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We conducted a study to determine the dose-limiting toxicity of an extended dosing schedule of temozolomide (TMZ) when used with a fixed dose of BCNU, or 1,3-bis(2-chloroethyl)-1-nitrosourea (carmustine), taking advantage of TMZ’s ability to deplete O6-alkylguanine-DNA-alkyltransferase and the synergistic activity of these two agents. Patients with malignant gliomas who had undergone radiation therapy were eligible. Patients were treated with TMZ for 28 days, followed by a 28-day rest (1 cycle). The TMZ was started at 50 mg/m2 and increased in 10-mg/m2 increments; a fixed dose of BCNU (150 mg/m2) was given within 72 h of starting TMZ. A standard phase 1 dose-escalation scheme was used with 3 patients per cohort. Fourteen glioblastoma patients and 10 anaplastic astrocytoma patients were treated. The dose-limiting toxicity was myelosuppression at 90 mg/m2 of TMZ. The total number of cycles given was 73 (median number was 2). Six patients (25%) required a dose reduction in BCNU, and six were removed from study for hematologic toxicity after cycle 1; three patients overlapped. The median time to progression and overall survival were, respectively, 82 and 132 weeks for anaplastic astrocytomas and 14 and 69 weeks for glioblastomas. We conclude that the combination of BCNU and the extended dosing schedule of TMZ is feasible and that the maximal tolerated dose of a 28-day course of TMZ is 80 mg/m2 when combined with a fixed dose of BCNU at 150 mg/m2. This is the recommended dose for phase 2, but myelosuppression after cycle 1 suggests that long-term treatment may be difficult
Malignant gliomas make up more than 50% of primary brain tumors and have a poor prognosis. Surgery and radiation therapy (RT)3 are the main therapeutic modalities in the treatment of these tumors. The addition of chemotherapy has an important but modest effect on survival (Fine et al., 1993; GMT, 2002). Nitrosoureas have been the mainstay of therapy, either as a single agent BCNU (carmustine) or CCNU (lomustine) or in combination (procarbazine, CCNU, and vincristine). Temozolomide is an alkylating agent that spontaneously converts to its active metabolite, MTIC—3-methyl-(triazen-1-yl)imidazole-4-carboxamide — at physiological pH (Friedman et al., 2000a). It is approved in the United States for the treatment of recurrent anaplastic astrocytoma (AA) but has activity in recurrent glioblastoma (GBM) and other glial tumors in the neoadjuvant or recurrent setting (Chinot et al., 2001; Gilbert et al., 2002; Quinn et al., 2003; Yung et al., 1999, 2000).
The mechanism of action of nitrosoureas is the formation of DNA interstrand cross-links between the O6 position of guanine and adjacent cytosine; the main mechanism of TMZ cytotoxicity is methylation of DNA at the O6 position of guanine (Gerson, 2002). The enzyme O6-alkylguanine-DNA-alkyltransferase (AGT) is one of the mechanisms responsible for the development of resistance to nitrosoureas and alkylating agents (Friedman et al., 2000a; Gerson, 2002; Pegg, 2000). This enzyme repairs DNA damage by removing the adduct at the O6 position. High levels of AGT correlate with increased resistance to the cytotoxic effects of these agents and inversely correlate with response rates (Belanich et al., 1996; Friedman et al., 1998; Jaeckle et al., 1998).
A chronic dosing schedule of TMZ leads to depletion or reduction in AGT levels in tumor cells (de Bono et al., 2001; Tolcher et al., 2003). A second benefit of a low-dose continuous schedule is the potential antiangiogenic effects (Browder et al., 2000; Folkman, 2003). When cells are pretreated with TMZ, the cytotoxicity of BCNU is enhanced, and TMZ can reverse resistance to chloroethylnitrosoureas (D’Incalci et al., 1991; Mitchell and Dolan, 1993). Additionally, blockade or depletion of AGT may enhance the cytotoxicity of nitrosoureas and alkylating agents (Gerson, 2002; Pegg, 2000; Wedge et al., 1996). Although the mechanism is unknown, the combination of TMZ and BCNU is synergistic in vitro (Plowman et al., 1994).
We designed a phase 1 trial of dose-escalating, extended, low-dose TMZ in combination with BCNU as adjuvant post-RT for patients with malignant gliomas. Our rationale for this combination was that extended, low-dose TMZ would significantly deplete AGT and enhance the cytotoxicity of BCNU, a high enough dose of TMZ could be delivered to have single-agent activity, and the combination of these agents would be synergistic.
