Introduction When a new cancer drug first enters the clinic, its development typically proceeds empirically by defining the maximum tolerated dose, then assessing clinical activity across a range of diseases. In the era of molecularly targeted cancer therapy, this approach has been questioned, because it is anticipated that these agents will be effective primarily in those patients whose tumors are dependent on the molecular lesion that is specifically targeted by the new agent [1–3]. However, target-focused clinical development is challenging, because clearly defined, validated molecular criteria to select patients for clinical trials must be established. Inability to access tumor tissue in most patients with solid tumors presents further difficulties. One approach is to conduct small pilot studies in which the targeted agent is administered to patients prior to a scheduled tumor resection to ensure access to tissue during treatment. Such neoadjuvant studies have been successfully implemented with hormonal agents alone or in combination with kinase inhibitors in breast cancer [4,5]. Current technologies permit analyses of gene copy number, mutation status, and mRNA and protein expression from small tissue samples, thereby allowing for the collection of high–molecular content datasets that can guide further clinical development. We have used this approach to study the targeted agent rapamycin in a molecularly defined subset of patients with recurrent glioblastoma. Inhibitors of the mammalian target of rapamycin (mTOR) have received regulatory approval as immunosuppressive agents for the treatment of allograft rejection and as antitumor agents for kidney cancer [6,7]. Rapamycin and its analogs (CCI-779, RAD001) have shown antitumor activity across a variety of human cancers in clinical trials, but molecular determinants of drug response are currently unknown [8]. Previous work by our group [9] and others [10–15] demonstrated that mutational activation of the phosphatidyl-inositol-3-kinase (PI3K) pathway through loss of PTEN (phosphatase and tensin homolog deleted on Chromosome 10) or activation of the serine/threonine kinase Akt sensitizes tumor cells to the antiproliferative activity of mTOR inhibitors in preclinical models. These findings provided the rationale to explore the antitumor activity of mTOR inhibitors in patients with PTEN-deficient tumors. Glioblastoma is one model disease to address this question, because PTEN inactivation occurs in ∼40% of patients. Furthermore, salvage surgical resection is often part of the clinical management of patients who relapse after standard up-front therapy (which typically consists of surgical resection followed by adjuvant radiation and chemotherapy). This second resection is an opportunity to collect tumor tissue to assess the molecular effects of treatment administered pre-operatively. Indeed, others have used this salvage surgery to define the dose of O6-benzylguanine required to deplete the DNA-repair protein AGT, which is associated with resistance to temozolamide [16]. Importantly, the antitumor effects of mTOR inhibition in many preclinical models are cytostatic, raising the possibility that traditional radiographic clinical endpoints of tumor shrinkage may not be observed. Glioblastoma may be suitable for assessing cytostatic activity, because these tumors are highly proliferative. Therefore, short-term effects of treatment on growth kinetics could be detectable by immunohistochemical analysis. Finally, clinical benefit can be assessed by measuring time-to-tumor progression after surgery. For these reasons, we conducted a neoadjuvant clinical trial of rapamycin in patients with relapsed, PTEN-negative glioblastoma undergoing salvage resection, with the primary goals of defining a dose required for mTOR target inhibition and assessing potential antiproliferative effects on tumor cells. Methods Participants This Phase I trial was registered with http://www.ClinicalTrials.gov (#NCT00047073) (see also http://www.cancer.gov/search/ViewClinicalTrials.aspx?cdrid=257255&version=patient&protocolsearchid=3718462). The clinical trial protocol (#02-03-078–11) was approved by the Institutional Review Board of the University of California Los Angeles. Enrollment was restricted to patients with a histological diagnosis of glioblastoma (GBM), radiographic evidence for disease recurrence after standard GBM therapy (surgery, radiation, temozolamide), evidence for PTEN loss in tumor tissue (see below), and no previous mTOR inhibitor therapy. Other enrollment criteria included age > 18 y, Karnofsky performance score (KPS) ≥ 60, life expectancy ≥ 8 wk, adequate bone marrow function (white blood cell [WBC] > 3,000/μl, absolute neutrophil count [ANC] > 2,000/μl, platelets > 100,000/μl, hemoglobin > 10 gm/dl), adequate liver and renal function (serum glutamic oxaloacetic transaminase [SGOT] and bilirubin 50% inhibition of S6 phosphorylation for at least one of the two examined phosphosites (p 20% of tumor cells) [37]. Discussion 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 [9–15]. 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 [27,28], 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 S8A). 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 S8B). 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 [40,41]. 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 [38,42]. 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 [18,46–48]. Another possibility is combined PI3K/mTOR blockade to prevent rapamyin-induced activation of Akt caused by loss of negative feedback [27,28]. 