Originally published as JCO Early Release 10.1200/JCO.2007.14.5482 on March 10 2008

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Dose- and Schedule-Dependent Inhibition of the Mammalian Target of Rapamycin Pathway With Everolimus: A Phase I Tumor Pharmacodynamic Study in Patients With Advanced Solid Tumors

Josep Tabernero, Federico Rojo, Emiliano Calvo, Howard Burris, Ian Judson, Katharine Hazell, Erika Martinelli, Santiago Ramon y Cajal, Suzanne Jones, Laura Vidal, Nicholas Shand, Teresa Macarulla, Francisco Javier Ramos, Sasa Dimitrijevic, Ulrike Zoellner, Pui Tang, Michael Stumm, Heidi A. Lane, David Lebwohl, José Baselga

From the Medical Oncology and Pathology Departments, Vall d’Hebron University Hospital, Barcelona, Spain; Medical Oncology Department, Sarah Cannon Cancer Center, Nashville, TN; Medical Oncology Department, Royal Marsden Hospital, London, United Kingdom; Novartis Oncology, Basel, Switzerland; and Novartis Oncology, Florham Park, NJ

Corresponding author: José Baselga, MD, Medical Oncology Department, Vall d’Hebron University Hospital, P. Vall d’Hebron, 119-129, 08035, Barcelona, Spain; e-mail: jbaselga{at}vhebron.net

	ABSTRACT

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Purpose Everolimus is a selective mammalian target of rapamycin (mTOR) inhibitor with promising anticancer activity. In order to identify a rationally based dose and schedule for cancer treatment, we have conducted a tumor pharmacodynamic phase I study in patients with advanced solid tumors.

Patients and Methods Fifty-five patients were treated with everolimus in cohorts of 20, 50, and 70 mg weekly or 5 and 10 mg daily. Dose escalation depended on dose limiting toxicity (DLT) rate during the first 4-week period. Pre- and on-treatment steady-state tumor and skin biopsies were evaluated for total and phosphorylated (p) protein S6 kinase 1, eukaryotic initiation factor 4E (elF-4E) binding protein 1 (4E-BP1), eukaryotic initiation factor 4G (eIF-4G), AKT, and Ki-67 expression. Plasma trough levels of everolimus were determined on a weekly basis before dosing during the first 4 weeks.

Results We observed a dose- and schedule-dependent inhibition of the mTOR pathway with a near complete inhibition of pS6 and peIF-4G at 10 mg/d and 50 mg/wk. In addition, pAKT was upregulated in 50% of the treated tumors. In the daily schedule, there was a correlation between everolimus plasma trough concentrations and inhibition of peIF4G and p4E-BP1. There was good concordance of mTOR pathway inhibition between skin and tumor. Clinical benefit was observed in four patients including one patient with advanced colorectal cancer achieving a partial response. DLTs occurred in five patients: one patient at 10 mg/d (grade 3 stomatitis) and four patients at 70 mg/wk (two with grade 3 stomatitis, one with grade 3 neutropenia, and one with grade 3 hyperglycemia).

Conclusion Everolimus achieved mTOR signaling inhibition at doses below the DLT. A dosage of 10 mg/d or 50 mg/wk is recommended for further development.

	INTRODUCTION

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The mammalian target of rapamycin (mTOR) is a serine/threonine kinase, downstream of the phosphatidyl inositol 3'-kinase (PI3K)-AKT signaling pathway.^1,2 mTOR is activated in response to different stimuli such as nutrients and growth factor receptors.³ With the involvement of the PI3K-AKT pathway, mTOR relays a signal to translational regulators, specifically enhancing the translation of mRNAs encoding proteins essential for cell growth and cell cycle progression through G1 to S phase.^2,4,5 As a result of its central position within this signal transduction pathway, mTOR has been considered an important target for new anticancer drug development.^2,6,7 In support of its role in cancer, the mTOR pathway is aberrantly activated in around half of human tumors^1,4,8 and plays a critical role in angiogenesis.^9-14

mTOR signals to at least two downstream effectors, the translational repressor protein eukaryotic initiation factor 4E (elF-4E) binding protein 1 (4E-BP1) and the ribosomal protein S6 kinase 1 (S6K1).^15-17 Binding of 4E-BP1 to the translational activator eIF-4E is modulated by mTOR-dependent phosphorylation of multiple specific serine and threonine residues.^18,19 After a final phosphorylation at Ser65, 4E-BP1 dissociates from eIF4E, thereby allowing for the reconstitution of a translationally competent initiation factor complex with the involvement of eIF-4F²⁰ and eIF-4G.^21-23 eIF-4F activation results in the translation of a subset of capped mRNAs containing highly structured 5'-untranslated regions and encoding proteins involved in the G1 to S transition.^24,25

