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Introduction= =20
Methods=20
Results=20
Discussion=20
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RESEARCH ARTICLE

Open=20 Access=20

Everything we publish is freely available online throughout the = world, for=20 you to read, download, copy, distribute, and use (with attribution) any = way you=20 wish. No permission required. Read a detailed = definition of=20 open access.

Antitumor Activity of Rapamycin in a Phase I Trial for Patients with = Recurrent PTEN-Deficient Glioblastoma

Tim F. Cloughesy¹⁼, Koji Yoshimoto²^=
=C2=A4= , Phioanh Nghiemphu¹⁼, Kevin Brown³,=20 Julie Dang²,=20 Shaojun Zhu²,=20 Teli Hsueh⁴,=20 Yinan Chen⁴,=20 Wei Wang⁵,=20 David Youngkin³,=20 Linda Liau⁶,=20 Neil Martin⁶,=20 Don Becker⁶,=20 Marvin Bergsneider⁶,=20 Albert Lai¹,=20 Richard Green⁷,=20 Tom Oglesby⁵,=20 Michael Koleto⁵,=20 Jeff Trent³,=20 Steve Horvath⁸,=20 Paul S. Mischel^2,4⁼, Ingo K. Mellinghoff⁴⁼, Charles L. Sawyers⁹^=
*⁼

1 = Department of=20 Neurology, Jonsson Comprehensive Cancer Center, David Geffen School of = Medicine,=20 University of California Los Angeles, Los Angeles, California, United = States of=20 America, 2 Department of = Pathology and=20 Laboratory Medicine, Jonsson Comprehensive Cancer Center, David Geffen = School of=20 Medicine, University of California Los Angeles, Los Angeles, California, = United=20 States of America, 3 = Translational=20 Genomics Research Institute, Phoenix, Arizona, United States of America, = 4 Department of Molecular and = Medical=20 Pharmacology, Jonsson Comprehensive Cancer Center, David Geffen School = of=20 Medicine, University of California Los Angeles, Los Angeles, California, = United=20 States of America, 5 = Taylor=20 Technology, Princeton, New Jersey, United States of America, 6 Department of Neurosurgery, Jonsson=20 Comprehensive Cancer Center, David Geffen School of Medicine, University = of=20 California Los Angeles, Los Angeles, California, United States of = America, 7 Department of Neurology, = Kaiser=20 Permanente; Los Angeles, California, United States of America, 8 Department of Biostatistics and Human = Genetics,=20 Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, = University=20 of California Los Angeles, Los Angeles, California, United States of = America, 9 Department of Human = Oncology and=20 Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, = New=20 York, United States of America

Background=20

There is much discussion in the cancer drug development community = about how=20 to incorporate molecular tools into early-stage clinical trials to = assess target=20 modulation, measure anti-tumor activity, and enrich the clinical trial=20 population for patients who are more likely to benefit. Small, = molecularly=20 focused clinical studies offer the promise of the early definition of = optimal=20 biologic dose and patient population.

Methods and Findings=20

Based on preclinical evidence that phosphatase and tensin homolog = deleted on=20 Chromosome 10 (PTEN) loss sensitizes tumors to the inhibition of = mammalian=20 target of rapamycin (mTOR), we conducted a proof-of-concept Phase I = neoadjuvant=20 trial of rapamycin in patients with recurrent glioblastoma, whose tumors = lacked=20 expression of the tumor suppressor PTEN. We aimed to assess the safety = profile=20 of daily rapamycin in patients with glioma, define the dose of rapamycin = required for mTOR inhibition in tumor tissue, and evaluate the = antiproliferative=20 activity of rapamycin in PTEN-deficient glioblastoma. Although = intratumoral=20 rapamycin concentrations that were sufficient to inhibit mTOR in vitro = were=20 achieved in all patients, the magnitude of mTOR inhibition in tumor = cells=20 (measured by reduced ribosomal S6 protein phosphorylation) varied = substantially.=20 Tumor cell proliferation (measured by Ki-67 staining) was dramatically = reduced=20 in seven of 14 patients after 1 wk of rapamycin treatment and was = associated=20 with the magnitude of mTOR inhibition (p =3D 0.0047, Fisher = exact test)=20 but not the intratumoral rapamycin concentration. Tumor cells harvested = from the=20 Ki-67 nonresponders retained sensitivity to rapamycin ex vivo, = indicating that=20 clinical resistance to biochemical mTOR inhibition was not = cell-intrinsic.=20 Rapamycin treatment led to Akt activation in seven patients, presumably = due to=20 loss of negative feedback, and this activation was associated with = shorter=20 time-to-progression during post-surgical maintenance rapamycin therapy=20 (p < 0.05, Logrank test).

Conclusions=20

Rapamycin has anticancer activity in PTEN-deficient glioblastoma and = warrants=20 further clinical study alone or in combination with PI3K pathway = inhibitors. The=20 short-term treatment endpoints used in this neoadjuvant trial design = identified=20 the importance of monitoring target inhibition and negative feedback to = guide=20 future clinical development.

Trial registration: http://www.ClinicalTrials.gov = (#NCT00047073).

Funding. Funding = for this=20 investigator-initiated clinical trial was provided by the Doris Duke = Foundation=20 and Howard Hughes Medical Institute (CLS), Accelerate Brain Cancer Cure = (IKM),=20 the Goldhirsh Foundation (CLS), the National Cancer Institute (CA108633 = to PSM),=20 the National Institute for Neurological Disorders and Stroke (NS050151 = to PSM),=20 UCLA GCRC (M01-RR0865), and the Brain Tumor Funders' Collaborative. = Further=20 funding was provided by the Harry Allgauer Foundation through the Doris = R.=20 Ullmann Fund for Brain Tumor Research Technologies, the Henry E. = Singleton Brain=20 Cancer Program at UCLA, the Ziering Family Foundation in memory of Sigi = Ziering,=20 the Art of the Brain, the Roven Family Fund in memory of Dawn Steel, and = the=20 Joseph Drown Foundation in memory of Mark Schackman.The funders played = no role=20 in study design, data collection, data analysis, decision to publish, or = preparation of the manuscript.

Competing = Interests: The=20 authors have declared that no competing interests exist.

Academic Editor: Robert J. Weil, Brain = Tumor=20 Institute, Cleveland Clinic Foundation, United States of America

Citation: Cloughesy TF, Yoshimoto = K, Nghiemphu P, Brown K, Dang J, et al. (2008) Antitumor = Activity=20 of Rapamycin in a Phase I Trial for Patients with Recurrent = PTEN-Deficient=20 Glioblastoma. PLoS Med 5(1): e8 doi:10.1371/journ= al.pmed.0050008

Received: March 22, 2007;=20 Accepted: November 15, 2007; = Published:=20 January 22, 2008

Copyright: =C2=A9 2008 Cloughesy et = al. This is an=20 open-access article distributed under the terms of the Creative Commons=20 Attribution License, which permits unrestricted use, distribution, and=20 reproduction in any medium, provided the original author and source are=20 credited.

Abbreviations: CGH, = comparative=20 genomic hybridization; GBM, glioblastoma; IHC, immunohistochemistry; = KPS,=20 Karnofsky performance score; mTOR, mammalian target of rapamycin; PTEN,=20 phosphatase and tensin homolog deleted on Chromosome 10; S1, surgery 1; = S2,=20 surgery 2; TTP, time to progression

* To whom correspondence should be = addressed.=20 E-mail: sawyersc@mskcc.org

These authors contributed equally to this work.

=C2=A4 Current address: Department = of Neurosurgery,=20 Graduate School of Medical Sciences, Kyushu University, Fukuoka, = Japan.

Editors' Summary

Background.

