Intravesical gemcitabine for superficial bladder cancer: rationale for a new treatment option
Paolo Gontero, Clinica Urologica, University of Piemonte Orientale, Novara, Italy.
superficial bladder cancer
transurethral resection of bladder tumour
carcinoma in situ
European Organization for Research and Treatment of Cancer
Superficial bladder cancer (SBC) represents nearly 70% of all bladder cancers at first presentation and comprises a heterogeneous population of tumours that do not invade the muscularis propria. The 5-year recurrence rate after complete transurethral resection of bladder tumour (TURBT) is estimated to be as high as 80%, whereas progression to muscle-invasive disease occurs in 4–30%. The presence or absence of lamina propria invasion (T1 and Ta) and the grade of differentiation (G1 to G3) allow the identification of a spectrum of categories with different prognoses. Low-grade noninvasive papillary tumours carry a small risk of tumour progression. In contrast, high-grade T1 tumours have a 30–50% risk of progression . Carcinoma in situ (CIS), a flat and noninvasive lesion of the surface mucosa that can occur alone or in association with papillary tumours, is also considered a high-risk SBC for its potential to develop into a muscle-invasive disease in 20–80% of cases .
Based on histology and other prognostic factors, the European Organization for Research and Treatment of Cancer (EORTC) and the European Guidelines  distinguish three risk categories for SBC. Single Ta, G1 lesions <3 cm in diameter are considered as low-risk whereas high-risk tumours are represented by T1, G3 or CIS. All other tumours, i.e. Ta, T1, G1-G2, multifocal, recurrent, or >3 cm in diameter, fall into the intermediate-risk group.
While observation after complete endoscopic eradication has been advocated for low-risk SBC, several intravesical drugs have been proposed for intermediate- and high-risk diseases in an attempt to reduce or delay both recurrence and progression. Intermediate risk SBC is initially managed with prophylactic intravesical chemotherapy, whereas BCG immunotherapy has become the standard treatment for high-risk SBC, including T1G3, CIS and some recurrent Ta diseases. Significant limitations in efficacy and tolerability for the most widely used intravesical agents across all risk categories of SBC have favoured the search for new treatment alternatives. The present overview will address the rationale and the initial results of intravesical gemcitabine as a novel chemotherapeutic agent for SBC.
THE NEED FOR NEW TREATMENT OPTIONS FOR SBC
There are limitations in efficacy of intravesical treatments for intermediate-risk SBC; conventional intravesical chemotherapy (i.e. doxorubicin, mitomycin C) or BCG are used as a prophylaxis to prevent recurrence after complete tumour resection. Early recurrence can be decreased by half after mitomycin C, while long-term recurrence rates seem to be reduced to a lesser extent (54% vs 41%) . Other authors have reported even worse results. According to Lamm  the short-term recurrence rate cannot be reduced by more than 15–20%, and the long-term risk of recurrence by 6%. In a median follow-up of 26 months, 46% of 1328 patients treated with mitomycin C developed recurrence, according to Bohle et al.. Recent meta-analyses have shown that BCG is superior to intravesical chemotherapeutic agents in reducing recurrences , although a 2-year recurrence rate of 40% has to be expected .
There are also limitations in efficacy of intravesical treatments for high-risk SBC; at present, the role of intravesical treatment for high-grade stage T1 SBC remains controversial. The role of mitomycin C in this risk category seems to be confined to a reduction of the recurrence rate with no effect on tumour progression . By contrast, two recent meta-analyses showed that BCG is able to reduce the risk of progression to muscle-invasive disease , provided maintenance treatment is used . However, most of the studies available included mixed series where only a minority of the recruited patients had high-risk SBC.
Although BCG is currently considered the most effective conservative treatment for T1G3 SBC, its real efficacy remains controversial. Peyromaure et al. reported a 42% and 28% recurrence and progression rate, respectively, in 57 patients followed up for 53 months. Shahin et al. found that 70% of their patients recurred after BCG, vs 75% treated with TURBT alone. These results have led many authors to advocate early cystectomy for T1G3 SBC, particularly in the presence of unfavourable prognostic factors . However, radical surgery will undoubtedly be an over-treatment for a substantail proportion of patients and hence there is an urgent need for alternative treatment options for this highly recurrent and progressive disease.