Everolimus (RAD001), an oral rapamycin derivative, has demonstrated potent antiproliferative effects against a variety of mammalian cell lines. Everolimus inhibits cytokine-driven lymphocyte proliferation,²⁶ as well as the proliferation of human tumor-derived cells, both in vitro in cell culture and in vivo in animal xenograft models.^27-31 As a result of these properties, everolimus has been developed as an immunosuppressant^32-34 and is now being developed as an anticancer agent. In the syngenic CA20948 rat pancreatic tumor model, everolimus has been shown to inhibit 4E-BP1 phosphorylation and S6K1 signaling in tumor and normal tissue.^29,35,36 Treatment with everolimus demonstrated equivalent activity with daily and intermittent schedules, this activity being dose-dependent for the two administration schedules.²⁹

Based on these considerations, the objectives of this trial were to assess the optimal dose and schedule of orally everolimus, administered weekly or daily, based on the safety profile and pharmacodynamic (PD) effects on mTOR dependent pathways in sequential tumor and skin biopsies. In addition, the effects of everolimus on tumor and skin specimens were correlated to plasma trough concentrations of the drug.

	PATIENTS AND METHODS

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Patient Population
Main inclusion criteria were histologically confirmed advanced tumors, unresponsive to standard therapy; presence of disease accessible to repetitive biopsies; age 18 years; life expectancy 12 weeks; WHO performance status of 0 to 2; and adequate bone marrow, hepatic, and renal function. All patients gave informed consent, and approval was obtained from the ethics committees at the participating institutions and regulatory authorities. The study followed the Declaration of Helsinki and good clinical practice guidelines.

Treatment and Dose Escalation Criteria
Everolimus was administered either as a single weekly oral dose (20, 50, and 70 mg) or as a continuous daily oral dose (5 and 10 mg) until progression. Initially, six to eight patients were to be enrolled to each dose level to have a minimum of four fully assessable patients for the PD end points of the study. Dose escalation proceeded in the absence of more than one of six patients with dose-limiting toxicity (DLT) in the first 28 days of treatment. If two or more patients presented DLT at a dose level, enrollment of patients to that dose level was discontinued and the immediately preceding dose level was considered the maximum tolerated dose for a given schedule. DLT was defined as any one of the following drug-suspected toxicities: grade 3 or higher National Cancer Institute Common Toxicity Criteria version 3.0 hematologic toxicity and grade 3 or higher nonhematologic toxicity despite the use of adequate/maximal medical intervention and/or prophylaxis.

Safety and Response Assessments
Routine clinical and laboratory assessments were conducted on a weekly basis during the first 4 weeks of treatment and thereafter every 2 weeks; after 6 months, assessments were conducted once per month. ECG monitoring was performed at baseline and at the fourth week of treatment after dosing. Adverse events were recorded, graded using the National Cancer Institute Common Toxicity Criteria version 3.0, and assessed by the investigator for any relationship with everolimus treatment. Objective measurement of tumor mass was assessed in accordance with the Response Evaluation Criteria in Solid Tumors criteria³⁷ after 8 weeks on treatment, and thereafter every 8 weeks.

Pharmacokinetic Analysis
A significant linear correlation between steady-state trough concentrations and overall exposure (area under the curve) to everolimus had been previously found when the drug was administered daily.³⁸ In this context, steady-state trough blood levels were chosen as a convenient monitoring pharmacokinetic (PK) parameter for this trial. Plasma levels of everolimus were determined on a weekly basis before dosing during the first 4 weeks of treatment.

The concentration of everolimus in whole blood was determined by liquid chromatography-mass spectrometry after liquid-liquid extraction. This method for blood sample has a lower limit of quantification of 0.3 ng/mL. Trough concentrations were reported as mean and standard deviation.