Glioblastoma is a highly malignant tumor of the brain. As with other = tumors,=20 it can result from a number of different molecular changes. Traditional=20 chemotherapy does little more than contain these tumors, and cannot cure = it. An=20 alternative approach to the treatment of such tumors is to target = specific=20 molecular changes in the tumor. Obviously such targeted treatment will = work only=20 in patients who have the specific molecular defect being targeted. = Hence,=20 traditional clinical trials, which include a large variety of different = patients=20 and tumors with different genetic changes, may be an inappropriate way = to test=20 how effective targeted treatments are.

One specific change that has been identified in around 40% of = patients with=20 glioblastoma is inactivation of a gene known as PTEN, which = acts as a=20 tumor suppressor gene. When PTEN is inactivated it has = previously been=20 shown to make cells more sensitive to a class of drugs known as mTOR=20 inhibitors=E2=80=94one of which is rapamycin (trade name Sirolimus). = mTOR is a protein=20 that is involved in the regulation of a number of cellular processes = including=20 growth and proliferation. Drugs active against mTOR are currently being = tested=20 for effectiveness against other cancers and as immunosuppressive = agents.

Why Was This Study Done?

This was a Phase I study=E2=80=94that is, the earliest type of a drug = study that is=20 done in humans=E2=80=94which aimed to look at the safety of rapamycin in = a selected=20 group of patients who were undergoing surgery after recurrence of = glioblastoma,=20 and whose tumors did not express PTEN. In addition, the authors also = wanted to=20 assess the feasibility of incorporating detailed molecular studies of = the action=20 of this drug into such a Phase I study and whether these molecular = studies could=20 predict whether patients were more or less likely to respond to = rapamycin.

What Did the Researchers Do and Find?

A total of 15 patients were treated with rapamycin at differing doses = for one=20 week before surgery and then again after surgery until there was = evidence that=20 the tumors were progressing. There was no evidence of very severe = toxicity in=20 any of the patients, though there were some adverse effects that = required=20 treatment. When samples from the patients were tested after surgery, = seven of=20 them showed a reduction in how rapidly the tumor cells divided, and this = reduction was associated with how much inhibition there was of mTOR. Two = of=20 these patients showed evidence on scans of a reduction in tumor mass. = Cells from=20 tumors that appeared resistant to rapamycin in patients were sensitive = to=20 rapamycin in tissue culture, suggesting that the lack of response was = due to the=20 drug not being able to penetrate the tumor. A second, unfortunate effect = of=20 rapamycin was to cause activation of another intracellular protein, Akt, = in some=20 patients; when this activation occurred, patients had a shorter time = between=20 surgery and a return of their disease.

What Do These Findings Mean?

The detailed molecular studies within this Phase I trial allow a = better=20 understanding of how this targeted drug works. These findings suggest = that the=20 rapamycin can reduce the proliferation rate of glioblastoma cells, and = that this=20 reduction appears to be related to how well the drug is able to = penetrate the=20 tumor and inhibit mTOR. However, in some patients the activation of a = second=20 pathway can speed up the course of the disease, so further trials should = incorporate inhibitors of this second pathway.

Additional Information.

Please access these Web sites via the online version of this summary = at http://dx.doi.org/10.1371/journal.pmed.0050008.

The US National Cancer = Institute=20 provides information on all aspects of cancer (in English and Spanish)
The UK charity Cancerbackup=20 provides information on brain tumors
Wikipedia has a page on mTOR (note that = Wikipedia is a=20 free online encyclopedia that anyone can edit; available in several=20 languages)

Introduction

When a new cancer drug first enters the clinic, its development = typically=20 proceeds empirically by defining the maximum tolerated dose, then = assessing=20 clinical activity across a range of diseases. In the era of molecularly = targeted=20 cancer therapy, this approach has been questioned, because it is = anticipated=20 that these agents will be effective primarily in those patients whose = tumors are=20 dependent on the molecular lesion that is specifically targeted by the = new agent=20 [1=E2=80=933].=20 However, target-focused clinical development is challenging, because = clearly=20 defined, validated molecular criteria to select patients for clinical = trials=20 must be established. Inability to access tumor tissue in most patients = with=20 solid tumors presents further difficulties. One approach is to conduct = small=20 pilot studies in which the targeted agent is administered to patients = prior to a=20 scheduled tumor resection to ensure access to tissue during treatment. = Such=20 neoadjuvant studies have been successfully implemented with hormonal = agents=20 alone or in combination with kinase inhibitors in breast cancer [4,5].=20 Current technologies permit analyses of gene copy number, mutation = status, and=20 mRNA and protein expression from small tissue samples, thereby allowing = for the=20 collection of high=E2=80=93molecular content datasets that can guide = further clinical=20 development. We have used this approach to study the targeted agent = rapamycin in=20 a molecularly defined subset of patients with recurrent = glioblastoma.

Inhibitors of the mammalian target of rapamycin (mTOR) have received=20 regulatory approval as immunosuppressive agents for the treatment of = allograft=20 rejection and as antitumor agents for kidney cancer [6,7].=20 Rapamycin and its analogs (CCI-779, RAD001) have shown antitumor = activity across=20 a variety of human cancers in clinical trials, but molecular = determinants of=20 drug response are currently unknown [8].=20 Previous work by our group [9]=20 and others [10=E2=80=9315]=20 demonstrated that mutational activation of the = phosphatidyl-inositol-3-kinase=20 (PI3K) pathway through loss of PTEN (phosphatase and tensin homolog = deleted on=20 Chromosome 10) or activation of the serine/threonine kinase Akt = sensitizes tumor=20 cells to the antiproliferative activity of mTOR inhibitors in = preclinical=20 models. These findings provided the rationale to explore the antitumor = activity=20 of mTOR inhibitors in patients with PTEN-deficient tumors.

Glioblastoma is one model disease to address this question, because = PTEN=20 inactivation occurs in 40% of patients. Furthermore, salvage surgical resection is = often part=20 of the clinical management of patients who relapse after standard = up-front=20 therapy (which typically consists of surgical resection followed by = adjuvant=20 radiation and chemotherapy). This second resection is an opportunity to = collect=20 tumor tissue to assess the molecular effects of treatment administered=20 pre-operatively. Indeed, others have used this salvage surgery to define = the=20 dose of O6-benzylguanine required to deplete the DNA-repair protein AGT, = which=20 is associated with resistance to temozolamide [16].=20 Importantly, the antitumor effects of mTOR inhibition in many = preclinical models=20 are cytostatic, raising the possibility that traditional radiographic = clinical=20 endpoints of tumor shrinkage may not be observed. Glioblastoma may be = suitable=20 for assessing cytostatic activity, because these tumors are highly=20 proliferative. Therefore, short-term effects of treatment on growth = kinetics=20 could be detectable by immunohistochemical analysis. Finally, clinical = benefit=20 can be assessed by measuring time-to-tumor progression after surgery. = For these=20 reasons, we conducted a neoadjuvant clinical trial of rapamycin in = patients with=20 relapsed, PTEN-negative glioblastoma undergoing salvage resection, with = the=20 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=20 (#NCT00047073) (see also http://www.cancer.gov/search/ViewClinicalTrials.aspx?cdrid=3D257255&version=3Dpatient&protocolsearchid=3D3718462). The = clinical trial protocol=20 (#02-03-078=E2=80=9311) was approved by the Institutional Review Board = of the University=20 of California Los Angeles. Enrollment was restricted to patients with a=20 histological diagnosis of glioblastoma (GBM), radiographic evidence for = disease=20 recurrence after standard GBM therapy (surgery, radiation, = temozolamide),=20 evidence for PTEN loss in tumor tissue (see below), and no previous mTOR = inhibitor therapy. Other enrollment criteria included age > 18 y, = Karnofsky=20 performance score (KPS) 60, life expectancy 8 wk, adequate bone marrow function (white blood cell [WBC]=20 > 3,000/=CE=BCl, absolute neutrophil count [ANC] > = 2,000/=CE=BCl,=20 platelets > 100,000/=CE=BCl, hemoglobin > 10 gm/dl), = adequate=20 liver and renal function (serum glutamic oxaloacetic transaminase [SGOT] = and=20 bilirubin < 2.5=C3=97 upper limits of normal, creatinine < 1.5 = mg/dl), plasma=20 cholesterol < 350 mg/dl, and plasma triglycerides < 400 mg/dl. = Irradiation=20 and/or chemotherapy were discontinued for 4 wk before trial entry ( 6 wk if prior therapy included a nitrosourea compound). All = 15=20 patients enrolled in the clinical trial gave written informed consent to = participate in these evaluations.

