Dose-dense temozolomide regimens

Antitumor activity, toxicity, and immunomodulatory effects

Authors


Abstract

Temozolomide is an oral alkylating agent with established antitumor activity in patients with primary brain tumors and melanoma. The originally approved temozolomide dosing regimen is 150 to 200 mg/m2 per day (Days 1 to 5 every 28-day cycle [5 of 28 days]). However, extended dosing regimens (eg, 7 of 14 days, 21 of 28 days, 6 of 8 weeks, or continuously daily) allow for administration of a higher cumulative dose per cycle and have been shown to deplete O6-methylguanine-DNA methyltransferase, which may enhance cytotoxic activity. This article reviews efficacy and safety data from studies that investigated dose-dense temozolomide regimens in patients with recurrent glioma and advanced metastatic melanoma. The clinical benefits of these dose-dense regimens compared with the standard 5 of 28-day regimen have yet to be established. Although the toxicity profile of dose-dense temozolomide is generally similar to that of the standard 5 of 28-day regimen, it is associated with an increased incidence and severity of lymphocytopenia. The clinical management of temozolomide-associated lymphodepletion and the potential risks and benefits of extended dosing with temozolomide are discussed. Preclinical and clinical evidence suggests that temozolomide-associated lymphodepletion may enhance the host immune response to tumor-associated antigens and/or immunotherapy and may overcome tumor-mediated immunosuppression. Further studies exploring the clinical implications of lymphodepletion are warranted. Cancer 2010. © 2010 American Cancer Society.

Temozolomide is a second-generation, orally administered, DNA alkylating agent with excellent bioavailability in the central nervous system. Phase 2 studies in patients with recurrent high-grade glioma and advanced metastatic melanoma established 150 to 200 mg/m2 per day on Days 1 to 5 of each 28-day cycle (5 of 28 days) as a clinically active and generally well-tolerated regimen.1-6 This regimen has been used widely for the treatment of patients with recurrent high-grade glioma and advanced metastatic melanoma since the late 1990s. The major dose-limiting toxicity of the standard dosing regimen is myelosuppression, mainly grade 3/4 thrombocytopenia with an incidence of 7% to 17% across all studies, and neutropenia to a lesser extent.2, 5, 7, 8 Unlike nitrosoureas, treatment with temozolomide is not associated with cumulative hematologic toxicity, and the occurrence of severe myelosuppression prohibiting the continuation of temozolomide treatment is rare.

Rationale for Alternate Regimens

The cytotoxic effect of temozolomide is mediated primarily via methylation at the O6 position of guanine, and the predominant mechanism of tumor resistance to temozolomide is overexpression of O6-methylguanine-DNA methyltransferase (MGMT).9 Evidence supporting the role of MGMT in determining response to temozolomide comes from clinical studies demonstrating that epigenetic inactivation of MGMT by hypermethylation of the promoter is associated with improved tumor response and survival in patients with glioblastoma.10-12 Other studies have extended this observation to include patients with anaplastic glioma and anaplastic (oligo)astrocytoma.13, 14

Although MGMT can effectively repair temozolomide-mediated DNA damage, the enzyme is consumed in the process.15 This led to exploration of extended dose-dense temozolomide regimens, with the idea that they could potentially deplete MGMT in tumor cells by overwhelming the cells' ability to synthesize MGMT, which might enhance therapeutic activity. The concept of MGMT depletion was validated by Tolcher et al,16 who showed that MGMT enzyme activity was depleted in peripheral blood mononuclear cells (PBMCs) during treatment with temozolomide for either 7 consecutive days every 14 days (7 of 14-day regimen) or 21 consecutive days every 28 days (21 of 28-day regimen). The hypothesis that these dosing schedules that deplete MGMT could result in improved antitumor activity presupposes that MGMT depletion in tumor cells is similar to that observed in PBMCs. However, the extent of MGMT depletion in tumor cells has not been directly demonstrated and might not parallel the effect observed in PBMCs. Therefore, it remains unclear whether dose-dense temozolomide regimens can deplete MGMT in tumors and overcome MGMT-mediated resistance.

