Rapamycin and other mTOR inhibitors have shown great promise as anticancer drugs in a spectrum of preclinical models, but it has been difficult to demonstrate convincing clinical activity in single-agent trials using conventional radiographic and clinical criteria for response [38
]. Potential explanations include the largely cytostatic action of these drugs in the laboratory, uncertainty over dose and schedule, and lack of studies to evaluate the drug in subsets of patients most likely to respond based on molecular phenotypes defined preclinically. The goal of this study was to evaluate directly rapamycin in patients whose tumors have defects in PTEN, based on preclinical findings originally generated by our group and others showing mTOR dependence in such models [9
]. In designing the clinical experiment, we sought to validate the use of a PTEN assay for patient selection, document mTOR inhibition in tumor tissue (of particular importance for brain cancers), and gain preliminary evidence of antitumor activity. Glioblastoma was selected based on the high frequency of PTEN loss (~40%), the clinical opportunity to collect tumor tissue at the time of salvage surgical resection, and the high proliferative index of these tumors, providing a robust endpoint for assessing antitumor effect. The intent was to generate information that could be used for more focused hypothesis testing in subsequent trials.
In the present study 165 patients were screened for PTEN status after initial surgical resection, then followed until relapse. Fifteen patients whose initial surgical samples stained negative for PTEN by immunohistochemistry were treated with rapamycin for about 1 wk before a planned salvage surgical resection. Short-term effects of rapamycin on mTOR inhibition in tumor cells and on the tumor proliferation index were determined by comparing immunohistochemical measures of these indices in the initial surgical sample (surgery 1 or S1) to the salvage resection sample (surgery 2 or S2). Rapamycin treatment led to substantial inhibition of tumor cell proliferation in seven of 14 patients, which correlated with the greatest magnitude of mTOR inhibition in tumor tissue. As predicted from preclinical studies [27
], rapamycin also led to the activation of Akt in some cases, and this activation was significantly correlated with shorter time-to-tumor progression.
The primary findings from this neoadjuvant rapamycin trial are evidence of antitumor activity using a short-term endpoint, novel insights into the importance of achieving sufficient target inhibition, and clinical evidence for evaluating combination PI3-kinase/mTOR therapy to address negative feedback. All three findings should guide future clinical development of mTOR inhibitors in this disease. The Ki-67 response data demonstrate that rapamycin has clear antitumor activity in a subset of patients with PTEN loss. In addition to effects on tumor cell proliferation, two patients also had radiographic evidence of response. Patient 8 received an extended course of neoadjuvant rapamycin (25 d) due to an intercurrent upper respiratory infection and had >50% tumor regression by magnetic resonance imaging prior to surgery (Figure S8
A). Patient 11 showed continued radiographic improvement during the postoperative phase of rapamycin treatment and died without evidence of tumor recurrence 538 d after starting rapamycin (Figure S8
B). The experience with patient 8 might justify a longer neoadjuvant treatment period to gain radiographic response data on all patients in subsequent trials. While our trial was underway, a single-arm phase II study of the mTOR inhibitor CCI-779 reported that 20 of 65 patients with recurrent glioblastoma (36%) had radiographic improvement [39
]. Of note, these patients were not evaluated prospectively for PTEN status (no molecular selection criteria), and CCI-779 was delivered weekly rather than daily based on a phase I experience that defined a maximum tolerated dose using this schedule [40
]. In light of our findings about the magnitude of mTOR inhibition required for response (discussed below), this schedule raises concerns about the presumed lack of target coverage during nontreatment days. Nonetheless, the fact that both trials showed evidence of antitumor activity provides confidence that further investigation of mTOR inhibitors is warranted. The role of PTEN loss in defining sensitivity could be determined using a trial design in which all patients are initially eligible but sufficient numbers of PTEN negative versus PTEN positive are accrued to allow subset analysis.