Interventions

Fifteen patients with PTEN-deficient tumors, who also met all other=20 eligibility criteria, were enrolled at the time of tumor recurrence and = received=20 neoadjuvant oral daily rapamycin (2 mg, 5 mg, or 10 mg/d) for = approximately 1 wk=20 (median: 6 d, mean: 7.5 d) prior to salvage surgical resection (S2). = After=20 recovery from surgery, patients resumed daily rapamycin treatment at the = neoadjuvant dose until clinical and/or radiographic evidence for tumor=20 progression was found.

Objectives

The primary goals of this phase I trial were as follows: (1) to = define in=20 PTEN-deficient glioblastoma the dose of rapamycin required for mTOR = inhibition;=20 (2) to establish in PTEN-deficient glioblastoma the antiproliferative = activity=20 of rapamycin; and (3) to define the safety profile of daily rapamycin in = patients with glioma.

Outcomes

Effect of rapamycin on mTOR activity and tumor cell = proliferation in=20 tumor tissue. To quantify mTOR activity in matched S1 and S2 = samples,=20 we measured phosphorylation of S6 ribosomal protein by = immunohistochemistry=20 using two distinct phosphosite antibodies directed against Ser235/236 or = Ser240/244. To determine if 7 d of rapamycin treatment had any antitumor = activity, we assessed the proliferation rate of matched S1 and S2 = samples by=20 measuring the Ki-67 labeling index. Immunohistochemistry (IHC) scoring = is=20 described in detail in Text=20 S1.

Rapamycin concentrations in peripheral blood and tumor=20 tissue. Determination of rapamycin concentrations in peripheral = whole=20 blood was performed by the UCLA Medical Center Clinical Laboratory using = high-performance liquid chromatography/mass spectrometry (HPLC/MS).=20 Quantification of intratumoral rapamycin levels was performed by SFBC = Taylor=20 (Princeton, New Jersey) using the following protocol: Fresh frozen = tissue=20 samples ranging in mass between 50 and 250 mg were homogenized in water = to yield=20 a tissue homogenate concentration of 0.200 g tissue/ml. Rapamycin-free = control=20 tissue was homogenized in a similar fashion. Calibrants were prepared = from=20 aliquots of the control tissue homogenate pool by spiking with rapamycin = to=20 appropriate levels. Desmethoxyrapamycin was used as an internal standard = and was=20 spiked into 1.00 ml aliquots of samples and standards (if needed samples = were=20 prediluted to 1.00 ml with control tissue homogenate). The homogenates = were then=20 extracted with 1-chlorobutane. The extracts were isolated, dried, and=20 reconstituted to a final volume of 100 =CE=BCl. Forty =CE=BCl of the = extracts were=20 analyzed by liquid chromatography/atmospheric pressure chemical = ionization/mass=20 spectrometry/mass spectrometry (LC/APCI/MS/MS) in positive ion mode.=20 Chromatography was performed at a temperature of 50 =C2=B0C on a YMC = ODS-AQ C18=20 column (Waters), 2.0 =C3=97 100 mm, 5 =CE=BCm column using a Paradigm = pump (Michrom=20 Bioresources) Mobile phases were 20 mM ammonium acetate and 0.0005% = acetic acid=20 in water, 20 mM ammonium acetate and 0.0005% acetic acid in methanol. = Detection=20 was performed on a Finnigan TSQ Quantum Ultra AM mass spectrometer.

Tolerability of neoadjuvant and postoperative = rapamycin.=20 Adverse events were evaluated according to the National Cancer Institute = Common=20 Toxicity Criteria, version 2 (http://ctep.info.nih.gov/reporting/index.html).

Genomic studies. Tumor cell DNA was isolated from=20 microdissected fresh frozen clinical tumor samples using the Qiagen = DNeasy Kit=20 (Qiagen). Bidirectional full length sequencing of PTEN (exons = 2=E2=80=939) was=20 performed by Agencourt, and sequence traces were analysed using Mutation = Surveyor software (Softgenetics). For gene copy number determination, = labeled=20 tumor DNA was hybridized to Agilent 44A comparative genomic = hybridization (CGH)=20 microarrays consisting of 40,000 oligonucleotide probes (Agilent Technologies) and = scanned on an=20 Agilent DNA microarray scanner. Raw log₂ ratio data were = calculated=20 using Agilent Feature Extraction 9.1 software. Log₂ ratios = for PTEN=20 were generating using the ADM1 aberration calling algorithm implemented = in=20 Agilent's CGH Analytics 3.4 software. A detailed description of = array-CGH=20 methodology is provided in Text=20 S1.

Sample Size

One hundred and sixty five patients were screened for PTEN status = after=20 initial surgical resection, then followed until relapse. Fifteen = patients whose=20 initial surgical samples stained negative for PTEN by = immunohistochemistry were=20 treated with rapamycin for about 1 wk prior to a planned salvage = surgical=20 resection. Tumor samples from nine glioblastoma patients who underwent = S1 and S2=20 surgeries at UCLA but did not receive rapamycin served as controls for = changes=20 in phosphoS6 and Ki-67 staining ratio from S1 to S2. For additional = comparisons,=20 Ki-67 staining was also measured in S2 samples from an additional 12 = patients=20 whose tumors showed reduced PTEN staining but who did not receive = rapamycin.=20 (Matched S1 samples were not available for this latter group of = tumors).

Statistical Methods

Since time to progression (TTP) was uncensored, we were able to use=20 (multivariate) linear regression models to relate TTP to the molecular = and=20 clinical variables. Since TTP was highly skewed, we log transformed it = to=20 satisfy the normality assumption of a linear regression model. We used = Kaplan=20 Meier plots to visualize the TTP distributions for different patient = strata and=20 used the log rank to test for differences. To test for median = differences across=20 different patient groups, we used nonparametric group comparison tests=20 (Wilcoxon, Kruskal Wallis test). For example, we used the Wilcoxon test = to=20 compare Ki67% among the different patient and control groups. We used = the Fisher=20 exact test to test the independence of rows and columns in a contingency = table.

Reagents

The following antibodies were used for IHC: anti-PTEN (6H2.1, = #ABM-2052,=20 Cascade BioScience; 1:400 dilution), anti-Ki-67 (MIB-1, M7240, = DakoCytomation;=20 1:100 Dilution), anti-phospho Ser235/236 S6 (91B2, #4857, Cell = Signaling; 1:50=20 dilution), anti phospho Ser240/244 S6 (#2215, Cell Signaling; 1:200 = dilution),=20 anti-phospho-PRAS 40 (#44=E2=80=931100G, Biosource; 1:200 dilution), = anti-phospho Ser473=20 Akt (736E11, #3787, Cell Signaling; 1:50 dilution), anti-phospho Thr389 = p70 S6=20 Kinase (#9205, Cell Signaling; 1:100 Dilution), and anti phospho Ser1108 = eIF4G=20 at 1:400 (#2441, Cell Signaling; 1:400 dilution). Antigen retrieval was=20 performed using 0.01 M citrate buffer, pH 6.0 for 30 min in an oven and=20 peroxidase activity was quenched with 3% hydrogen peroxide in water. = Primary=20 antibodies were diluted in PBS /2% bovine serum albumin/2% normal horse = serum=20 (for anti-PTEN) or TBS/0.1% Tween/5% normal goat serum (for = phosphosite-specific=20 antibodies) and applied overnight at 4 =C2=B0C. Biotinylated secondary = antibodies=20 (Vector) were applied at 1:200 dilution for 45 min, and the = avidin-biotin=20 complex (Elite ABC, Vector) for 30 min. Vector NovaRed was used as the = enzyme=20 substrate to visualize specific antibody localization. For Ki-67 = staining,=20 antigen retrieval was performed with a 1mM EDTA buffer (pH 8.0) for 13 = min in a=20 pressure cooker microwave (power level: 80%). All slides were = counterstained=20 with Harris hematoxylin.