In addition, other mechanisms may further contribute to the antitumor activity of extended temozolomide dosing schedules. First, a growing body of evidence suggests that daily temozolomide may act as a radiosensitizer when coadministered with radiotherapy.17-19 Second, protracted, low-dose, daily chemotherapy, also referred to as metronomic chemotherapy,20 may exert antiangiogenic effects via induction of endogenous angiogenesis inhibitors,21, 22 apoptosis of tumor-associated endothelial cells,23 and/or decreased mobilization and viability of circulating endothelial progenitor cells.24

Dose-Dense Temozolomide Regimens

Initial attempts to modify the standard temozolomide dosing regimen examined the safety of continuous daily administration for up to 7 weeks. In a preliminary phase 1 study, temozolomide administered continuously for 6 or 7 weeks at a dose of 75 mg/m2/d to patients with malignant glioma was well tolerated.25 The demonstration that continuous daily dosing with temozolomide was feasible paved the way for combining it with fractionated radiation therapy (RT) in patients with newly diagnosed glioblastoma. After promising survival data emerged from a phase 2 study of RT combined with concomitant temozolomide (75 mg/m2/d) followed by 6 cycles of adjuvant temozolomide (at the standard dose of 150-200 mg/m2/d on the 5 of 28-day schedule),26 a randomized phase 3 trial (European Organization for Research and Treatment of Cancer [EORTC] 26,981-22,981/CE.3) established this regimen as the new standard of care for initial treatment of glioblastoma.27 This landmark trial demonstrated improved overall survival (OS) in patients treated with RT plus temozolomide compared with RT alone (hazard ratio, 0.6; 95% confidence interval, 0.5-0.7; P < .0001), and combination therapy did not adversely affect quality of life.28, 29

Recurrent high-grade glioma (World Health Organization grade III and IV)

Given that many patients with high-grade glioma receive temozolomide as part of their initial treatment and typically experience disease progression during or within a few months after completing first-line therapy, standard-dose temozolomide is not a viable option at recurrence. Therefore, several alternative dose-dense temozolomide regimens have been explored in patients with recurrent high-grade glioma. The most widely studied regimens in this setting include a continuous daily schedule at a dose of 50 mg/m2/d,30, 31 the 7 of 14-day schedule at a dose of 150 mg/m2/d (also referred to as the “1 week on/1 week off” schedule),32 and the 21 of 28-day schedule at a dose of 75 to 100 mg/m2/d.16, 25 These dosing regimens increase the cumulative dose of temozolomide over a 28-day cycle by approximately 2-fold compared with the standard 5 of 28-day schedule.2, 5, 6, 30, 32-38 Dose-dense temozolomide regimens have been investigated in patients with recurrent high-grade glioma, with reported 6-month progression-free survival (PFS) rates ranging 30% to 44% in patients with glioblastoma and 56% in patients with anaplastic oligoastrocytoma (Table 1).30, 32, 34-37 Studies investigating the activity of these regimens for the treatment of glioma have been reviewed recently.39 The results of these various uncontrolled studies suggest that retreatment with a dose-dense regimen has activity in some glioblastoma patients who experience disease progression during or shortly after completing adjuvant treatment with the 5 of 28-day regimen (after initial therapy with RT plus concomitant temozolomide), thus lending support to the notion that dose-dense temozolomide regimens may, to some extent, overcome resistance that develops during standard-dose adjuvant temozolomide therapy.

Table 1. Results From Selected Studies Using Extended Dosing Temozolomide Regimens
StudyTemozolomide RegimenHistologyNo.Previous CT, %PR+CR, %6-Month PFS Rate, %OS, mo
  • CT indicates chemotherapy; PR, partial response; CR, complete response; PFS, progression-free survival; OS, overall survival; GBM, glioblastoma; AA, anaplastic astrocytoma; AOA, anaplastic oligoastrocytoma; HGG, high-grade glioma; NR, not reported; LGG, low-grade glioma; O, oligodendroglioma.

  • a

    Patients with recurrence during the first 3 to 6 months of adjuvant temozolomide therapy.

  • b

    Patients with recurrence after >6 months of adjuvant temozolomide therapy.

  • c

    Patients who recurred after stopping adjuvant treatment.

  • d

    Twelve-month PFS.

  • e

    Retrospective study.

  • f

    Excludes minor response.