Similar conclusions can be drawn for CIS, which is usually considered highly responsive to BCG treatment. Although 70% of cases are indeed initially cured, the 5-year progression rate remains as high as 35%.
T1G3 and CIS that fail to respond to BCG are defined as BCG-refractory . Current options for such cases are total cystectomy or alternative intravesical therapy. Various immunological and chemotherapeutic regimens have been evaluated for this purpose but their efficacy is still a matter of debate. Encouraging initial results were reported with salvage intravesical protocols combining interferon-α2B and BCG , but long-term follow-up and larger studies are needed to validate this treatment schedule. Alternating mitomycin C and BCG has been proposed with the aim of improving the cure rate of CIS, although preliminary data from a Nordic study failed to show any advantage over BCG monotherapy . Until new agents can be shown to decrease recurrence rates and delay progression of high-risk SBC, cystectomy remains the best option for such patients .
Side-effect burden of currently used intravesical agents
Local side-effects can be experienced by up to 90% of patients treated with BCG. Cystitis is by far the most common complaint, which is described as moderate to severe by nearly half of patients. More seriously, BCG can cause severe systemic side-effects like ‘BCG-osis’ and sepsis that have resulted in several deaths. A fever of >39°C may occur in 14–30% of cases, leading to cessation of treatment in 10%. There is an emerging role for BCG maintenance as a crucial requirement for optimum efficacy. Although an increase in toxicity with this treatment schedule has not been shown clearly , usually ≤ 15% of patients complete the maintenance cycles . The assumption that BCG would work better in the presence of side-effects has recently been found to be inconsistent  and therefore BCG toxicity should be viewed as a significant limitation to treatment compliance.
Chemotherapeutic agents such as mitomycin C and doxorubicin, despite the low probability of systemic side-effects, can give rise to severe forms of chemical cystitis .
A new treatment option for SBC would be suitable for a large population of treatable patients, e.g. with intermediate- risk SBC recurring after conventional treatment, primary T1G3 patients (where conventional treatment is still controversial) and with BCG-refractory high-risk SBC who are unfit for and/or refusing radical surgery.
THE RATIONALE FOR USING INTRAVESICAL GEMCITABINE
Mechanism of action
Gemcitabine (2′,2′-difluorodeoxycytidine) is a novel deoxycytidine analogue with a broad spectrum of antitumour activity. After transport into the cell it is phosphorylated and incorporated into the DNA and RNA. This causes inhibition of cell growth and triggers apoptosis . Gemcitabine is then deactivated by deamination into 2′,2′-difluorodeoxyuridine (dFdU) and transported out of the cell.
Several characteristics make gemcitabine a promising candidate for intravesical therapy for superficial TCC:
(i) When given systemically there is significant activity as a single agent against invasive bladder cancer, yielding response rates of 27–38%. These results have led to the assessment of gemcitabine combined with cisplatin, which gave excellent response rates. Also, in a phase II study on 35 patients with metastatic bladder cancer previously treated with cisplatin, 1200 mg/m2 of gemcitabine resulted in an overall response rate of 22.5%.
(ii) Absorption of chemotherapy drugs from the bladder is related to several factors. A molecular weight of <300 Da is one of the primary determining features. Gemcitabine has a molecular weight of 299 Da, lower than that of commonly used intravesical chemotherapeutic agents such as mitomycin C (389 Da) and doxorubicin (589 Da). This may enable gemcitabine to penetrate the bladder mucosa, with beneficial effects in the treatment of early invasive bladder cancer (T1 disease). At the same time the molecular weight is high enough to prevent significant systemic absorption in an intact bladder. The pH of the solution and the polarity of the drug is another key element which influences the rate of absorption. The pKa of gemcitabine is 3.6 and its reconstitution results in a solution with a pH of 2.7–3.2. This will favour negligible ionisation of the drug at the usual urinary pH of 6.0–7.0.
(iii) Its pharmacokinetic properties make gemcitabine an ideal candidate for regional therapy. When given i.v. it is rapidly deaminated into the inactive metabolite, resulting in a high total body clearance .
In vitro studies
The in vitro cytotoxic effect of gemcitabine in intravesical therapy was investigated by Kilani et al. using a co-cultured spheroid model composed of bladder cancer cells and fibroblasts. Gemcitabine produced selective cytotoxicity to human and rodent TCC cell lines with relative sparing of fibroblasts, suggesting that intravesical exposures sufficient to induce the death of SBC cells might produce tolerable levels of normal bladder tissue toxicity.
