvement may be hoped for when induction chemotherapy (potentially leading to a reduction in distant failure rate) is combined with chemoradiation (enhancing locoregional control). Data from a Cancer and Leukemia group B study (CALGB 9431) were quite promising in that respect [108]. In principle, such an approach is also possible by combining chemoradiation with adjuvant chemotherapy [112]. However, in some specific tumor types, e.g., head and neck cancer, that approach has shown to be difficult to tolerate, leading to poor compliance [113].

In our review of the available clinical data, the concurrent administration of gemcitabine and radiation has a high potential for improving the outcome of treatment in several advanced solid tumors. However, very often the toxicity of this combination also appears to be significant. To date, it is difficult to draw any definitive conclusions about which of all the tested combined treatment schedules are optimal with a positive balance between efficacy and tolerance.


   CONCLUSIONS
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 Learning Objectives
 Abstract
 Introduction
 Gemcitabine as a Single...
 Combination of Gemcitabine and...
 Conclusions
References
 
A body of experimental evidence shows that gemcitabine is a potent radiosensitizer in vitro and in vivo. However, the observations reported so far indicate that various mechanisms of radiosensitization might play a role in tumor and normal cells, translating into potential differences in radiosensitization [50, 70].

The in vitro data suggest that most rodent and human tumor cells are sensitized by gemcitabine. This enhancement effect seems to depend on the concentration and duration of exposure and the timing of irradiation. Radiosensitization occurred at noncytotoxic concentrations and the maximal radiosensitizing effect was observed when gemcitabine exposure preceded radiation without any delay. Despite extensive studies, the molecular mechanisms of interaction between gemcitabine and ionizing radiation are only partially resolved. In essence, the current evidence supports the notion that gemcitabine-mediated radiosensitization occurs through a combination of mechanisms that appears to include nucleotide pool perturbations, cell cycle redistribution, reduction of apoptotic threshold, inhibition of DNA synthesis, and reduction of DNA repair.

All the in vivo studies showed an increased antitumor effect when radiotherapy was given concurrently with gemcitabine and that normal tissues recover from the radiosensitizing effects of gemcitabine more quickly than do tumors. Maximal effect was observed when gemcitabine was administered 24 hours or more prior to radiation. Data provided suggest that the effect of the drug is dependent not only on the schedule but also on the duration of the infusion.

Although it is often difficult to transfer experimental data to the clinic, these studies offer the possibility to develop an improved schedule of administration for patient treatment, based on rational evidence in tumor biology. The clearest observation from the clinical data is the amount of acute toxicity from the combination of gemcitabine and radiation. The amount of acute toxicity seems strongly related to the dose and schedule of gemcitabine administration, as well as to the radiation field size. There appears to be an inverse linear relationship between the maximum tolerated gemcitabine dose and the radiation dose used [99]. The observed impact of timing of administration of gemcitabine related to radiation in the preclinical setting has not yet been confirmed by clinical data. Also important but less clear is the infusion rate of gemcitabine as it relates to the systemic efficacy of the drug. The saturation in the rate of dFdCTP thereby seems to play a crucial role [114]. In that respect, observations in early clinical studies were of importance, showing that the rate of dFdCTP formation was optimized using dose rates approximating to 10 mg/m2/minute [115, 116]. The improved antitumor activity with the fixed dose-rate infusion of gemcitabine [117] in patients with advanced and metastatic pancreatic cancer so far has not been tested in the chemoradiation context.

Nevertheless, gemcitabine is one of the most promising agents to combine with radiotherapy, not only in the laboratory but also in the clinic (Table 3Go). Comparative, multi-institutional trials are needed to draw conclusions about important issues such as the optimal dose, schedule, and combination of agents to use with radiotherapy. Major attention should be given to strategies that might reduce in particular the incidence of severe acute side effects. Modern techniques of radiation delivery, such as three-dimensional conformal radiotherapy, could potentially decrease normal tissue toxicity and allow radiation dose escalation. This may lead to further improvement in local control of the disease and cure for more patients. The current series of ongoing trials will be of great importance in that and in the elucidation of the mechanism involved in cell- or tissue-dependent nucleoside analogue radiosensitization.


   ACKNOWLEDGMENT
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 Learning Objectives
 Abstract
 Introduction
 Gemcitabine as a Single...
 Combination of Gemcitabine and...
 Conclusions
 References
 
This study was financially supported by a grant from the Foundation Emmanuel van der Schueren.


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 Introduction
 Gemcitabine as a Single...
 Combination of Gemcitabine and...
 Conclusions
 References
 

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Received May 17, 2004; accepted for publication September 3, 2004.




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