Recurrent high-grade glioma       
 Wick 200732150 mg/m2/d 7/14 dGBM90641543.89.5
 Neyns 200836100 mg/m2/d 21/28 dAA, AOA190105612.9
 Strik 200837100 mg/m2/d 21/28 dGBM1810022399.1
 Berrocal 20063385 mg/m2/d 21/28 dHGG291006.7NR5.9
 Brandes 20063475 mg/m2/d 21/28 dGBM33093010
 Perry 20083050 mg/m2/d continuousGBM60100NRNRNR
  Cohort 1a   23 
  Cohort 2b   7 
  Cohort 3c   35 
 Khan 20023575 mg/m2/d 42/70 dGBM35770197.7
Low-grade glioma       
 Tosoni 20084475 mg/m2/d 21/28 dLGG3003073dNR
 Pouratian 200742e75 mg/m2/d 21/28 dLGG25852f74dNR
 Soffietti 200843150 mg/m2/d 7/14 dO31NR32NRNR

Low-grade gliomas (World Health Organization grade II)

Protracted low-dose chemotherapy has been suggested as an alternative to RT to reduce the risk of radiation encephalopathy and cognitive deficits in patients with low-grade gliomas (LGGs) that recur or progress after surgery. The standard 5 of 28-day temozolomide regimen is clinically active in LGGs, with 1-year PFS rates in chemo- and RT-naive patients ranging from 73% to >90%,40, 41 potentially offering a therapeutic option to delay RT.

In the context of low-grade tumors, which have a limited number of cells in the proliferation phase at any given time, chronic exposure to lower doses of cytotoxic drugs may be a more effective therapeutic strategy than achieving high peak drug levels. Thus, in low-grade tumors, extended low-dose temozolomide regimens might provide additional benefit compared with the standard 5 of 28-day regimen. A limited number of nonrandomized studies have evaluated the efficacy of protracted temozolomide schedules in patients with LGG (Table 1).42-44 Two studies evaluating the 21 of 28-day regimen reported 1-year PFS rates of 72% and 73%.42, 44 However, the incidence of grade III lymphopenia ranged from 20% to 48%. Another study evaluating the 7 of 14-day regimen in patients with oligodendroglioma or mixed oligoastrocytoma reported an objective response rate of 32%, and 80% of patients were progression-free at a median follow-up of 14 months.43 Again, the incidence of grade III lymphopenia was high (45%), and 15% of patients required dose interruption or transfer to the standard temozolomide regimen (5 of 28 days). On the basis of the small number of studies and the heterogeneity of the patient populations, it is difficult to assess differences in the clinical benefit of dose-dense regimens compared with the standard 5 of 28-day regimen. However, it is clear that dose-dense regimens are associated with increased myelotoxicity, especially chronic lymphopenia. Prospective randomized studies comparing these regimens are warranted to determine whether dose-dense temozolomide regimens can improve clinical outcomes in this setting. The potential benefit of temozolomide as an alternative to RT is currently being evaluated in an ongoing EORTC randomized phase 3 trial comparing temozolomide (75 mg/m2/d; 21 of 28-day schedule) for a maximum of 12 cycles with RT as primary treatment.

Melanoma

Several phase 2 and phase 3 trials have also evaluated the clinical benefit of temozolomide monotherapy in patients with unresectable metastatic melanoma. Whereas initial trials used the standard 5 of 28-day dosing regimen,3, 45-49 more recent studies have evaluated dose-dense regimens, most commonly 75 mg/m2/d for 6 or 7 weeks followed by a 2- to 4-week break.50, 51 Response rates reported in these studies ranged from 10% to 13%, and median OS ranged from 6 to 10 months. In general, both standard and dose-dense temozolomide monotherapy have demonstrated modest activity (response rates <20% and median survival times <8 months) in advanced melanoma. Most recently, the EORTC 18,032 phase 3 trial demonstrated similar benefit associated with the 7 of 14-day temozolomide regimen (150 mg/m2/d) compared with dacarbazine (1000 mg/m2 every 21 days) in patients with advanced melanoma.52 Thus, the antitumor activity of single-agent temozolomide appears to be similar to single-agent dacarbazine, regardless of the dosing schedule. A large number of phase 2 studies have also investigated the combination of temozolomide with interferon-α (IFN-α) or thalidomide in patients with advanced melanoma and have reported promising activity, particularly when dose-dense temozolomide regimens were used (reviewed by Hwu et al53).

Association of MGMT with therapeutic efficacy of dose-dense temozolomide

A retrospective subset analysis of MGMT promoter methylation status in glioblastoma patients enrolled in the randomized phase 3 National Cancer Institute of Canada (NCIC)–EORTC 26,981-22,981/CE.3 trial suggested that the survival benefit associated with temozolomide was primarily restricted to patients with methylated MGMT promoter sequences in their tumor tissue; 93 (45%) of 206 assessable cases were methylated.11 Within this subset, RT plus temozolomide significantly improved OS compared with RT alone (21.7 months vs 15.3 months, respectively; P = .007). In contrast, there was a negligible survival benefit among patients whose tumors had unmethylated MGMT promoter sequences.