Pharmacodynamic Assessments
The PD effects by everolimus in tumor and skin were determined in all the patients included in the study. The timing of tumor and skin tissue biopsies was different in the two schedules: in the daily schedule at baseline and before dosing at day 2 in week 4; and in the weekly schedule at baseline, 24 hours and at day 6 postdose administration in week 4. The aim of the third biopsy in the weekly schedule was to assess whether everolimus-related inhibition was sustained between dosing. Processing of the samples, immunohistochemistry, and statistical analysis were performed as previously described (online-only Appendix A1).^39,40

Briefly, immunohistochemical analysis of total AKT, phosphorylated AKT at Ser473 (pAkt), total 4E-BP1, phosphorylated 4E-BP1 at Thr70 (p4E-BP1), phosphorylated eIF-4G at Ser1108 (peIF-4G), total S6, phosphorylated S6 at Ser235/236 and at Ser240/244 (pS6), and proliferation marker Ki-67 were performed in formalin-fixed paraffin-embedded sections from tumor and skin samples. Qualitative changes in the expression of markers were assessed in a blinded fashion. For quantitative analysis, the histochemical score (Hscore) was calculated to evaluate complete tumor sections and epidermis on skin samples at high magnification using a light microscopy, as previously described. Paired pretherapy and on-therapy samples were analyzed using the Wilcoxon rank test by SPSS Data Analysis Program version 10.0 (SPSS Inc, Chicago, IL). Statistical tests were conducted at the two-sided .05 level of significance. Pearson linear correlation was employed to examine the potential relationships between trough concentration values of everolimus and PD effects in tumor and skin samples.

	RESULTS

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Characteristics of the 55 patients included in the weekly schedule (n = 31) and the daily schedule (n = 24) are listed in Table 1. The distribution of patients across dose levels is present in Table 2.

Antitumor Activity
There was one partial response in a patient with a heavily pretreated metastatic colorectal cancer treated at 20 mg weekly that lasted 5.3 months (disease control during 9 months). Three patients presented stabilization of their disease for more than 5 months: one patient with an advanced renal cell cancer treated at 50 mg weekly lasting 14.6+ months and two patients with advanced breast cancer treated at 70 and 20 mg weekly lasting 10.7 and 5.6+ months, respectively.

PK
Table 3 depicts the trough concentrations of everolimus according to the different schedules and doses. Unweighted linear regression analysis of the relationship between dose and pharmacologic exposure was performed. Everolimus exhibited a linear dose-trough concentration relationship in the daily schedule (regression slope 0.96; 90% CI, 0.25 to 1.66), whereas this relationship could not be found in the weekly schedule due to insufficient serum sampling.

View larger version (17K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. Pharmacodynamic effects after everolimus treatment in all the patients included in the study with (A) assessable paired tumor (n = 30) and (B) skin samples (n = 43). Box-plots showing the expression at baseline and on-treatment for the following markers: pS6Ser235/6, pS6Ser240/4, peIF4GSer1108, p4E-BP1Thr70, pAktSer473 and Ki-67. Boxes indicate 90% of the values. Bold lines indicate the mean of the values. External lines indicate the complete range when beyond 90% of the values. Day 0: baseline sample; day 22: sample obtained at fourth week 24 hours after dosing.

View larger version (36K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 2. (A-E) Tumor pharmacodynamic effects in patients treated in the daily (treated at 5 and 10 mg) and in the weekly schedules (treated at 20, 50, and 70 mg). The lines show the individual evolution of the expression of the biomarkers (pS6Ser235/6, pS6Ser240/4, peIF4GSer1108, p4E-BP1Thr70, pAktSer473, and Ki-67) at baseline and on-treatment. In the daily schedule samples were obtained on day 0 (baseline) and on day 22 (on-treatment). In the weekly schedules samples were obtained on day 0 (baseline), on day 22 (on-treatment, 24 hours after dosing) and on day 27 (on-treatment, 144 hours after dosing). The aim of this third biopsy was to assess the persistent effect of everolimus administered on a weekly basis.

View larger version (91K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 3. Biomarker expression changes in the tumor (pS6Ser235/6, peIF4GSer1108, p4E-BP1Thr70, and pAktSer473) and in the skin (pS6Ser235/6, peIF4GSer1108, and p4E-BP1Thr70) between baseline (day 0) and on-treatment (day 22) in two selected patients with advanced breast cancer treated at 10 mg daily and 50 mg weekly.