Results

Design of a Neoadjuvant Glioblastoma Trial with Short-Term, = Tissue-Based=20 Endpoints

Our primary motive in conducting this single-arm study was to follow = up on=20 the compelling preclinical activity of mTOR inhibitors in PTEN-null = cancer=20 models by designing a small clinical trial focused on measuring = antitumor=20 activity using short-term endpoints. To enhance the probability of = success based=20 on the preclinical hypothesis, we restricted enrollment to those = patients with=20 recurrent glioblastoma whose tumors had evidence of PTEN loss based on = an=20 analysis of tissue obtained from the initial resection (S1) (Figure=20 1). Eligibility was also limited to those patients scheduled to = undergo=20 salvage surgical resection (S2) so that tumor tissue would be available = for=20 assessing the endpoints of mTOR inhibition and tumor cell proliferation, = as well=20 as intratumoral rapamycin concentrations. By mandating access to pre- = and=20 posttreatment samples for each patient, this trial design allows = intrapatient=20 comparison of molecular endpoints, thereby enhancing the statistical = power to=20 detect changes in a small sample size. To provide confidence that any = S1-to-S2=20 changes could be attributed to rapamycin treatment, we conducted an = identical=20 set of measurements using S1 and S2 samples from nine glioblastoma = patients who=20 did not receive rapamycin (controls).

Figure 1. Clinical Trial = Design

Enrollment in the Phase I clinical trial was restricted to patients = whose=20 initial tumor resection (=E2=80=9Csurgery 1=E2=80=9D) specimen was = PTEN-deficient by=20 immunohistochemistry. Patients were enrolled after failing standard = therapy with=20 radiation and chemotherapy (i.e., =E2=80=9Ctumor recurrence=E2=80=9D). = Prior to the scheduled=20 salvage tumor resection (=E2=80=9Csurgery 2=E2=80=9D), patients received = a short course (mean:=20 7.5 d) of oral rapamycin. Rapamycin was resumed after recovery from = surgery=20 until patients developed clinical and/or radiographic evidence of = treatment=20 failure. The effects of rapamycin on tumor cell proliferation and mTOR = signaling=20 in tumor tissue were determined by comparing the tumor tissue collected = during=20 salvage resection (=E2=80=9Csurgery 2=E2=80=9D) with a sample of the = same tumor collected during=20 the initial tumor resection (=E2=80=9Csurgery 1=E2=80=9D). = Time-to-progression (TTP) was defined=20 as the interval between start of rapamycin therapy and postoperative = treatment=20 failure.

Patients whose tumors had PTEN loss were identified using a = previously=20 reported semi-quantitative scoring system that evaluates PTEN expression = in=20 tumor cells relative to adjacent vascular endothelial cells [17,18].=20 We screened tumor samples obtained at the time of initial surgery (S1) = from 165=20 glioblastoma patients followed at our institution for subsequent = neuro-oncology=20 care. Either complete (43/165) or partial (24/165) loss of PTEN = immunoreactivity=20 was shown in 67/165 (40.6 %) of tumors. Fifteen patients with = PTEN-deficient=20 tumors, who also met all other eligibility criteria (see Methods,=20 Texts=20 S2 and S3),=20 were enrolled at the time of tumor recurrence and received neoadjuvant = oral=20 daily rapamycin (2 mg, 5 mg, or 10 mg per day) for approximately 1 wk = (median: 6=20 d, mean: 7.5 d) (Table=20 1) prior to salvage surgical resection (S2). Matching S1 and S2 = samples were=20 used to evaluate the effects of rapamycin on tumor cell proliferation = and mTOR=20 activity. After recovery from surgery, patients resumed daily rapamycin=20 treatment at the neoadjuvant dose until clinical and/or radiographic = evidence=20 for tumor progression was found.

Table 1.

Clinical Characteristics of Rapamycin Study Patients

Rapamycin Tissue and Blood Levels and S6 Kinase Inhibition in Tumor=20 Tissue

Because rapamycin is a macrolide natural product whose size could = prevent=20 distribution across the blood=E2=80=93brain barrier, we measured = rapamycin=20 concentrations by mass spectrometry in an aliquot of tumor tissue = obtained at=20 S2. Rapamycin was detected in 14 of 14 tumors (insufficient tissue was = available=20 from patient 11) at concentrations ranging from 0.3=E2=80=9336.3 nM (Figure=20 2A). Rapamycin concentrations known to confer antiproliferative = activity in=20 PTEN-null cell lines in vitro are typically 1 nM [9,11].

Figure 2. Rapamycin Crosses the = Blood=E2=80=93Brain=20 Barrier and Blocks mTOR in Tumor Tissue

(A) Rapamycin concentrations in tumor tissue (filled squares) and = peripheral=20 blood (empty circles) grouped by rapamycin dose cohorts (2 mg, 5 mg, or = 10 mg=20 per os daily). Intratumoral rapamycin concentration for patient 11 could = not be=20 determined due to insufficient frozen tumor material. The last = preoperative dose=20 of rapamycin was given on the day of craniotomy and peripheral blood was = collected within 24 h of surgery.

(B) Quantification of mTOR activity in tumor tissue by = immunohistochemistry.=20 The cartoon to the left depicts the S6 kinase 1 branch of the mTOR = signaling=20 pathway resulting in phosphorylation of S6 ribosomal protein at serine = 235/236=20 and serine 240/244. The panel to the right shows a comparison between=20 immunoblotting (top) and IHC (bottom) for the determination of S6=20 phosphorylation in tumor tissue from rapamycin patients 1, 2, and 3. The = fold=20 change in serine 235/236 phosphorylation between S2 and S1 for patients = 1, 2,=20 and 3 were 0.45, 1.01, and 0.45, respectively (see Figure=20 S2A).

(C) Changes in S6 phosphorylation between S2 and S1 (y-axis: = ratio=20 of S6 phosphorylation in S2 sample to S6 phosphorylation in S1 sample) = for all=20 patients for whom matched S1 and S2 samples were available (14/15 = rapamycin=20 patients and 9/9 patients who did not receive rapamycin). S6 = phosphorylation was=20 determined by IHC using phosphosite-specific antibodies against serine = 235/236=20 (left) and serine 240/244 (right). Please see Figures=20 S1 and S2=20 for details regarding IHC scoring method and results for individual = tumors.=20 p-values for the difference in mean S2/S1 ratios for each group = were=20 determined using the Kruskal Wallace test.

To quantify mTOR activity, we measured phosphorylation of S6 = ribosomal=20 protein by immunohistochemistry. S6 is a direct substrate of S6 kinase = 1, a=20 downstream effector of mTOR action, and has been widely used as a=20 pharmacodynamic readout of mTOR inhibition in preclinical studies. To = ensure=20 specificity, we used two distinct phosphosite antibodies directed = against=20 Ser235/236 or Ser240/244 (Figure=20 2B). Both sites are directly phosphorylated by S6 kinase 1, but = Ser235/236=20 can also be phosphorylated by p90 ribosomal S6 kinase (RSK) and other = kinases=20 [19].=20 All measurements were quantified by digital readout of = 2,500=E2=80=935,000 tumor cells=20 per slide cut from paraffin-embedded tissue (Figure=20 S1). We attempted to assess mTOR activity using phosphosite-specific = antibodies against Thr389 of S6 kinase 1, a direct mTOR site, and = eukaryotic=20 initiation factor 4G (serine 1108), but the performance characteristics = of these=20 antibodies on paraffin sections, in our hands, were inadequate for = reliable=20 quantification (unpublished data).