Unfortunately, based on the available data, it is not yet clear whether dose-dense temozolomide regimens can overcome MGMT-mediated resistance or improve clinical outcome compared with the standard 5 of 28-day regimen. The only study that prospectively compared the standard 5 of 28-day regimen with a dose-dense regimen is the British MRC-B12 trial.54 This trial randomized 447 patients with recurrent malignant glioma to treatment with standard temozolomide (5 of 28-day), dose-dense temozolomide (21 of 28-day), or procarbazine, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, and vincristine (PCV) chemotherapy. Median survival was 8.5 months with the 5 of 28-day regimen, 6.5 months with the 21 of 28-day regimen, and 6.7 months with PCV.54 Several studies have examined whether dose-dense regimens can improve survival in patients with an unmethylated MGMT promoter. In the single-arm UKT-05 trial,55 patients with newly diagnosed glioblastoma (n = 41) received sequential treatment with 2 cycles of neoadjuvant temozolomide (150 mg/m2, 7 of 14-day regimen), standard RT with concomitant temozolomide, and adjuvant therapy with the 7 of 14-day regimen for 16 weeks. In addition, patients received 25 mg of the radiosensitizing agent indomethacin (twice daily) throughout the treatment period. The authors concluded that the clinical outcome in patients with an unmethylated MGMT promoter (median OS, 15.9+ months; median PFS, 7.6 months) was similar to that observed in the overall patient population in the NCIC-EORTC trial treated with the standard 5 of 28-day regimen in the adjuvant setting,27 which compares favorably with the outcomes for patients with an unmethylated MGMT promoter in that trial.56

The correlation between MGMT promoter methylation status and the antitumor activity of dose-dense temozolomide regimens has also been assessed in 2 prospective phase 2 trials and 1 retrospective study in patients with recurrent glioma.13, 32, 34 In these studies, patients whose tumors had a methylated MGMT promoter at initial diagnosis had numerically improved survival rates compared with patients whose tumors had an unmethylated promoter; however, these differences, within a relatively small series of patients, did not achieve statistical significance. In the largest of these studies, reported by Wick et al32 (n = 90), among the subset of patients evaluable for MGMT methylation status (n = 36), median PFS was 19 weeks in patients with an unmethylated MGMT promoter compared with 27 weeks in patients with a methylated MGMT promoter. In the study reported by Brandes et al,34 33 patients with glioblastoma were treated with the 21 of 28-day regimen, and no correlation was observed between MGMT promoter methylation status and overall response rate, time to progression, or survival. This observed lack of correlation could be interpreted as evidence that dose-dense regimens may, to some extent, overcome MGMT-mediated resistance and improve outcomes in the unmethylated subset. However, these studies may not have been sufficiently powered, and multiple factors could have confounded the analysis.

Thus, further study of dose-dense temozolomide regimens is needed to clarify whether they can overcome MGMT-mediated resistance. Larger randomized studies in which patients are prospectively stratified on the basis of MGMT promoter methylation status are required to establish whether dose-dense temozolomide regimens can increase the sensitivity of unmethylated tumors to temozolomide. The recently completed, phase 3 Radiation Therapy Oncology Group 0525/EORTC trial (n = 1169) was prospectively designed to address this question (Fig. 1).

Figure 1.

A schematic representation of the Radiation Therapy Oncology Group 0525/European Organization for Research and Treatment of Cancer 26,052/22,053 phase 3 trial compares conventional adjuvant temozolomide with dose-intensive temozolomide in patients with newly diagnosed glioblastoma (NCT00304031). KPS indicates Karnofsky performance score.