In another in vitro study, gemcitabine sensitivity was compared to adriamycin, epirubicin and mitomycin C for relative potency on TCC cell cultures . Gemcitabine at 10 mg/mL resulted in a more robust cytotoxic activity (90% lethality in all cell lines) than the other chemotherapeutic agents. Based on these experimental findings the assessment of the antitumour activity of gemcitabine when administered intravesically appears feasible.
Pre-clinical studies of intravesical gemcitabine
Three preclinical animal studies were reported with the specific aims of assessing organ-specific toxicity and identifying the possible systemic absorption of gemcitabine following intravesical administration. In the first , three groups of dogs received 100 mg, 350 mg (equivalent to the 1000 mg/m2 human dose) or 1 g of gemcitabine in 50 mL for 1 h three times a week for 4 weeks. Two further dogs were treated with a 3.5 g dose. The 100 and 350 mg doses were well tolerated with no clinical signs of side-effects. There was a slight decrease in platelet count and white cell count in the 350 mg group. At necroscopy there were no apparent toxic effects, particularly in the bladder and the bone marrow. Both the 1 and 3.5 g doses caused severe clinical toxicity, myelosuppression and multi-organ toxicity on necroscopy, and high serum concentrations of gemcitabine. Plasma levels of gemcitabine after intravesical use of 350 mg were less high, at ≈ 30 µmol/L, a concentration usually found in human plasma after i.v. administration of a full dose. The authors concluded that, despite significant systemic absorption, it was possible to deliver a clinically active dose with no local or systemic toxicity.
By contrast, Witjes et al. detected no measurable plasma concentrations of either gemcitabine or dFdU when 350 mg of the active drug, diluted in 50 mL, was instilled in five female pig bladders for 2 h every week for 6 weeks. The marked difference between these findings and those of Cozzi et al. are probably explained by the much more frequent schedule (three times a week) used in the latter study.
Matera et al. assessed the intravesical administration of gemcitabine in New Zealand male rabbits, in view of the high sensitivity of rabbit bladder to toxic agents and of the histological resemblance to the human bladder. Twenty-four rabbits were assigned to three groups in which saline solution, 25 or 50 mg/kg gemcitabine were instilled. In the group treated with 25 mg/kg (equivalent to a 1575 mg/m2 human dose) there were no clinical symptoms indicative of toxicity. By contrast, rabbits receiving 50 mg/kg (equivalent to 3150 mg/m2 human dose) developed severe symptomatic toxicity leading to death in three. There was mild myelosuppression only at the highest dose, although systemic absorption was very low at 1 and 2 h after dosing, and undetectable after 24 h.
In conclusion, these studies, albeit limited by the use of animal models, provided useful information for estimating the maximum dose to be used in a potential phase I human study. Drug concentrations of >50 mg/kg, according to Matera et al., may be above the threshold at which increased morbidity is likely to occur. Another important finding was the absence of bladder-specific toxicity and negligible systemic absorption, even at high doses of 50 mg/kg in rabbits (equivalent to ≈ 3150 mg/m2 in humans) , and at 350 mg in dogs (equivalent to 1000 mg/m2 in humans) .
PHASE I HUMAN STUDIES: DEFINITION OF THE OPTIMUM DOSAGE
Pharmacokinetics of intravesical gemcitabine
After standard i.v. administration of 1000 mg/m2 gemcitabine in 30 min, peak plasma concentrations of 30 µmol/L for the drug and 100 µmol/L for its inactive metabolite dFdU were recorded . In the early report by Dalbagni et al. gemcitabine was administered intravesically starting at the 500 mg dose (300 mg/m2 in a 1.7 m2 individual) which is ≈ 20% of the maximum-tolerated dose found in a previous animal study (1000 mg/m2) . In a dose-escalation protocol, 18 high-risk patients with BCG-refractory SBC received 500, 1000, 1500 and 2000 mg gemcitabine diluted in 100 mL for 1 h twice weekly for 3 weeks, with each course separated by a week of rest. The levels of gemcitabine were undetectable up to 1500 mg, whereas at the 2000 mg dose two of six patients had measurable plasma concentrations (including one with grade 3 thrombocytopenia and neutropenia).
