Phase I study of low-dose metronomic temozolomide for recurrent malignant gliomas

Unlike the conventional schedule of TMZ at 150–200 mg/m²/day for 5 days, mTMZ is typically given continuously at a dose of 25 to 50 mg/m²/day. Such lower daily dosage may not be myelotoxic enough to cause significant leukopenia or thrombocytopenia while retaining antitumor efficacy and, when given over a longer period of time, the cumulative dose from mTMZ could be higher than the dose from conventional schedule. In the current study, TMZ given in metronomic doses was well tolerated by our subjects with recurrent malignant gliomas. The side effects observed were primarily hematological and gastrointestinal in nature, and nearly all of them were in the grade 1 or 2 severity category. This is consistent with findings in past phase II trials and retrospective series where others observed mild lymphopenia, neutropenia, thrombocytopenia and liver enzyme elevation [25–27].

Chronic daily dosing of cytotoxic chemotherapies have been in use as salvage treatment in oncology. Fulton et al. [28] reported the use of metronomic oral etoposide for recurrent malignant gliomas and noted an objective response rate of 18% (8 of 46 patients) and a median time to tumor progression of 8.8 weeks, while side effects consisted of manageable neutropenia and thrombocytopenia. Compared to pulsed intravenous administration of etoposide, metronomic oral etoposide has similar or even better bioavailability [29]. In addition, daily capecitabine is indicated for metastatic colon cancer and taxane-refractory breast cancer [30, 31]. Compared to intravenous 5-fluorouracil and leucovorin, capecitabine has less frequent hematological toxicity but more hepatic enzyme elevation, probably due to its first pass in the liver when taken orally [31].

Multiple mechanisms likely contribute to the antitumor efficacy of mTMZ. Our trial is the first to incorporate both neuroimaging and CSF correlative studies to help elucidate the underlying antitumor mechanisms of mTMZ. Arterial spin labeling MRI was used to measure blood flow at the site of disease and elsewhere in the brain. It is important to note that our current method of visualization of brain tumors relies on leakage of gadolinium from highly permeable vasculature into the brain parenchyma and thus this process delays its clearance. However, malignant gliomas are highly infiltrative and vascular breakdown is typically not present at the invasive front of the tumor. Therefore, gadolinium-enhanced MRI demonstrates only a part of the tumor that has permeable vasculature. Pirzkall et al. [32] used multivoxel MR spectroscopy to demonstrate the presence of non-enhancing gliomas in areas that has elevated choline signals but no leakage of gadolinium. Similarly, arterial spin labeling can demonstrate regions of malignant gliomas without gadolinium enhancement, which is probably a result of the elevated metabolic demand of the tumor that requires increased blood flow. Furthermore, unlike measurement of the antiangiogenesis effect of mTMZ using cerebral blood volume maps [25], which are calculated values that can be altered by steroid’s effect on vascular permeability, arterial spin labeling has another advantage because it does not require a contrast agent. Instead, this technique utilizes magnetic field gradients and radiofrequency fields to label the endogenous water of blood and, because water is freely diffusible within the brain even without damage to the blood brain barrier, it allows for a quantitative analysis of blood flow in regions that include the malignant glioma [19, 33]. The absolute quantification of blood flow may be limited by regional heterogeneity of the tumor and slight variability may appear in data acquired at different time points. As shown by our data, a blood flow ratio in the tumor normalized to a reference part of the brain may reflect more accurately changes over time.

Alterations in the blood flow ratio have been detected in some of our subjects during treatment with mTMZ. Kerbel et al. [12, 34] demonstrated in an experimental setting that metronomic cyclophosphamide, an alkylator similar to temozolomide but requiring first pass hepatic metabolism to its active agent, delayed or prevented the growth of xenografted tumors in mice. This antitumor effect was most likely mediated by a reduction in the circulating endothelial precursor cells, which are thought to be more sensitive to cytotoxic chemotherapy, and this effect is not specific to cyclophosphamide but also other agents such as cisplatin, vinblastine and vinorelbine [35]. However, in patients with recurrent glioblastomas treated with mTMZ and an antiangiogenic adjuvant celecoxib, immunostaining of CD31-positive endothelial cells of resected tumors before treatment showed high variability in microvessel density [36]. Furthermore, microvessel density did not correlate with patient outcome [36]. Nevertheless, an objective response rate of 5 to 14% and a PFS at 6 months of 17 to 57% were observed in patients treated with mTMZ, suggesting other mechanisms of action may be relevant.

Invasion is a major hallmark of malignant gliomas and antiangiogenesis therapy can bias the tumor towards an invasive phenotype [5, 37, 38]. These invasive glioma cells are thought to possess stem-like cellular characteristics [39]. In this process, MMPs are activated and, in particular, the expression of MMP-2 and MMP-9 is upregulated within the tumor microenvironment [40]. Furthermore, both MMP-2 and MMP-9 activities can also be measured in the CSF, and MMP-9 activity in particular was noted to correlate with disease activity in recurrent glioblastoma [41, 42]. We used activation isoform-specific ELISA as a proxy for MMP-2 and MMP-9 activity within the CSF. In our subjects, the average aMMP-9 level did not correlate with either PFS or OS, but average aMMP-2 level did appear to correlate with OS. This may indicate that the source of aMMP-2, which is constitutively expressed in the brain, may come from sources other than the tumor or the brain parenchyma. Specifically, immune cells can also secrete MMP-2 and MMP-9, and the elevated MMP-2 activity that correlated with OS may indicate an antitumor inflammatory response as a part of innate immunity in the host [43]. However, given the weak correlations between aMMP-2 and aMMP-9 in the CSF and outcomes, it is not clear that mTMZ exerts an anti-angiogenic or anti-invasive effect. In fact, the insignificant changes in metalloproteinases during mTMZ treatment suggest that mTMZ may not work by an anti-angiogenic mechanism and that immunogenic or alkylating effects may have greater relevance.

mTMZ can modulate the immune system to elicit an antitumor response by selective depletion of Tregs [9]. It is notable that at doses given to our subjects that are not cytotoxic to tumor cells, mTMZ still produced a response rate of 14%. This antitumor effect may be the result of Treg depletion that effectively reduces immune suppression within the tumor microenvironment [9, 10, 25–27]. Specifically, Tregs can suppress T lymphocyte activation by inhibiting IL-2 production [44]. Indeed, a high serum level of sIL-2Rα in patients with metastatic melanoma is strongly correlated with poor outcomes from anti-CTLA-4 treatment, which requires concomitant IL-2-mediated immune activation [23]. Likewise, sIL-2Rα modulates IL-2-mediated immune response in patients with follicular lymphoma [24]. Using CSF, we observed that baseline sIL-2Rα was inversely correlated with time to first recurrence of glioblastomas prior to mTMZ treatment, and that the two subjects having elevated baseline levels possessed the longest PFS while the other two with undetectable levels exhibited the shortest PFS. These data suggest a potential contribution of T-cell biology to mTMZ benefit and that patients with elevated CSF sIL-2Rα at baseline have more aggressive disease but they may benefit more from the immunomodulatory effect of mTMZ.

In our cohort, mTMZ was well tolerated and without serious side effects. Although gadolinium enhancement on T1 is observed at the region of the tumor, blood flow as measured by arterial spin labeling showed high variability across individuals and time, with some tumor blood flow increased briefly before subsiding while others showed a gradual decrease or stabilization. The correlation of aMMP-2 with OS and baseline sIL-2Rα with OS both suggest that mTMZ may exhibit a T-cell-dependent immune modulatory effect in patients with recurrent malignant gliomas.