Regimen-Dependent Toxicity of Temozolomide

The greater dose intensity achieved with alternative temozolomide regimens does not appear to significantly increase the frequency of thrombocytopenia or neutropenia. Available data from phase 2 trials indicate a high incidence of lymphopenia, particularly in patients with recurrent glioma treated with the 21 of 28-day regimen (Table 2).32-36, 56, 57 Moreover, the incidence of severe lymphopenia appears to correlate with cumulative dose.36, 57 Importantly, some patients treated with the 21 of 28-day regimen at an initial dose of 100 mg/m2 have developed profound and prolonged myelosuppression after the first treatment cycle, with blood counts recovering to normal only after 12 weeks.36 Therefore, at least in the recurrent setting, a lower initial dose may be advisable in the first treatment cycle to identify patients with lower tolerance. In comparison, the 7 of 14-day regimen appears to be associated with a lower incidence of lymphopenia. However, in the EORTC 18,032 trial, the incidence of lymphopenia was significantly higher with the 7 of 14-day dosing regimen compared with dacarbazine monotherapy.58

Table 2. Grade 3/4 Myelotoxicity in Selected Phase 2 Studies Investigating Dose-Dense Temozolomide Regimens in Patients With Glioma
StudyTemozolomide RegimenThrombocytopenia, %Neutropenia, %Lymphopenia, %
Grade 3Grade 4Grade 3Grade 4Grade 3Grade 4
  • NR indicates not reported.

  • a

    Overall, 13 (14.4%) of 90 patients experienced hematologic Common Terminology Criteria for Adverse Events grade 4 toxicity. There were no opportunistic infections or toxic deaths in the study.

Tosoni 20065775 mg/m2/d 42/70 d03.93.93.925.51.9
Khan 20023575 mg/m2/d 42/70 d2.802.80170
Brandes 20063475 mg/m2/d 21/28 d036624.20
Berrocal 20063385 mg/m2/d 21/28 d10.402.1027.10
Neyns 200836100 mg/m2/d 21/28 d01611115347
Chinot 200756150 mg/m2/d 7/14 d317314173
Wick 200732a150 mg/m2/d 7/14 dNRNRNRNRNR12

As a result of lymphopenia, patients treated with dose-dense temozolomide regimens are at increased risk for Pneumocystis carinii pneumonia (PCP) and other opportunistic infections. Although the reported incidence of grade 3/4 lymphopenia is high in some studies (Table 2), the reported incidence of lethal or life-threatening opportunistic infections has remained relatively low, highlighting the importance of proactively addressing infectious complications. Physicians treating patients with dose-dense temozolomide regimens should actively monitor pulmonary health and should consider PCP prophylaxis. Trimethoprim-sulfamethoxazole at a dose of 1 single-strength tablet daily or 1 double-strength tablet 3× per week is the standard prophylactic therapy for PCP. Pentamidine 300 mg monthly by aerosol is an alternative.58 Additional clinical variables such as older age, concomitant RT, concurrent corticosteroids, and poor performance status are also likely to increase risk for infections. In addition, prophylactic antifungal and antiviral (mainly antiherpetic) drugs might be needed to control the risk of infection or viral reactivation.

Furthermore, because temozolomide is a DNA-damaging agent, secondary malignancies are a potential cause for concern. Although the 5 of 28-day regimen has no known cumulative side effects, and the number of patients reported to have developed secondary myelodysplasia, leukemia, or aplastic anemia is low,59-62 the increased total dose delivered with dose-dense regimens could potentially increase the risk for delayed toxicity or secondary malignancies. This concern is particularly relevant in populations with a better prognosis, such as patients with LGG. A recent case report on 3 patients who developed non-Hodgkin lymphoma after treatment with temozolomide exemplifies this risk.63 Notably, 2 of these patients received dose-dense temozolomide (≥12 cycles of 21 of 28-day regimen).

Immunomodulatory Effects of Lymphodepletion

Intuitively, it might be expected that lymphodepletion associated with chemotherapy would suppress the immune response to tumor antigens and compromise the therapeutic potential of immunomodulatory strategies. This is particularly relevant in melanoma, where cytotoxic agents, including temozolomide, are often investigated in combination with immunomodulatory agents such as IFN-α, interleukin (IL)-2, thalidomide, or cytotoxic T lymphocyte-associated antigen-4–blocking antibodies.