In the dose-finding study of Laufer et al., 15 patients received 500, 1000, 1500 and 2000 mg in 100 mL (in four the 2000 mg dose was diluted in 50 mL) once a week for 6 weeks, with an instillation time of 2 h. The pharmacokinetics were assessed with the first dose; blood samples were obtained at 15, 30, 60, 90 and 120 min after the instillation and at 5, 10, 15, 30 and 60 min after voiding. Very little gemcitabine was found in plasma; there were low plasma concentrations of gemcitabine (≤1 µmol/L) in the four patients receiving 2000 mg in 50 mL; dFdU was detectable only at doses >1500 mg in some patients. Most importantly, plasma concentrations of gemcitabine decreased during the time that gemcitabine was left in the bladder, indicating that it is the initial influx of gemcitabine into the bladder, rather than the presence of the drug in the bladder, which may be critical for systemic absorption.
In a similar study by De Berardinis et al., gemcitabine resulted in plasma concentrations always below the detection limit when instilled in the bladder for 2 h at dosages up to 40 mg/mL (2000 mg/50 mL), and the concentrations of its inactive metabolite dFdU were also remarkably low. In the same study, the activity of the activating enzyme deoxycytidine kinase (dCK) and of the deactivating enzyme deoxycytidine deaminase were determined using tumour samples collected during follow-up cystoscopy. dCK levels were lower than those in other sensitive tumours, in keeping with the possible low chemosensitivity of a recurrent tumour. Interestingly, dCK expression increased in one patient receiving various courses of gemcitabine, thus supporting a potential rationale for maintenance treatment cycles.
In the study by Witjes et al. six weekly instillations with 1000, 1500 and 2000 mg for 1 h were given in three, four and three patients, respectively. Blood samples were taken at 5, 30, 60 and 120 min after instillations 1, 3 and 6. The highest plasma level of gemcitabine (0.91 µmol/L) was detected in the only patient (dose 1000 mg) who had a grade 1 myelosuppression at the first instillation. All other levels were unmeasurable. The same patient was the only one to show measurable levels of the active metabolite 2′,2′-difluorodeoxycytidine-TP. Plasma levels of dFdU were measurable in all patients, with a peak of 4.19 µmol/L in a patient treated with the 1500 mg dose.
Finally, Palou et al. assessed the pharmacokinetics and safety of an early single intravesical instillation of gemcitabine at dosages of 1500 and 2000 mg in 10 patients. Blood samples were taken at several intervals starting 15 min from the onset of the instillation up to 3 h after urine voiding. Plasma concentrations of gemcitabine were generally low but with a high inter-patient variability. Remarkably, the highest concentrations (4.5 and 6.1 µmol/L) were found in the two patients in whom an undetected bladder perforation was suspected because of the recovery of less than the instilled volume after bladder voiding. Similarly, overall maximum dFdU concentrations were also low, with a mean value of 7.8 µmol/L.
In conclusion, pharmacokinetic data from different phase I studies show clearly that systemic absorption of intravesical gemcitabine at up to 40 mg/mL (2000 mg in 50 mL), when kept in the bladder for up to 2 h, is minimal and transient, and thus unlikely to produce clinically significant adverse events. The presence of low levels of the inactive metabolite further reinforce the concept of the relative impermeability of bladder mucosa to the drug. Based on plasma drug concentrations, early intravesical instillation might be a feasible treatment option, provided there is no significant bladder perforation.
In Table 1[26,33–36] all side-effects reported in the currently available phase I studies are listed and graded according to the Common Terminology Criteria for Adverse Events . In agreement with the pharmacokinetic data, systemic toxicity was absent in the study of De Berardinis et al. and did not go beyond grade 1 in two other studies [35,36]. Overall, no systemic toxicity exceeding grade II was recorded in any of the five phase I studies, except for one case of grade 3 myelosuppression and thrombocytopenia reported at 20 mg/mL by Dalbagni et al.. In that study, two factors in the design may have promoted an increased systemic absorption resulting in significant haematological toxicity: first, the drug was administered twice a week, a rather unusual schedule for an intravesical agent; second, the low pH of the gemcitabine solution, which was adjusted to 5.5–7.0 to prevent bladder irritation . The resulting increased non-ionic form of the drug may have facilitated its diffusion through the bladder mucosa . Notably, intravesical instillation administered within a few hours after bladder resection proved to be well tolerated, even when the bladder was perforated .