The protocol and consent were reviewed and approved by the Memorial Sloan-Kettering Cancer Center Institutional Review Board. All patients were 18 years of age with a Karnofsky performance score of 60 and were required to sign informed consent prior to enrolling on the study. All patients had to have histopathologic confirmation of a GBM, gliosarcoma, malignant glioma not otherwise specified, AA, anaplastic mixed oligoastrocytoma, or anaplastic oligodendroglioma. All patients must have completed RT at least 2 weeks prior to starting treatment. Patients were eligible for treatment as long as their postradiation MRI did not reveal >25% increase in their tumor. Prior treatment with chemotherapy, immunotherapy, or biologic therapy was not allowed. Patients with a secondary malignancy (excluding surgically cured carcinoma in situ of the cervix and basal or squamous cell carcinoma of the skin), known human immunodeficiency virus positivity, poor medical risk or psychiatric illness that would interfere with protocol compliance, and those who were pregnant or nursing were ineligible. Patients were required to have adequate hematologic function (absolute neutrophil count 1500/mm3, platelets 100,000/mm3, and hemoglobin 10 g/dl) as well as a blood urea nitrogen and serum creatinine <1.5 times the upper limit of normal (ULN), a total and direct bilirubin <1.5 times ULN, an alanine aminotransferase and aspartate aminotransferase <3 times ULN, and an alkaline phosphatase <2 times ULN. A complete physical and neurologic exam and a contrast-enhanced MRI or CT scan were performed prior to treatment. All patients had pulmonary function tests within 14 days of beginning therapy and were required to have a diffusion capacity >50.
All patients were treated with a fixed dose of BCNU at 150 mg/m2 up to a maximum cumulative dose of 1500 mg/m2. BCNU was given intravenously over 1 to 3 h according to our institution’s guidelines. Temozolomide was orally administered on an empty stomach 30 min after antiemetic prophylaxis; redosing was not allowed if vomiting occurred. There was no intrapatient dose escalation of TMZ. The starting dose of TMZ was 50 mg/m2, and it was increased in 10-mg/m2 increments. Temozolomide was taken for 28 consecutive days followed by a 28-day rest period; one cycle was 56 days. BCNU was administered within 72 h of the first dose of TMZ in each cycle. When the maximal dose of BCNU was reached, patients could remain on the single agent TMZ.
A minimum of 3 patients and a maximum of 6 were enrolled per cohort. A dose-limiting toxicity (DLT) was defined as grade 3 or 4 nonhematologic or grade 4 hematologic toxicity, based on the National Cancer Institute Common Toxicity Criteria (NCI, 1999), during cycle 1. If 1 of 3 patients experienced a DLT, an additional three patients were added at that level. If 2 of the 6 patients experienced a DLT, that level was declared too toxic, and an additional 3 patients were added at the prior dose level. The maximal tolerated dose (MTD) was defined as the dose where 0/3 or 1/6 patients experienced a DLT. We observed the patients in each cohort for 8 weeks from day 1 of treatment before enrolling patients at the next dose level. After cycle 1, if there was grade 3 or 4 hematologic toxicity, the BCNU was reduced in 25-mg/m2 increments to a minimum dose of 100 mg/m2. If the grade 3 or 4 toxicity persisted, patients were removed from the study.
All patients had a complete physical examination and a contrast-enhanced MRI or CT scan at baseline and before beginning each new cycle. Each patient had a complete blood count every week and electrolyte and liver function tests every 4 weeks. Pulmonary function tests were repeated after every 2 cycles. Radiographic response was assessed by using the Macdonald criteria (Macdonald et al., 1990). Overall survival (OS) and time to progression (TTP) were calculated by the Kaplan-Meier product limit method. Overall survival was calculated from date of diagnosis to date of death or last follow-up. Time to progression was calculated from the start of chemotherapy until radiographic progression, clinical deterioration, or last follow-up. Progression-free survival (PFS) was calculated at 6 and 12 months.
Twenty-four patients were enrolled onto the study between December 29, 1999, and October 23, 2002. There were 14 women and 10 men with a median age of 52 (range, 37–71). Fourteen patients had a GBM and 10 had an AA (one with gliomatosis cerebri). The median Karnofsky performance score was 80 (range, 60–100). A total of 73 cycles of chemotherapy were administered to 24 patients, including two who did not complete cycle 1 because of tumor progression. The median number of treatment cycles was 2 for all patients (range, 1–11), 2 for GBM (range, 1– 7), and 2.5 for AA (range, 1–11). One patient was treated to the maximum cumulative dose of BCNU and was then treated with TMZ alone. The median time from completion of radiation to starting chemotherapy was 30 days.