For the three patients in the 2-mg cohort, sufficient frozen tissue = from S1=20 and S2 was available to directly compare immunoblot and IHC measures of = S6=20 phosphorylation (Figure=20 2B). Although the magnitude of pS6 reduction measured by immunoblot = was more=20 dramatic, both methods were in agreement and thereby provide reassurance = that=20 the IHC approach could be used across the sample set. Total ribosomal S6 = levels=20 were also reduced in patients 1 and 3, consistent with the fact that = translation=20 of S6 is mTOR-dependent.

When examined in aggregate, the level of S6 phosphorylation in S2 = samples=20 from all three cohorts was reduced at both phosphosites compared to = matched S1=20 samples (S2/S1 ratio < 1.0, Figure=20 2C). In contrast, a similar analysis of S1 and S2 samples collected = from the=20 nine glioblastoma patients who did not receive rapamycin showed no = change in S6=20 phosphorylation. In comparing the performance of the two antibodies, it = is=20 noteworthy that three patients had an increase (or no change) in S6=20 phosphorylation in the S2 sample when measured using the serine 235/236 = antibody=20 (patients 2, 5, and 13) (Figure=20 S2B), whereas the same samples showed reduced S6 phosphorylation = using the=20 serine 240/244 antibody (Figure=20 S3A and Table=20 2). This paradox might be explained by mTOR-independent = phosphorylation of=20 the 235/236 site [20,21]=20 and suggests that the 240/244 site may be a preferred endpoint for = assessment of=20 mTOR activity. It is also noteworthy that the magnitude of pS6 = phosphorylation=20 did not correlate with the intratumoral rapamycin concentration. Indeed, = some=20 patients with adequate drug concentrations in tumor tissue had = relatively modest=20 reduction reductions in pS6 (e.g., patients 2 , 12, and 15) (Table=20 2), indicating that some patients/tumors exhibit apparent = biochemical=20 resistance to mTOR inhibition (addressed further below).

Table 2.

Molecular and Clinical Parameters in the Cohort of Rapamycin-Treated=20 Patients

Inhibition of Tumor Cell Proliferation Is Correlated with Magnitude = of mTOR=20 Inhibition

To determine if 7 d of rapamycin treatment had any antitumor = activity, we=20 assessed the proliferation rate of matched S1 and S2 samples by = measuring the=20 Ki-67 labeling index. S2 samples from the rapamycin-treated patients had = a=20 significantly lower labeling index than the matched S1 tumors (median: = 2.1 %=20 versus 21.3 %, p < 0.005 by Wilcoxon test), whereas the same = S1/S2=20 comparison in patients who did not receive rapamycin showed no change = (Figure=20 3A). Because these untreated patients were not selected for PTEN = deficiency,=20 we measured proliferation in 12 additional nonstudy patients following = salvage=20 resection whose tumors were matched for PTEN status. (Matching S1 = samples from=20 these 12 patients were not available for intrapatient comparison.) The = Ki-67=20 labeling index in these PTEN-null S2 samples (no rapamycin) was = comparable to=20 that of the S1 and S2 samples from the control patients and = significantly higher=20 than the S2 samples from the rapamycin-treated patients (Figure=20 3A). Remarkably, the reduction in Ki-67 labeling index was = attributable to=20 nearly complete inhibition of tumor cell proliferation in half (7/14) of = the=20 patients (Figure=20 3B), suggestive of at least two subgroups (rapamycin-sensitive and=20 rapamycin-resistant) within this patient population.

Figure 3. Rapamycin Inhibits Tumor = Cell=20 Proliferation in Subsets of PTEN-Deficient GBMs

(A) Ki-67 labeling index of GBMs before (S1) and during (S2) = rapamycin=20 therapy compared with tumor samples from patients who did not receive = rapamycin=20 (see text for detail). The horizontal line inside a box plot shows the = median=20 value. The lower and upper end of the box corresponds to the 25th and = 75th=20 percentile, respectively. The whiskers extend to the 95% range. The = Wilcoxon=20 nonparametric group comparison test was used to calculate = p-values for=20 the differences between different patient groups.

(B) Changes in tumor cell proliferation between S1 and S2 for 14/15 = rapamycin=20 patients. The horizontal line inside the box indicates the median value. = The=20 lower and upper border of the box corresponds to the 25th and 75th = percentile,=20 respectively. The whiskers extend to the 95% range. Paraffin blocks from = patient=20 14 were not sufficient for quantification. The median Ki-67 labeling = index of=20 the S2 specimen from patients 1, 3, 8, 9, 11, and 13 was 0 (indicated by = asterix). N.S. indicates that the difference in Ki-67 labeling index not = statistically significant at the 0.05 level according to the Wilcoxon = test.

(C) Relationship between the magnitude of S6 inhibition and Ki-67 = response in=20 S2 tumor samples from rapamycin-treated patients. p-values were = determined by Fisher Exact test for different thresholds of pS6 = inhibition.

(D) Ex vivo rapamycin response of short-term cultures derived from = tumors=20 with in vivo S6 response (patients 1 and 3) or resistance (patients 2 = and 12).=20 Shown are S6 and p85 (loading) immunoblots of whole cell lysates 8 h = after=20 treatment with vehicle, 0.3 nM rapamycin, and 3 nM rapamycin.

In examining the molecular determinants of rapamycin sensitivity, we = noted=20 that the magnitude of mTOR inhibition was highly correlated with Ki-67 = response=20 using a cutoff of >50% inhibition of S6 phosphorylation for at = least=20 one of the two examined phosphosites (p < 0.0047) (Fisher = exact=20 test) (Figure=20 3C). In comparing the two antibodies, changes in pSer235/236 were=20 statistically more significantly linked to Ki-67 response, even though = this site=20 is believed to be less specific for S6K1 activity than pSer240/244 due = to=20 phosphorylation by other kinases [20].=20 This finding may reflect true biological differences in the input to = these=20 distinct phosphorylation sites or may be due to the relative sensitivity = of the=20 antibodies for detecting quantitative differences in mTOR inhibition. = [In our=20 hands, the staining intensity with the pSer240/244 antibody is generally = less=20 intense than with the pSer235/236 antibody.]

Whereas this analysis highlights the importance of achieving = sufficient mTOR=20 inhibition, it fails to address the fact that adequate intratumoral = rapamycin=20 concentrations did not translate into mTOR inhibition in some patients. = Such=20 biochemical resistance could be cell-intrinsic (mutation of the drug = target,=20 expression of a drug efflux pump in tumor cells, etc.) or host-related = (drug=20 bound to serum proteins, sequestration in specific cell types or = tissues, etc.).=20 To distinguish between these two categories, we examined the sensitivity = of=20 tumor cells removed at S2 from four study patients, two of whom were = sensitive=20 and the other two resistant to rapamycin, after short-term propagation = in=20 culture. If clinical rapamycin resistance is cell-intrinsic, these cells = should=20 be similarly resistant ex vivo, whereas sensitivity should be restored = if host=20 mechanisms are at play. Remarkably, S6 phosphorylation was inhibited = equally in=20 rapamycin-sensitive (patients 1 and 3) and rapamycin-resistant (patients = 2 and=20 12) samples at 0.3 and 3.0 nM concentrations (Figure=20 3D), indicating that the failure to inhibit mTOR in these patients = is not=20 cell-intrinsic. Rather, the data indicate that delivery of rapamycin to = tumor=20 cells is impaired in some patients despite achieving adequate = concentrations in=20 resected brain tumor tissue. One possibility, based on the fact that = rapamycin=20 is sequestered in red blood cells [22],=20 is that the high intratumoral concentrations of rapamycin observed in = these=20 resistant patients reflect red cell pooling in highly vascular tumors. = Indeed,=20 tumors from resistant patients showed abundant immunohistochemical = staining for=20 the vascular marker CD31 (Figure=20 S4) but the sample size is too small to make definitive conclusions. = Alternative explanations include variations in penetration of the = blood=E2=80=93brain=20 barrier or tumor hydrostatic pressure among patients.