	DISCUSSION

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This phase I study was aimed at identifying a recommended dose and schedule of everolimus in patients with cancer defined by the achievement of a complete and sustained inhibition of mTOR dependent-signaling pathways on tumor and skin. Our approach was based on the existing correlation in preclinical models between antitumor activity and mTOR-signaling inhibition. In a CA20948 syngenic rat pancreatic tumor model, doses of everolimus that inhibited tumor growth also dramatically inhibited mTOR signaling in tumor, skin, and peripheral blood mononuclear cells (PBMCs).²⁹ In this model, a decrease in p4E-BP1 (Thr70) and an increase in eIF-4E and 4E-BP1 association were consistently observed in all three tissues. Striking reductions in pS6 (Ser240/244) were demonstrated only in tumor, as baseline pS6 levels in skin and PBMCs were too low for immunoblot detection. By contrast, in vitro kinase assay, using 40S ribosomal subunits as a substrate, revealed a significant and consistent inhibition of S6K1 in tumor, skin, and PBMCs.²⁹ The relationship between S6K1 inhibition in tumor and PBMCs has also been demonstrated in vivo with temsirolimus.⁴¹ Taken together, these data demonstrate that both 4E-BP1 and S6K1 pathways are affected in tumor, skin, and PBMC samples obtained from preclinical models after treatment with mTOR inhibitors. We, therefore, included PD evaluation of molecular markers exploring the 4E-BP1 pathway (total and p4E-BP1, peIF-4G) and the S6K1 pathway (total and pS6) in the tumor and in the skin. In addition, it has been shown that mTOR inhibition can induce upstream insulin-like growth factor 1 receptor (IGF-1R) signaling resulting in AKT activation in cancer cells.^39,42 This phenomenon has been suggested to play a role in the attenuation of cellular responses to mTOR inhibition.^2,43 In order to study whether AKT activation was also observed in vivo, we incorporated an assessment of total and pAKT as well as the effects of treatment on proliferation.

There are several key findings in our study. We observed a tight correlation in the degree of everolimus-induced inhibition of mTOR signaling in the skin and in the tumors. Hence, skin may be useful as a surrogate tissue for PD evaluation with mTOR inhibitors. This has implications for future studies with novel agents interfering with the mTOR pathway. We identified a daily and a weekly dose level that resulted in maximal inhibition of the pathway. In the daily schedule, pS6 was completely inhibited at the 5- and 10-mg doses. However, inhibition of peIF-4G was only complete at the 10-mg dose level. Although p4E-BP1 expression was also lower at the higher dose level, it is felt to be a marker less reproducible of mTOR inhibition (W. Sellers, personal communication, January 2006).⁴² In the weekly schedule, complete pS6 inhibition was again seen at all the studied dose levels. However, complete and prolonged inhibition of peIF-4G was observed only at doses 50 mg. Because peIF-4G, together with pS6, have been suggested as the best indicators of mTOR blockade,^35,36 we considered optimal dose levels of everolimus those that inhibited both markers in full. The upregulation of tumor AKT phosphorylation that was observed with both daily and weekly everolimus schedules raises the question on whether it may attenuate the clinical activity of this agent. It should be noted that the increase in pAKT is not tumor specific, as we observed it also in skin. In addition, it did not occur in the tumors of all patients (approximately 50% in this study) and it was not always sustained in between doses in patients treated on the weekly schedule. Upregulation of AKT signaling occurs in experimental tumor models known to be very sensitive to mTOR inhibition, including the CA20948 model described earlier.²⁹ Nevertheless, it is likely that strategies aimed at preventing the activation of this IGF-1R mediated feed loop could result in an enhancement of the antitumor activity of mTOR inhibitors. We have observed that inhibition of the IGF-1R pathway with an anti-IGF-1R antibody given in combination with mTOR inhibitors is highly synergistic^39,42 and we are planning to explore this combination in the clinic.

Oral everolimus is characterized by a rapid and a moderate oral absorption (oral availability of approximately 30%), and the terminal half-life is around 30 hours.^{32-34,38,44,45} We have evaluated potential relationships between trough levels and treatment-related changes in the activated signaling markers in both tumor and skin samples in patients treated in the daily schedule. There was a trend for more profound decreases in tumor peIF-4G and p4E-BP1 levels in those patients with higher plasma exposure to everolimus. These data, together with the more profound dose effect in tumor discussed earlier, suggest that monitoring the 4E-BP1/eIF-4G downstream module of mTOR signaling, rather than the S6K1 pathway, may be more valuable for dose evaluation in the clinic.