Toxicity data in Phase I human studies on intravesical gemcitabine
|||500–2000 in 100 mL||Twice weekly for 3 weeks (1 week rest after each week)||1||18||n = 3 1 G1 nausea||n = 6 4 G2 UF 1 G3 UF 3 G2 HU 1 G2 asthenia||n = 3 1 G2 UF 1 G3 UF 1 G2 HU 1 G2 UTI||n = 6 1 G2 HU 1 G2 asthenia 1 G2 nausea 1 G2 vomiting 1 G3 Th + Np|
|||500–2000 in 50 or 100 mL||Once weekly for 6 weeks||2||15||n = 3 1 G1 Th 1 G1 dysuria 1 G2 UF 3 G1 HU 2 G1 fatigue/ headache 1 G1 arthralgia||n = 3 1 G1 LP 1 G1 HU 1 G1 dysuria 2 G1 HU 1 G1 pruritus 1 G1 dizziness 2 G1 myalgia 1 G3 urine retention||n = 3 1 G1 dysuria 3 G1 HU 1 G1 UF 1 G1 Bl spasm 1 G1 fatigue 1 G2 proteinuria 2 G1 headache 1 G1 nausea 1 G1 arthralgia||n = 6† 1 G1 LP 2 G1 HU 1 G1 incontinence 1 G1 fatigue 2 G1 chills 1 G3 UF‡|
|||500–2000 in 50 mL||Once weekly for 6 weeks||2||12||n = 3 None||n = 3 None||n = 3 None||n = 3 1 G1 dysuria|
|||1000–2000 in 50 mL|| ||1||10||–||n = 3 1 G1 MS¶||n = 4 ¶||n = 3 ¶|
|||1500–2000 in 100 mL||<2–3 h from TURBT||1||10||–||–||n = 5 2 G1 hypogastric discomfort||n = 5 1 G1 Bl spasms 2 G1 liver toxicity|
Local toxicity was minimal and generally described as rapidly self-resolving. With the possible exception of three cases of grade 3 urinary frequency (one in  at 2000 mg and the other two in  after 1000 and 1500 mg doses), genitourinary side-effects were usually confined to grade 1 toxicity.
Based on these results it appears that the 2000 mg dose of gemcitabine in 50 mL normal saline, when administered intravesically for up to 2 h once a week for 6 weeks, has no remarkable systemic and local side-effects, and therefore should be considered the most convenient schedule. Higher drug concentrations produce clinically relevant side-effects and do not allow optimum drug solubility, whereas a higher volume may not be appropriate, as it might exceed the maximum bladder capacity of many patients.
ACTIVITY OF INTRAVESICAL GEMCITABINE
The marker-lesion concept
The ability of a new chemotherapeutic agent to prevent superficial TCC tumour formation relies on its cytotoxic efficacy on bladder cancer cells. To assess the activity of new agents in treating intermediate-risk SBC, some sort of ‘indicator’ lesion is necessary, and this can be achieved by a marker-tumour study [39,40]. The EORTC Genitourinary Group and the UK Medical Research Council have conducted trials using marker tumours, which provided evidence that this method is safe, logical and ethically acceptable [41,42]. This type of study allows researchers to test the ablative activity of the investigational drug on one papillary marker lesion, and to assess the incidence and severity of early side-effects in relatively few patients and in a relatively short period of treatment. Several marker-lesion studies evaluated the ablative activity of different drugs alone or combined (mitomycin C, epirubicin, BCG) with a complete response rate of 50–60%[43–45]. These findings constitute the reference value of activity for any new intravesical agent.
Phase II studies on intermediate and high risk SBC
The ablative efficacy of intravesical gemcitabine on a marker-lesion tumour is currently being investigated by several authors (Table 2) [46–50]. The first published study  was designed as a two-stage phase II trial with the possibility of stopping the trial early if there was evidence that the drug had insufficient activity. Based on previous marker lesion series assessing other intravesical agents [43–45], it was determined that the treatment tested in that study would deserve further attention if it could produce responses in at least 21 of 39 entered patients. Overall, there was a complete response in 22 of 39 patients (56%), meaning that intravesical gemcitabine has cytoreductive activity in intermediate-risk SBC. The authors underlined that almost 60% of cases were recurrences after previous BCG treatment.