Safety Profile of Daily Rapamycin in Patients with Glioma

No grade 3 or 4 toxicities were observed during preoperative = rapamycin=20 treatment. Of particular importance, there were no perioperative = bleeding=20 complications. Five of 15 patients had grade 3 adverse events = (hypokalemia,=20 hypercholesterolemia, and cytopenias) during postoperative rapamycin = treatment,=20 which were managed with supportive care and did not require treatment=20 discontinuation (Table=20 S1).

Impact of Rapamycin-Induced Akt Activation on Clinical Outcome

Physiologic activation of the Akt pathway is regulated, in part, by a = negative feedback loop involving phosphorylation of insulin receptor = substrate 1=20 (IRS1) by the mTOR effector molecule S6 kinase 1 (Figure=20 4A) [23=E2=80=9326].=20 mTOR inhibition by rapamycin can cancel this negative feedback and = activate Akt=20 in some cancer cell lines and tumor samples, but the potential clinical = impact=20 is unknown [8,27,28].=20 We assessed Akt activity in S1 and S2 samples in the rapamycin-treated = patients=20 using phosphosite-specific antibodies against the serine/threonine = kinase Akt=20 (serine 473) and its downstream substrate PRAS 40 (threonine 246), which = serves=20 as a biomarker for Akt activity (Figure=20 4A). PRAS40 has also been recently shown to inhibit mTOR, and this=20 inhibition is relieved by Akt phosphorylation [29=E2=80=9331].=20 Seven of 14 (50%) patients had a statistically significant (p = <=20 0.05, Wilcoxon test) increase in PRAS40 phosphorylation in their S2 = sample (Figure=20 4B). Of note, one patient (11) had a significant decrease in PRAS40=20 phosphorylation (and pS473 Akt) at S2 (Figure=20 4B), which could reflect the potential inhibition of the TORC2 mTOR = complex=20 (implicated as the pS473 Akt kinase) by rapamycin after prolonged = exposure [32,33].

Figure 4. Induction of Akt = Signaling in a=20 Subset of Rapamycin-Treated Tumors

(A) Determination of Akt activation in tumor tissue during mTOR = inhibitor=20 therapy. The left panel is a cartoon illustrating the mTOR/S6K1 = dependent=20 feedback loop of the PI3k-Akt pathway. The right panel shows changes in=20 phosphorylation of the Akt-substrate PRAS40 (threonine 246) during = rapamycin=20 therapy correspond to changes in Akt phosphorylation (serine 473). = Tumors from=20 patients 2 and 5 show an increase in pAkt and pPRAS40 immunostaining = during=20 rapamycin treatment, whereas the tumor from patient 11 shows a decrease = in the=20 same markers on rapamycin.

(B) Changes in PRAS40 phosphorylation between S1 and S2 for 14/15 = rapamycin=20 patients. The horizontal line inside the box indicates the median value. = The=20 lower and upper border of the box corresponds to the 25th and 75th = percentile,=20 respectively. The whiskers extend to the 95% range. Paraffin blocks from = patient=20 14 were not sufficient for quantification. N.S. indicates that the = difference in=20 pPRAS40 staining intensity not statistically significant at the 0.05 = level=20 according to the Wilcoxon test.

Because activation of Akt may attenuate the antitumor activity of = rapamycin,=20 we examined the relationship between pPRAS 40-induction and = time-to-progression=20 during the postsurgical maintenance phase of rapamycin treatment. = Induction of=20 pPRAS40 was statistically significantly associated with a shorter=20 time-to-progression (p =3D 0.049, Logrank test) (Figure=20 4C). Given that only 14 patients led to this p-value, it is = plausible that a study with more patients could lead to a more = statistically=20 robust finding. In contrast, there was no statistically significant = relationship=20 between time-to-progression and age (p =3D 0.57), Karnofsky = performance=20 status (p =3D 0.91), number of tumor recurrences prior to = enrollment=20 (p =3D 0.12), rapamycin levels in tumor (p =3D 0.45) = or plasma=20 (p =3D 0.25), inhibition of S6 phosphorylation at serine 240/44 = (p =3D 0.42) or serine 235/236 (p =3D 0.65), basal = phosphorylation=20 of S6 at serine 240/244 (p =3D 0.24) or serine 235/236 = (p =3D=20 0.54), or Ki-67 response (p =3D 0.42) (all p-values = determined=20 by Logrank test). pPRAS40 induction remained an independent significant=20 predictor (p < 0.05) even after adjusting for age and KPS in = a=20 multivariate regression model.

To explore which genetic lesions might be associated with PRAS40 = induction=20 during mTOR inhibitor therapy, we hybridized tumor cell DNA collected = during=20 rapamycin therapy (S2) to CGH microarrays consisting of 40,000 oligonucleotide probes. Overall, our data set showed = many=20 chromosomal aberrations typically found in glioblastoma [34],=20 including frequent gains of chromosome 7, loss of chromosome 10, and = focal=20 amplifications of EGFR, PDGFRA, CDK4, and MDM2 (Figure=20 S5). Compared to tumors without pPRAS40 induction, tumors with = pPRAS40=20 induction more commonly showed focal amplifications of MDM2 (4/7 versus = 1/5) and=20 the receptor tyrosine kinases EGFR or PDGFRA (4/7 versus 1/5) (Figure=20 S6). While these observations lacked statistical power due to the = small=20 sample size, it is interesting to note that EGFR has been reported to = induce=20 expression of IRS1, the mediator of the negative mTOR/S6K1 feedback = loop, in=20 breast cancer cells [35].=20 Furthermore, PDGFR is an additional target of mTOR negative feedback = which can=20 be overcome by enhanced PDGFR=CE=B2 expression [36].

Comparison of S1 and S2 PTEN Status

One critical issue that can complicate the use of molecular = biomarkers for=20 clinical trial eligibility is whether a tissue sample obtained much = earlier than=20 the treatment intervention can be used to make an accurate assessment of = the=20 relapsed tumor. We addressed this question by comparing the PTEN status = using=20 the S1 and S2 samples from the 15 patients on this study. Although = eligibility=20 was determined on the basis of a semiquantitive manual PTEN score [17,18],=20 we later refined our PTEN scoring methodology by using digital image=20 quantification software. In comparing the two scoring methods on 44 = archival=20 glioblastoma samples, we found a high Spearman correlation between the = two=20 scoring methods (rho =3D 0.82, p =3D 8.6 =C3=97 = 10^=E2=88=9212) (Figure=20 S7A). When applied to the study patients, the digital method = confirmed that=20 PTEN immunoreactivity in the S1 sample was comparable to that of the S2 = sample=20 (all samples scored less than 35) and could be used to infer PTEN status = at=20 relapse (Figure=20 S7B).

To explore the molecular basis for loss of PTEN expression, we = searched for=20 PTEN mutations and/or gene loss in tumor cell DNA = microdissected from=20 all available (13/15) frozen tumor samples. 3/13 (23.1%) tumors harbored = missense mutations in the PTEN coding sequence. One of these = mutations=20 (R173C) has previously been described in glioblastoma, and the other two = missense mutations (H61Y, V217F) map to codons previously reported in = other=20 human cancer samples (http://www.sanger.ac.uk/genetics/CGP/cosmic). 9/13 (69.2 %) tumors showed evidence of = DNA copy-number=20 loss (log₂ ratio =E2=88=920.3) at the PTEN locus in at least one = tumor aliquot, as=20 measured by oligonucleotide array-based comparative genome hybridization = (Table=20 S2). At least one PTEN allele was thus found to be = inactivated in=20 10/13 (76.9 %) tumors, confirming that our study population was indeed = enriched=20 for PTEN deficient glioblastomas. Our failure to identify PTEN=20 mutations and/or PTEN DNA copy-number loss in some tumors with = PTEN=20 deficiency by IHC might be due to epigenetic mechanisms of PTEN = silencing,=20 incomplete sequencing coverage of the PTEN gene (exon 1 and the = 5=E2=80=B2=20 untranslated region [UTR] not sequenced) or the insensitivity of these = methods=20 when using heterogeneous tumor samples (the definition of PTEN = deficiency used=20 for the IHC test was >20% of tumor cells) [37].