In an effort to understand better the potential clinical implications of lymphodepletion, investigators have examined whether temozolomide selectively depletes specific subsets of lymphocytes such as effector (CD8+) T cells or regulatory (CD4+/CD25+/FOXP3+) T cells. Depletion of effector T cells might be expected to cause immunosuppression, whereas depletion of regulatory T cells might be expected to enhance antitumor immunity. Indeed, a recent study reported that metronomic cyclophosphamide (50 mg 3× per day) induced profound and selective reduction of regulatory T cells in patients with advanced cancer, thereby relieving suppression of cytotoxic T cells and natural killer (NK) cells and restoring their innate antitumor activity.64 These observations are also supported by recent preclinical data in a glioma model suggesting that low-dose metronomic temozolomide (corresponding to 3 mg/m2/d) depletes regulatory T cells and may ameliorate their immune suppressive activity.65 In contrast, other studies have provided some evidence that effector T cells may be depleted by dose-dense temozolomide regimens. For example, in a retrospective analysis of 97 melanoma patients treated with dose-dense temozolomide (75 mg/m2/d) either as monotherapy or in combination with thalidomide or low-dose IFN-α, lymphopenia (defined as absolute lymphocyte count [ALC] ≤800/μL) occurred in 60% of patients, and preferential depletion of CD4+ and CD8+ T cells was observed.66 Further analysis of 3 patients with severe lymphopenia (ALC ≤500/μL) demonstrated marked suppression of CD4+/CD25+ T-cell subsets, whereas B-cell (CD3/CD19+) and NK-cell (CD3/CD56+/CD16+) subsets were within the lower limits of normal. Patients in this retrospective cohort also experienced a high incidence of opportunistic infections (22%), indicative of T-cell dysfunction. In our own unpublished study in melanoma patients who were treated with the 21 of 28-day regimen (100 mg/m2/d), global depletion of all lymphocyte subsets was observed when lymphocyte counts fell below 500/μL (≥grade 3). At present, there is no conclusive evidence that temozolomide can be used to predictably deplete any particular lymphocyte subset at any given dose or schedule. Further research is, therefore, needed to determine how metronomic low-dose schedules can potentially be used to preferentially deplete regulatory T cells.

In addition, preclinical studies have elucidated physiologic rebound responses to global lymphodepletion that may enhance antitumor immune responses.67, 68 Possible mechanisms underlying these rebound responses include elimination of endogenous cytokine sinks for homeostatic cytokines such as IL-7 and IL-15, depletion of regulatory T cells, and enhanced proliferation of effector lymphocytes. IL-7 and IL-15 are important for CD8+ T-cell function and antitumor activity. After lymphodepletion or other myeloablative conditioning regimens, cells that normally compete for IL-7 and IL-15 are eliminated, increasing availability of these cytokines and lowering the threshold for T-cell activation. After lymphodepletion, antigen-activated effector T and B lymphocytes also proliferate rapidly. Furthermore, lymphodepletion appears to erase immune memory (as evidenced by a loss of recall responses to common antigens) and may reduce tumor-mediated tolerance to tumor antigens.69 The result is a more robust response to tumor-associated antigens. This is particularly relevant in patients with glioblastoma and melanoma, based on evidence of increased infiltration of lymphocytes (including regulatory T cells) into tumor tissue, which is correlated with poor prognosis.70, 71 Thus, lymphodepletion may reset the host immune system and reduce or eliminate host tolerance to autologous tumor antigens. This has led to the hypothesis that the efficacy of antitumor vaccines could be enhanced if they are administered immediately after lymphodepleting chemotherapy. In light of the availability of well-characterized tumor antigens associated with glioma and melanoma,69, 72 this hypothesis could be readily tested.

Combination of temozolomide with antitumor vaccines and immune modulatory therapy

Few studies have examined the influence of temozolomide on the immune response to anticancer vaccines, but several proof of concept studies have been conducted. In a phase 1/2 study in melanoma, 81% of patients treated with the standard 5 of 28-day schedule of temozolomide and the telomerase peptide vaccine GV1001 (hTERT: 611-626) had an immune response to GV1001 at 12 weeks, and patients who received 9 cycles of temozolomide had a stable proliferative response to GV1001 throughout the treatment period.73 Another recent study in melanoma patients demonstrated significantly improved and durable CD8+ T-cell memory responses to HLA-2–restricted melanoma antigen A and gp100 peptides when the vaccines were administered 1 day after dacarbazine treatment.74 A recent study of temozolomide plus an anti–epidermal growth factor receptor variant III (anti-EGFRvIII) peptide vaccine (PEPvIII-KLH) provided preliminary evidence in support of the hypothesis that dose-dense temozolomide regimens might enhance response to an antitumor vaccine. In this phase 2 multicenter study (ACT II, reported at the American Society of Clinical Oncology 2008 annual meeting), patients with newly diagnosed glioblastoma whose tumors expressed EGFRvIII underwent complete surgical resection (>95%) followed by concomitant RT plus temozolomide.69 Twenty days after completing RT, patients received 3 intradermal immunizations (every 2 weeks) with the PEPvIII-KLH vaccine plus granulocyte-macrophage colony-stimulating factor followed by adjuvant temozolomide with either the standard 5 of 28-day regimen or the dose-dense 21 of 28-day regimen. Patients received further immunizations with the PEPvIII-KLH vaccine on Day 21 of each cycle of adjuvant temozolomide (Fig. 1).