Patients were accrued at 5 dose levels of TMZ: 6 at 50 mg/m2, 3 at 60 mg/m2, 3 at 70 mg/m2, 6 at 80 mg/m2, and 6 at 90 mg/m2. One patient at the first dose level of TMZ (50 mg/m2) developed a grade 4 neutropenia; according to protocol design, an additional 3 patients were added to that level. The DLT was grade 4 thrombocytopenia and neutropenia in one patient, each at 90 mg/m2 of TMZ; no DLT was seen in the additional 3 patients at the prior dose level. Hence, 80 mg/m2 of TMZ was declared the MTD. The only other significant toxicities in cycle 1 (for all patients) were one grade 3 dehydration and grade 3 and 4 thrombocytopenia, leukopenia, and neutropenia; all other toxicities were grade 1 and 2 (Table 1). Dose reduction to 125 mg/m2 of BCNU occurred in cycle 2 for four patients and cycle 3 and 7 in one patient each; two of these patients required further dose reduction to 100 mg/m2. Prolonged hematologic toxicity led to the removal of 6 patients (25%) from study, one after cycle 1, three after cycle 2, and one each after cycle 3 and cycle 5. Three of the patients removed from study had a reduction in BCNU dose. No patient suffered a toxic mortality, although one patient died 18 weeks after study withdrawal at an outside institution from an undetermined cause. None of the patients developed pulmonary toxicity.
Two patients were removed from study for presumed disease progression after cycles 2 and 3. Both had a rere-section. Pathology review revealed treatment effect without viable tumor; with Institutional Review Board approval these patients were placed back on study. One patient (GBM) received an additional 4 cycles before being removed for true tumor progression, and the other (AA) received a single cycle and was removed for hematologic toxicity.
For GBM patients the median TTP and OS were 14 weeks and 69 weeks, respectively, and PFS at 6 months and 12 months was 21% and 14%, respectively. For AA patients the median TTP and OS were 82 weeks and 132 weeks, respectively, and PFS at 6 months and 12 months was 70%.
As noted, 6 patients were removed from study for hematologic toxicity in the absence of tumor progression. One of the patients (GBM) was treated with TMZ alone and progressed at 8 weeks, and another (GBM) was treated with TMZ and cis-retinoic acid and progressed at 14 weeks, while one (AA) remains on TMZ without tumor progression (78 weeks); all patients were treated with the standard 5-day schedule of TMZ. Two patients (AAs) had no treatment until tumor progression at 36 and 53 weeks, and one (AA) has remained off treatment without recurrence (93 weeks). Two patients withdrew from study for personal reasons, one after cycle 2 who remained alive without therapy for 87 weeks and one after cycle 5 who died, as previously noted. One patient was removed from study after 22 months of therapy by the treating physician for absence of tumor on MRI. No patients remain on study; all but one patient died from tumor progression.
Chemotherapy plays an important role in treating patients with malignant gliomas, even though only modest benefits are gained from its use. While new biologic and chemotherapeutic agents are continuously being developed, to date none of these agents has significantly increased survival in patients with malignant gliomas. Until better therapies are available, attempts to optimize currently available agents by overcoming mechanism of resistance is an important therapeutic strategy.
We determined that the MTD of TMZ that could be given over a 28-day period with a fixed dose of BCNU (150 mg/m2) every 8 weeks was 80 mg/m2; the DLT at 90 mg/m2 was grade 4 neutropenia and thrombocytopenia in 1 patient each. The 28-day course of TMZ was chosen because the myelosuppressive effects of BCNU occur approximately 4 weeks after administration, and the 28-day off period allowed hematologic recovery. On the 28-day schedule, the amount of TMZ exposure to the tumor was 1.4 to 2.5 times more, depending on the dose level, than exposure on the standard 5-day schedule of 200 mg/m2. For patients who continued on in therapy, the major toxicity in subsequent cycles was hematologic; most other toxicities were grade 1 or 2, and pulmonary toxicity was not seen. The true benefit of this regimen cannot be assessed, as these patients had just completed RT; hence any responses may have been due to radiation. Additionally, any effects seen from this regimen could be similar to either agent alone or due to the 28-day dosing schedule of TMZ and not necessarily the combination of drugs or AGT depletion.