Discussion

Rapamycin and other mTOR inhibitors have shown great promise as = anticancer=20 drugs in a spectrum of preclinical models, but it has been difficult to=20 demonstrate convincing clinical activity in single-agent trials using=20 conventional radiographic and clinical criteria for response [38].=20 Potential explanations include the largely cytostatic action of these = drugs in=20 the laboratory, uncertainty over dose and schedule, and lack of studies = to=20 evaluate the drug in subsets of patients most likely to respond based on = molecular phenotypes defined preclinically. The goal of this study was = to=20 evaluate directly rapamycin in patients whose tumors have defects in = PTEN, based=20 on preclinical findings originally generated by our group and others = showing=20 mTOR dependence in such models [9=E2=80=9315].=20 In designing the clinical experiment, we sought to validate the use of a = PTEN=20 assay for patient selection, document mTOR inhibition in tumor tissue = (of=20 particular importance for brain cancers), and gain preliminary evidence = of=20 antitumor activity. Glioblastoma was selected based on the high = frequency of=20 PTEN loss (40%), the clinical opportunity to collect tumor tissue at the = time of=20 salvage surgical resection, and the high proliferative index of these = tumors,=20 providing a robust endpoint for assessing antitumor effect. The intent = was to=20 generate information that could be used for more focused hypothesis = testing in=20 subsequent trials.

In the present study 165 patients were screened for PTEN status after = initial=20 surgical resection, then followed until relapse. Fifteen patients whose = initial=20 surgical samples stained negative for PTEN by immunohistochemistry were = treated=20 with rapamycin for about 1 wk before a planned salvage surgical = resection.=20 Short-term effects of rapamycin on mTOR inhibition in tumor cells and on = the=20 tumor proliferation index were determined by comparing = immunohistochemical=20 measures of these indices in the initial surgical sample (surgery 1 or = S1) to=20 the salvage resection sample (surgery 2 or S2). Rapamycin treatment led = to=20 substantial inhibition of tumor cell proliferation in seven of 14 = patients,=20 which correlated with the greatest magnitude of mTOR inhibition in tumor = tissue.=20 As predicted from preclinical studies [27,28],=20 rapamycin also led to the activation of Akt in some cases, and this = activation=20 was significantly correlated with shorter time-to-tumor progression.

The primary findings from this neoadjuvant rapamycin trial are = evidence of=20 antitumor activity using a short-term endpoint, novel insights into the=20 importance of achieving sufficient target inhibition, and clinical = evidence for=20 evaluating combination PI3-kinase/mTOR therapy to address negative = feedback. All=20 three findings should guide future clinical development of mTOR = inhibitors in=20 this disease. The Ki-67 response data demonstrate that rapamycin has = clear=20 antitumor activity in a subset of patients with PTEN loss. In addition = to=20 effects on tumor cell proliferation, two patients also had radiographic = evidence=20 of response. Patient 8 received an extended course of neoadjuvant = rapamycin (25=20 d) due to an intercurrent upper respiratory infection and had >50% = tumor=20 regression by magnetic resonance imaging prior to surgery (Figure=20 S8A). Patient 11 showed continued radiographic improvement during = the=20 postoperative phase of rapamycin treatment and died without evidence of = tumor=20 recurrence 538 d after starting rapamycin (Figure=20 S8B). The experience with patient 8 might justify a longer = neoadjuvant=20 treatment period to gain radiographic response data on all patients in=20 subsequent trials. While our trial was underway, a single-arm phase II = study of=20 the mTOR inhibitor CCI-779 reported that 20 of 65 patients with = recurrent=20 glioblastoma (36%) had radiographic improvement [39].=20 Of note, these patients were not evaluated prospectively for PTEN status = (no=20 molecular selection criteria), and CCI-779 was delivered weekly rather = than=20 daily based on a phase I experience that defined a maximum tolerated = dose using=20 this schedule [40,41].=20 In light of our findings about the magnitude of mTOR inhibition required = for=20 response (discussed below), this schedule raises concerns about the = presumed=20 lack of target coverage during nontreatment days. Nonetheless, the fact = that=20 both trials showed evidence of antitumor activity provides confidence = that=20 further investigation of mTOR inhibitors is warranted. The role of PTEN = loss in=20 defining sensitivity could be determined using a trial design in which = all=20 patients are initially eligible but sufficient numbers of PTEN negative = versus=20 PTEN positive are accrued to allow subset analysis.

Although intuitive, the correlation we found between the magnitude of = mTOR=20 inhibition and Ki-67 response was not anticipated from preclinical = studies.=20 Nearly complete inhibition of S6 phosphorylation is typically achieved = with=20 rapamycin treatment in xenografts and other mouse model systems; = therefore, most=20 studies of response have focused on defining genetic lesions (Pten, Akt, = Tsc,=20 Vhl, etc.) that affect mTOR dependence of tumor cells [38,42].=20 The surprising finding in this trial is that despite using doses of = rapamycin=20 sufficient to give low nM intratumoral levels, such doses do not = translate into=20 mTOR inhibition in all patients. Through ex vivo analysis of tumor cells = isolated at salvage surgery, we established that resistance in these = patients is=20 not cell intrinsic. Consistent with an extrinsic mechanism of rapamycin=20 resistance, our genomic survey of S2 tumor samples failed to identify=20 significant copy-number alterations within genes in the mTOR pathway = (FKBP12, S6=20 kinase 1, RAPTOR, RHEB, Akt) that might explain the observed rapamycin=20 resistance in vivo. This result contrasts with mechanisms of resistance = to other=20 kinase inhibitors (in chronic myeloid leukemia, gastrointestinal stromal = tumors,=20 and EGFR-dependent lung cancer), which often occurs through point = mutations in=20 the kinase target in tumor cells [43]=20 and raises the possibility that a larger fraction of PTEN null = glioblastomas=20 could be rapamycin-sensitive if more significant mTOR inhibition could = be=20 achieved.

The more challenging question is whether strategies can be developed = to=20 improve delivery of rapamycin directly to tumor cells and maximize mTOR=20 inhibition broadly across all patients. Oral delivery of significantly = higher=20 daily doses is an unlikely solution due to problems with tolerability=20 (mucositis, thrombocytopenia) seen in other diseases. Invasive = approaches such=20 as convection-enhanced delivery or implantation of drug-impregnated = wafers have=20 been used to treat glioblastoma patients with chemotherapeutic agents = and may be=20 considered. Alternatively, a better understanding of the reason = underlying the=20 failure to achieve mTOR inhibition in selected patients could point to a = solution. For example, if rapamycin in these patients is sequestered in = red=20 cells due to enhanced tumor vascularity, antiangiogenic agents such as=20 bevacizumab (already known to have activity in glioblastoma) [44]=20 may prevent sequestration and allow more efficient drug delivery. = Evaluation of=20 all of these approaches requires quantitative assessment of mTOR = activity and=20 highlights the need to develop broadly useful clinical tools for = quantitative=20 analysis of target inhibition. In the short term, it may be possible to = identify=20 the early Ki-67 responders using PET tracers such as=20 3=E2=80=B2-deoxy-3=E2=80=B2-18F-fluorothymidine (FLT) that can read out = proliferation=20 noninvasively [45].=20 Although such identification would not itself improve rapamycin delivery = to the=20 tumor cells, it could at least identify the subset of tumors in which = rapamycin=20 delivery appears to be problematic. Success here would also obviate the = need for=20 salvage surgery and could greatly expand eligibility of patients for = larger=20 trials.