During the adjuvant phase, ALC decreased to a greater extent, more consistently, and in a cumulative fashion in patients treated with the 21 of 28-day regimen compared with the 5 of 28-day regimen. In the majority of patients who completed 6 cycles of adjuvant treatment with the 21 of 28-day regimen, lymphocyte counts were <500/μL (≥grade 3). Unexpectedly, anti-PEPvIII antibody titers were >10-fold higher in patients treated with the 21 of 28-day regimen. In addition, the delayed-type hypersensitivity (DTH) skin reaction to the vaccine also became increasingly stronger in this group, an effect that was not observed with the standard 5 of 28-day regimen. In contrast, the DTH response to a control antigen (Candida) did not increase in either group, but decreased only in patients treated with the 21 of 28-day regimen, suggesting a greater loss of memory T cells. Median time to progression and median OS for patients who received the EGFRvIII peptide vaccine in the ACT II trial compared favorably with matched historical controls treated with standard RT plus temozolomide. These preliminary results support the hypothesis that lymphodepletion associated with the dose-dense 21 of 28-day temozolomide regimen may enhance the immune response, and particularly the humoral immune response (production of antivaccinal antibodies) to tumor vaccines, which may translate into improved clinical benefit.

In patients with melanoma, the combination of immunotherapeutic strategies after lymphodepletion has also been explored, although not using temozolomide. Several studies have investigated the benefit of adoptive cell therapy, a process by which tumor-infiltrating lymphocytes are harvested, stimulated in vitro with tumor antigens, expanded, and then reinfused. Recent studies of adoptive cell therapy preceded by lymphodepletion with cyclophosphamide and fludarabine in patients with metastatic melanoma reported favorable response rates (49%-72%) compared with a study in which patients received minimal or no lymphodepletion before adoptive cell therapy (34%).75 These studies also reported elevation of serum levels of IL-7 and IL-15 after lymphodepletion. Therefore, there is evidence to suggest that controlled lymphodepletion may be used to generate positive immunomodulatory effects. Moreover, the use of dose-dense temozolomide to achieve lymphodepletion before adoptive cell therapy or therapeutic antitumor vaccination might have added benefits in patients with melanoma or glioma, because temozolomide has direct antitumor activity in these patient populations.

Conclusions and Future Directions

Dose-dense temozolomide regimens are being investigated widely based on their potential to enhance antitumor activity and overcome MGMT-mediated drug resistance. At present, however, definitive evidence that MGMT-mediated resistance to temozolomide can be overcome in tumor tissues remains elusive. Whether the potential benefits associated with these regimens will outweigh the added toxicity in patients with glioma and melanoma awaits confirmation from prospective randomized trials. For example, the recently concluded EORTC 18,032 study in patients with melanoma demonstrated that the efficacy of dose-dense temozolomide was similar to that of dacarbazine.52 Recently completed and ongoing trials evaluating the 21 of 28-day regimen as adjuvant therapy in patients with newly diagnosed glioblastoma and as primary treatment for LGG will no doubt provide further crucial insights into the therapeutic potential and safety of these regimens in patients with glioma. An additional intriguing aspect of dose-dense temozolomide regimens relates to the emerging evidence that temozolomide-associated lymphodepletion may have clinically important immunomodulatory effects and may enhance the response to antitumor vaccines or adoptive cell therapy strategies. These pilot observations warrant further investigation.

CONFLICT OF INTEREST DISCLOSURES

Financial support for medical editorial assistance was provided by Schering-Plough Corporation. We thank Jerome Sah, PhD, and Jeffrey Riegel, PhD, ProEd Communications, for their medical editorial assistance with the manuscript. B. Neyns has received financial compensation for public speaking from Schering-Plough and Merck/Serono; and research funding from Schering/Plough, Merck/Serono, and Pfizer. D. A. Reardon has received financial compensation as a member of a speakers' bureau and consultant for Schering-Plough and Merck/Serono.

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