Two phase 1 studies and one phase 2 study used O6-benzylguanine to modulate intratumoral AGT, in combination with BCNU, to treat malignant gliomas or other solid tumors (Friedman et al., 2000b; Quinn et al., 2002; Schilsky et al., 2000). The phase 1 trials were able to use only 40 mg/m2 of BCNU, a dose far lower than typically used (Friedman et al., 2000b; Schilsky et al., 2000). The DLT was grade 3 or 4 thrombocytopenia and neutropenia. The hematologic toxicity was cumulative and prevented some patients from continuing on therapy, which corresponds to in vitro data that O6-benzylguanine potentiates BCNU toxicity in hematopoietic stem cells (Westerhof et al., 2001). In the phase 1 and 2 studies for patients with recurrent or progressive malignant gliomas, myelosuppression was the major toxicity, and pulmonary toxicity was seen in 2 patients in the phase 1 trial (Friedman et al., 2000b; Quinn et al., 2002). None of the patients in either study responded to therapy, possibly because of the low dose of BCNU, a lack of reversal of AGT activity, or an alternate mechanism of resistance. Brandes et al. (2002) used procarbazine (100 mg/m2, days 1–5) to deplete AGT prior to treating with BCNU (80 mg/m2, days 3–5). They treated 58 chemotherapy-naïve patients with recurrent or progressive GBM post-RT. Several patients required dose reduction because of hematologic toxicity, and 5 patients had partially reversible pulmonary toxicity. Another group (Beith et al., 1997; Slancar et al., 1997) also used procarbazine to deplete AGT with a slightly different regimen (procarbazine 200 mg/m2 on days 1–5, BCNU 80 mg/m2 on days 3–5, and vincristine 1.4 mg/m2 on day 3). Significant pulmonary, hematologic, and hepatic toxicities were seen. Measurement of AGT levels in peripheral monocytes revealed a 54% decrease after 2 days of procarbazine therapy (Beith et al., 1997; Slancar et al., 1997).
A phase 1 trial of BCNU and TMZ was performed through the North American Brain Tumor Consortium, using the knowledge that this combination is synergistic in vitro. In that study, the MTD was BCNU at 150 mg/m2 followed by TMZ at 550 mg /m2, each as a single dose, every 6 weeks. As in our study, the main toxicity was myelosuppression, but 3 patients had pulmonary toxicity, at least one of which was likely due to BCNU (Schold et al., 2000). A follow-up study of this combination in patients with AA prior to RT also noted significant myelosuppression preventing 22% of the patients from completing the full 4 cycles prior to receiving RT (Chang et al., 2003).
As a phase 1 study, efficacy was not a primary end point. However, our results are comparable to other studies of adjuvant therapy. The Medical Research Council Brain Tumor Working Party (2001) found a median TTP and OS of 24 and 40 weeks, respectively, in patients with high-grade astrocytomas treated with RT and PCV. Levin et al. (2000) treated GBM patients post-RT with procarbazine, CCNU, and vincristine ± alpha-difluoromethylornithine and found a median TTP of approximately 18 weeks and OS of 57 to 61 weeks. Brandes et al. (2002) determined a median TTP of approximately 19 weeks and a PFS at 6 and 12 months of 42.3% and 15.4%, respectively, in chemotherapy-naïve GBM patients with progressive disease.
The combination of TMZ (80 mg/m2) given over 28 days and BCNU (150 mg/m2) given every 8 weeks is feasible. A phase 2 trial of this regimen at the MTD will determine the true efficacy of this regimen when compared to other trials of adjuvant therapy. However, the toxicity seen in our study following cycle 1 raises the issue of whether a lower dose of TMZ or BNCU might be more rational. Dose reductions in BCNU may alter the efficacy of this combination, and the cytotoxic effects of BCNU may decrease at a dose less than 100 mg/m2. Pulmonary toxicity was not a complication in our study and may have been due to the level of AGT reduction. Cumulative myelosuppression from this combination may affect long-term therapy for patients responding to treatment or alternative therapies for patients who have progressed. Post-cycle 1 toxicities should be assessed in all phase 1 studies, as these toxicities may impact present and future therapies.
1This work was supported in part by an unrestricted grant from Integrated Therapeutics.
3Abbreviations used are as follows: AA, anaplastic astrocytoma; AGT, O6-alkylguanine-DNA-alkyltransferase; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea, or carmustine; CCNU, N-(2-chloroethyl)-N′-cyclohexyl-N-nitrosourea, or lomustine; DLT, dose-limiting toxicity; GBM, glioblastoma; MTD, maximal tolerated dose; OS, overall survival; PFS, progression-free survival; RT, radiation therapy; TMZ, temozolomide; TTP, time to progression; ULN, upper limit of normal.