There seems little doubt from the time-to-progression curves reported = here=20 and in the CCI-779 study that combination therapy is required for = significant=20 clinical impact. The challenge, of course, lies in choosing the most = promising=20 second drug from an almost infinite number of possibilities. Based on = earlier=20 work from us and others, combined EGFR/mTOR blockade is one logical = choice,=20 because PTEN loss predicts for resistance to EGFR inhibitors in patients = with=20 the mutant EGFRviii variant [18,46=E2=80=9348].=20 Another possibility is combined PI3K/mTOR blockade to prevent = rapamyin-induced=20 activation of Akt caused by loss of negative feedback [27,28].=20 The time-to-progression analysis in our study suggests that the = prognosis of=20 these patients is worse, therefore inhibitors that act upstream of Akt = may be=20 useful to prevent this complication. Indeed, one dual PI3K/mTOR = inhibitor has=20 shown superiority to a pure mTOR inhibitor in preclinical models [49].

Although the findings reported here are directly relevant to mTOR = inhibitors=20 in glioblastoma, the implication is that these drugs will have activity = in a=20 broad range of cancers with PI3K/Akt pathway = dysregulation=E2=80=94through PTEN loss,=20 PI3K p110=CE=B1 mutation, AKT gene amplification, or other mechanisms. = Recently, mTOR=20 inhibitors have shown clinical activity in metastatic kidney cancer, = where the=20 frequency of PTEN loss is low [50].=20 The molecular basis for sensitivity in this disease is unknown, but loss = of the=20 von Hippel-Lindau (VHL) tumor suppressor and subsequent mTOR-dependent = HIF-1=CE=B1=20 expression is one postulated mechanism [51].=20 For reasons similar to those articulated above for glioblastoma, = mTOR-based=20 combination therapies are also under consideration in kidney cancer. The = neoadjuvant clinical trial design described here should be easily = exportable to=20 other cancers in which experimental drug delivery can be timed prior to = a=20 planned surgical excision of tumor, and such an approach is consistent = with=20 recent national efforts to speed clinical development through novel = trial=20 designs [52].

Supporting Information

Figure=20 S1. Digital Scoring of Immunohistochemical Stains

Adjacent tissue sections from each tumor were stained with antibodies = against=20 Ki-67, phospho S6 ribosomal protein (S6RP), phospho-PRAS40, and PTEN=20 (unpublished data). Five areas per slide, each representing = approximately=20 500=E2=80=931,000 tumor cells, were selected for digital scoring. Image = conversion and=20 scoring was performed using Soft Imaging System Software. The = distribution of=20 immunoreactivity within these 2,500=E2=80=935,000 cells was graphed for = each sample as a=20 box plot. The =E2=80=9Cfold change=E2=80=9D (F.C.) in S6 = immunoreactivity (Table=20 2 and Figure=20 S2) was calculated for each tumor as the ratio between median = staining score=20 in the S2 and the median staining score in the S1 sample.

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Figure=20 S2. S6 Phosphorylation at Ser 235/236 in Matched S1/S2 = Tumor Tissue=20 Pairs

(A) Representative IHC staining results for pSer 235/236 S6. Shown = are=20 examples for a tumor with biochemical mTOR inhibitor resistance (patient = 2)=20 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=20 S1/S2 tumor samples from 14 patients in the rapamycin clinical trial = cohort (B)=20 and nine glioblastoma patients who did not receive rapamycin prior to S2 = (C).=20 For additional information regarding IHC scoring methodology, see Figure=20 S1 and Text=20 S1.

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Figure=20 S3. S6 Phosphorylation at Ser 240/244 in Matched S1/S2 = Tumor Tissue=20 Pairs

IHC-based quantification of S6 phosphorylation at Ser 240/244 in (A) = matched=20 S1/S2 tumor samples from 14 patients in the rapamycin clinical trial = cohort and=20 (B) matched S1/S2 tumor samples from nine glioblastoma patients who did = not=20 receive rapamycin prior to S2.

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Figure=20 S4. CD31 Immunostaining of Representative Tumor Tissue = Sections=20 from Rapamycin-Treated Tumors with Low (Patients 1 and 3) Versus High = (Patients=20 5 and 15) Intratumoral Rapamycin Concentrations

Arrows indicate CD31 IHC-positive intratumoral vasculature.

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Figure=20 S5. Frequency of Genomic Aberrations in S2 Samples by=20 Array-CGH

Genomic DNA microdissected from fresh frozen tumor samples (S2) was = subjected=20 to oligonucleotide microarray-based CGH (aCGH) analysis. Aberrations = were scored=20 using CGH Analytics Software (Agilent) using the ADM1 algorithm = (parameters=20 listed in Text=20 S1), and filtered to retain only aberrations with a log₂ = ratio=20 less than =E2=88=920.3 or greater than 0.3. The frequency of DNA = copy-number increases=20 (red, right of the zero axis for each chromosome) and losses (green, = left of the=20 axis) within the genome for the samples profiled is plotted in terms of=20 percentage of the samples analyzed.

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Figure=20 S6. Gene Copy-Number Alterations Stratified by pPRAS40=20 Response

CGH data for biopsies following rapamycin treatment were plotted = using=20 Cluster and TreeView Software (available at http://rana.lbl.gov/EisenSoftware.htm). DNA copy-number losses are = represented in=20 green, while gains are represented in red. Patient samples are = stratified by the=20 presence or absence of significant induction of pPRAS40 following = rapamycin=20 therapy as determined by IHC (see Figure=20 4B and Table=20 2).

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Figure=20 S7. IHC Scoring of PTEN Expression in Clinical Tumor = Samples from=20 Rapamycin Study Patients Compared to a Group of 44 Archival Glioblastoma = Tissue=20 Samples

(A) Comparison of two PTEN IHC scoring methods (x-axis: = manual=20 score; y-axis: image-guided digital score) in 44 archival = glioblastoma=20 samples. The manual score compares PTEN expression in tumor cells to = PTEN=20 expression in adjacent endothelial cells and assigns scores of 0 (absent = in=20 tumor cells), 1 (reduced in tumor cells relative to endothelial cells), = and 2=20 (tumor cells staining similar to endothelial cells). Digital PTEN scores = are=20 based on absolute values of PTEN immunoreactivity as determined through = an=20 image-guided software scoring system (Figure=20 S1). Digital PTEN scores were determined by an independent reviewer = without=20 knowledge of the manual PTEN score.

(B) Mean digital PTEN IHC score of tumors from the rapamycin study = group=20 before (S1) and during (S2) rapamycin therapy.

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Figure=20 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)=20 due to an intercurrent upper respiratory infection and experienced = >50% tumor=20 regression by magnetic resonance imaging.

(B) Patient 11 showed radiographically stable disease for >16 mo=20 postoperatively and died without evidence for tumor recurrence.

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Table=20 S1. Rapamycin-Related Adverse Events

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Table=20 S2. PTEN Copy Number (Log₂ Ratio) and = Missense=20 Mutations in Tumor Tissue

Genomic DNA was extracted from microdissected tumor cells. N/A: no = fresh=20 frozen tumor aliquot available for analysis. Log₂ ratios =E2=88=920.3 are consistent with DNA copy-number loss.

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Text=20 S1. Supplementary Methods

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Text=20 S2. Protocol

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Text=20 S3. CONSORT Checklist

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Acknowledgments

We thank Peter Lamb (Exelixis) for help with pPRAS40=20 immunohistochemistry.

Author = contributions. TFC, PN,=20 JT, PSM, IKM and CLS designed the study. TFC, KY, PN, KB, JD, SZ, TH, = YC, WW,=20 DY, LL, NM, RG, TO, MK, and CLS collected the data. TFC, KY, PN, KB, TH, = DY, SH,=20 PSM, IKM and CLS analyzed the data. TFC, PN, LL, NM, DB, AL, RG, and CLS = enrolled patients. TFC, KY, PN, KB, DY, PSM, IKM, and CLS contributed to = writing=20 the paper.

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