Although intuitive, the correlation we found between the magnitude of mTOR inhibition and Ki-67 response was not anticipated from preclinical studies. Nearly complete inhibition of S6 phosphorylation is typically achieved with rapamycin treatment in xenografts and other mouse model systems; therefore, most studies of response have focused on defining genetic lesions (Pten, Akt, Tsc, Vhl, etc.) that affect mTOR dependence of tumor cells [38
]. The surprising finding in this trial is that despite using doses of rapamycin sufficient to give low nM intratumoral levels, such doses do not translate into mTOR inhibition in all patients. Through ex vivo analysis of tumor cells isolated at salvage surgery, we established that resistance in these patients is not cell intrinsic. Consistent with an extrinsic mechanism of rapamycin resistance, our genomic survey of S2 tumor samples failed to identify significant copy-number alterations within genes in the mTOR pathway (FKBP12, S6 kinase 1, RAPTOR, RHEB, Akt) that might explain the observed rapamycin resistance in vivo. This result contrasts with mechanisms of resistance to other kinase inhibitors (in chronic myeloid leukemia, gastrointestinal stromal tumors, and EGFR-dependent lung cancer), which often occurs through point mutations in the kinase target in tumor cells [43
] and raises the possibility that a larger fraction of PTEN null glioblastomas could be rapamycin-sensitive if more significant mTOR inhibition could be achieved.
The more challenging question is whether strategies can be developed to improve delivery of rapamycin directly to tumor cells and maximize mTOR inhibition broadly across all patients. Oral delivery of significantly higher daily doses is an unlikely solution due to problems with tolerability (mucositis, thrombocytopenia) seen in other diseases. Invasive approaches such as convection-enhanced delivery or implantation of drug-impregnated wafers have been used to treat glioblastoma patients with chemotherapeutic agents and may be considered. Alternatively, a better understanding of the reason underlying the failure to achieve mTOR inhibition in selected patients could point to a solution. For example, if rapamycin in these patients is sequestered in red cells due to enhanced tumor vascularity, antiangiogenic agents such as bevacizumab (already known to have activity in glioblastoma) [44
] may prevent sequestration and allow more efficient drug delivery. Evaluation of all of these approaches requires quantitative assessment of mTOR activity and highlights the need to develop broadly useful clinical tools for quantitative analysis of target inhibition. In the short term, it may be possible to identify the early Ki-67 responders using PET tracers such as 3′-deoxy-3′-18F-fluorothymidine (FLT) that can read out proliferation noninvasively [45
]. Although such identification would not itself improve rapamycin delivery to the tumor cells, it could at least identify the subset of tumors in which rapamycin delivery appears to be problematic. Success here would also obviate the need for salvage surgery and could greatly expand eligibility of patients for larger trials.
There seems little doubt from the time-to-progression curves reported here and in the CCI-779 study that combination therapy is required for significant clinical impact. The challenge, of course, lies in choosing the most promising second drug from an almost infinite number of possibilities. Based on earlier work from us and others, combined EGFR/mTOR blockade is one logical choice, because PTEN loss predicts for resistance to EGFR inhibitors in patients with the mutant EGFRviii variant [18
]. Another possibility is combined PI3K/mTOR blockade to prevent rapamyin-induced activation of Akt caused by loss of negative feedback [27
]. The time-to-progression analysis in our study suggests that the prognosis of these patients is worse, therefore inhibitors that act upstream of Akt may be useful to prevent this complication. Indeed, one dual PI3K/mTOR inhibitor has shown superiority to a pure mTOR inhibitor in preclinical models [49
Although the findings reported here are directly relevant to mTOR inhibitors in glioblastoma, the implication is that these drugs will have activity in a broad range of cancers with PI3K/Akt pathway dysregulation—through PTEN loss, PI3K p110α mutation, AKT gene amplification, or other mechanisms. Recently, mTOR inhibitors have shown clinical activity in metastatic kidney cancer, where the frequency of PTEN loss is low [50
]. The molecular basis for sensitivity in this disease is unknown, but loss of the von Hippel-Lindau (VHL) tumor suppressor and subsequent mTOR-dependent HIF-1α expression is one postulated mechanism [51
]. For reasons similar to those articulated above for glioblastoma, mTOR-based combination therapies are also under consideration in kidney cancer. The neoadjuvant clinical trial design described here should be easily exportable to other cancers in which experimental drug delivery can be timed prior to a planned surgical excision of tumor, and such an approach is consistent with recent national efforts to speed clinical development through novel trial designs [52