Phase II studies of intravesical gemcitabine for SBC
|||40||Weekly for 6 weeks||1||39||Ta–T1, G1–G2||Single, 0.5–1||22 (56)|
|||40||Weekly for 6 weeks||2||24||Ta–T1, G1–G2||Single, 1||12 (50)|
|||10–40||Weekly for 6 weeks||2||27||Ta–T1, G1–G2||1–3, 5–15|| 6 (22)|
|||20||Twice weekly for 3 weeks†||1||28||BCG-refractory, refused cystectomy||–||16 (57)|
|||20||Weekly for 6 weeks after TUR||–||29||Tis-T1||–||29 (100)|
Similar studies were recently reported in peered-reviewed abstracts. Responses were complete in half of a series of 24 patients with Ta-T1, G1-G2 SBC  and in 22% of 27 intermediate-risk SBC in another phase I-II trial . In the latter, a dose-escalation regimen and the adoption of multiple marker lesions may partly explain the lower level of chemoresection achieved.
Few attempts have been made to test the activity of intravesical gemcitabine in high-risk SBC. In a dose-escalation study, Dalbagni et al. achieved a complete response in seven of 18 BCG-refractory high-risk SBC, of which 14 were CIS. These results were further improved when a standard dose of 2000 mg in 100 mL was administered to another similar series of patients by the same authors  (Table 2). Intravesical gemcitabine was instilled once a week for 6 weeks in 29 high-risk chemo-naive patients with SBC after TURBT of all visible lesions. At the 1-year follow-up, all patients, including nine with CIS, were in complete remission . Notably, all phase II studies using the standard 2000 mg dose (in 50 or 100 mL dilution) have shown an excellent drug tolerability in both the once-weekly and twice-weekly regimens.
CONCLUSIONS: FUTURE TRENDS
Intravesical gemcitabine has so far shown an excellent safety profile and minimal toxicity at concentrations up to 40 mg/mL. This should therefore be the standard dose for any future study. Based on early phase I studies, an instillation over 2 h seems a feasible option and might be better than instillation for 1 h, which is usually recommended for traditional intravesical agents to minimize side-effects.
The standard scheme of weekly instillations of gemcitabine for 6 weeks as an induction course has shown an excellent safety profile and should be adopted when designing a treatment protocol for intermediate-risk SBC. The drug can be administered as early as 3 h after surgery, with no remarkable toxicity, provided there is no major bladder perforation. The question remains as to whether the six-instillation scheme, administered twice a week for 3 weeks, is more appropriate in trials on high-risk SBC, as this has been the only regimen studied so far.
Preliminary phase II marker-lesion studies show that the drug has antitumour activity on a marker lesion that is comparable to those in similar studies conducted on BCG. Additionally, response rates were promising in high-risk SBC that were BCG-refractory.
To date, gemcitabine seems to have fulfilled the requirements to be a promising new candidate for standard intravesical therapy in SBC. Moreover, several phase II trials are presently ongoing, either to confirm the activity of gemcitabine on marker lesions in patients with low-, intermediate- and high-risk SBC, or to compare the efficacy of gemcitabine vs mitomycin C or BCG in preventing recurrences in intermediate-/high-risk patients. The South-West Oncology Group will soon start a phase II study to assess the efficacy of intravesical gemcitabine given weekly for 6 weeks and then every 4 weeks for a maximum of 10 cycles, for complete response rates in patients with high-risk SBC who have failed two cycles of intravesical BCG. Also, concerning phase III trials, a German cooperative group study of immediate instillation after TURBT of gemcitabine vs saline in patients with newly diagnosed or occasionally recurring Ta/T1, G1-3 SBC has recently completed the enrollment of 328 patients. A similar study for a total of 340 patients has been planned by the South-West Oncology Group, to be started in the near future.
The ongoing phase II trials exploring the activity of gemcitabine on highly recurrent intermediate- or high-risk SBC would provide additional information to predict its efficacy in clinical practice, and thus constitute the framework for large comparative phase III trials.
CONFLICT OF INTEREST
L. Marini is an employee of Lilly.