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]. Supporting Information Figure S1 Digital Scoring of Immunohistochemical Stains Adjacent tissue sections from each tumor were stained with antibodies against Ki-67, phospho S6 ribosomal protein (S6RP), phospho-PRAS40, and PTEN (unpublished data). Five areas per slide, each representing approximately 500–1,000 tumor cells, were selected for digital scoring. Image conversion and scoring was performed using Soft Imaging System Software. The distribution of immunoreactivity within these 2,500–5,000 cells was graphed for each sample as a box plot. The “fold change” (F.C.) in S6 immunoreactivity (Table 2 and Figure S2) was calculated for each tumor as the ratio between median staining score in the S2 and the median staining score in the S1 sample. (135 KB PDF) Click here for additional data file. Figure S2 S6 Phosphorylation at Ser 235/236 in Matched S1/S2 Tumor Tissue Pairs (A) Representative IHC staining results for pSer 235/236 S6. Shown are examples for a tumor with biochemical mTOR inhibitor resistance (patient 2) compared to a tumor with marked mTOR inhibition in response to rapamycin (patient 8). (B and C) Quantification of S6 phosphorylation at Ser 235/236 in matched S1/S2 tumor samples from 14 patients in the rapamycin clinical trial cohort (B) and nine glioblastoma patients who did not receive rapamycin prior to S2 (C). For additional information regarding IHC scoring methodology, see Figure S1 and Text S1. (1.2 MB PPT) Click here for additional data file. Figure S3 S6 Phosphorylation at Ser 240/244 in Matched S1/S2 Tumor Tissue Pairs IHC-based quantification of S6 phosphorylation at Ser 240/244 in (A) matched S1/S2 tumor samples from 14 patients in the rapamycin clinical trial cohort and (B) matched S1/S2 tumor samples from nine glioblastoma patients who did not receive rapamycin prior to S2. (206 KB PPT) Click here for additional data file. Figure S4 CD31 Immunostaining of Representative Tumor Tissue Sections from Rapamycin-Treated Tumors with Low (Patients 1 and 3) Versus High (Patients 5 and 15) Intratumoral Rapamycin Concentrations Arrows indicate CD31 IHC-positive intratumoral vasculature. (159 KB PDF) Click here for additional data file. Figure S5 Frequency of Genomic Aberrations in S2 Samples by Array-CGH Genomic DNA microdissected from fresh frozen tumor samples (S2) was subjected to oligonucleotide microarray-based CGH (aCGH) analysis. Aberrations were scored using CGH Analytics Software (Agilent) using the ADM1 algorithm (parameters listed in Text S1), and filtered to retain only aberrations with a log2 ratio less than −0.3 or greater than 0.3. The frequency of DNA copy-number increases (red, right of the zero axis for each chromosome) and losses (green, left of the axis) within the genome for the samples profiled is plotted in terms of percentage of the samples analyzed. (56 KB PDF) Click here for additional data file. Figure S6 Gene Copy-Number Alterations Stratified by pPRAS40 Response CGH data for biopsies following rapamycin treatment were plotted using Cluster and TreeView Software (available at http://rana.lbl.gov/EisenSoftware.htm). DNA copy-number losses are represented in green, while gains are represented in red. Patient samples are stratified by the presence or absence of significant induction of pPRAS40 following rapamycin therapy as determined by IHC (see Figure 4B and Table 2). (53 KB PDF) Click here for additional data file. Figure S7 IHC Scoring of PTEN Expression in Clinical Tumor Samples from Rapamycin Study Patients Compared to a Group of 44 Archival Glioblastoma Tissue Samples (A) Comparison of two PTEN IHC scoring methods (x-axis: manual score; y-axis: image-guided digital score) in 44 archival glioblastoma samples. The manual score compares PTEN expression in tumor cells to PTEN expression in adjacent endothelial cells and assigns scores of 0 (absent in tumor cells), 1 (reduced in tumor cells relative to endothelial cells), and 2 (tumor cells staining similar to endothelial cells). Digital PTEN scores are based on absolute values of PTEN immunoreactivity as determined through an image-guided software scoring system (Figure S1). Digital PTEN scores were determined by an independent reviewer without knowledge of the manual PTEN score. (B) Mean digital PTEN IHC score of tumors from the rapamycin study group before (S1) and during (S2) rapamycin therapy. (18 KB PDF) Click here for additional data file. Figure S8 Radiographic Response to Rapamycin in 2/15 Study Patients Magnetic resonance imaging results for two patients who experienced a clinical response to single-agent rapamycin. (A) Patient 8 received an extended preoperative course of rapamycin (25 d) due to an intercurrent upper respiratory infection and experienced >50% tumor regression by magnetic resonance imaging. (B) Patient 11 showed radiographically stable disease for >16 mo postoperatively and died without evidence for tumor recurrence. (72 KB PDF) Click here for additional data file. Table S1 Rapamycin-Related Adverse Events (11 KB PDF) Click here for additional data file. Table S2 PTEN Copy Number (Log2 Ratio) and Missense Mutations in Tumor Tissue Genomic DNA was extracted from microdissected tumor cells. N/A: no fresh frozen tumor aliquot available for analysis. Log2 ratios ≤ −0.3 are consistent with DNA copy-number loss. (13 KB PDF) Click here for additional data file. Text S1 Supplementary Methods (42 KB DOC) Click here for additional data file. Text S2 Protocol (254 KB DOC) Click here for additional data file. Text S3 CONSORT Checklist (59 KB DOC) Click here for additional data file.