Oral everolimus was satisfactorily safe and well tolerated, with the exception of the weekly dose of 70 mg which had high frequency of grade 3 toxicities. Differences in reported toxicity between the daily and the weekly schedules were marginal and of little clinical relevance. The observed toxicity profile revealed the occurrence of well-known drug class effects of rapamycin and its derivatives, including stomatitis/oral mucositis, skin rash/erythema, and metabolic abnormalities.^46,47

In summary, this phase I study with oral everolimus has shown that this agent can be safely administered with the two different schedules. Based on PK/PD modeling efforts associated with another everolimus phase I trial in patients with advanced cancer,⁴⁸ where PBMC-derived S6K1 activity was used to establish a concentration-effect model, as well as the clinical safety profile and the tumor PD analysis presented herein, we recommend everolimus treatment at either 10 mg/d or 50 mg/wk. Moreover, also consistent with previous PK/PD modeling,⁴⁸ our tumor PD analysis shows a different pattern for the two schedules, with mTOR-pathway inhibition being more profound (and maintained) with the daily schedule. Whether these PD properties are critical enough to suggest a preference for the daily rather than the weekly schedule cannot be answered in the context of this phase I study. One point to consider is that flexibility of dosing could be extremely valuable in the context of drug combination scenarios which are being aggressively followed for mTOR inhibitors.^7,49,50 To our knowledge, this study is the first comprehensive study with an mTOR inhibitor, using a detailed tumor and skin PD analysis to provide evidence to support dosage and regimen in subsequent phase II-III studies. This study provides assurance of primary drug activity at dosages not necessarily limited by toxicity, providing direction for the further development of mTOR inhibitors like everolimus in patients with cancer.

	AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

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Although all authors completed the disclosure declaration, the following author(s) indicated a financial or other interest that is relevant to the subject matter under consideration in this article. Certain relationships marked with a "U" are those for which no compensation was received; those relationships marked with a "C" were compensated. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.

Employment or Leadership Position: Katharine Hazell, Novartis Pharmaceuticals (C), IT Operations (C); Nicholas Shand, Novartis Pharmaceuticals (C); Sasa Dimitrijevic, Novartis Pharmaceuticals (C); Ulrike Zoellner, Novartis Pharmaceuticals (C); Pui Tang, Novartis Pharmaceuticals (C); Michael Stumm, Novartis Pharmaceuticals (C); Heidi A. Lane, Novartis Pharmaceuticals (C); David Lebwohl, Novartis Pharmaceuticals (C) Consultant or Advisory Role: Josep Tabernero, Novartis Pharmaceuticals (C); Ian Judson, Novartis Pharmaceuticals (C); Suzanne Jones, Novartis Pharmaceuticals (C); José Baselga, Novartis Pharmaceuticals (C) Stock Ownership: Katharine Hazell, Novartis Pharmaceuticals, Novartis Pharmaceuticals; Nicholas Shand, Novartis Pharmaceuticals; Sasa Dimitrijevic, Novartis Pharmaceuticals; Ulrike Zoellner, Novartis Pharmaceuticals; Michael Stumm, Novartis Pharmaceuticals; Heidi A. Lane, Novartis Pharmaceuticals; David Lebwohl, Novartis Pharmaceuticals Honoraria: Howard Burris, Novartis Pharmaceuticals; Ian Judson, Novartis Pharmaceuticals Research Funding: Josep Tabernero, Novartis Pharmaceuticals; Howard Burris, Novartis Pharmaceuticals; Heidi A. Lane, Novartis Pharmaceuticals; José Baselga, Novartis Pharmaceuticals Expert Testimony: None Other Remuneration: None

	AUTHOR CONTRIBUTIONS

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Conception and design: Josep Tabernero, Federico Rojo, Ian Judson, Katharine Hazell, Nicholas Shand, Sasa Dimitrijevic, Ulrike Zoellner, José Baselga

Financial support: David Lebwohl

Administrative support: Katharine Hazell, Heidi A. Lane

Provision of study materials or patients: Josep Tabernero, Federico Rojo, Emiliano Calvo, Howard Burris, Erika Martinelli, Suzanne Jones, Laura Vidal, Teresa Macarulla, Francisco Javier Ramos, Michael Stumm, Heidi A. Lane, José Baselga

Collection and assembly of data: Josep Tabernero, Federico Rojo, Emiliano Calvo, Howard Burris, Katharine Hazell, Erika Martinelli, Santiago Ramon y Cajal, Suzanne Jones, Laura Vidal, Teresa Macarulla, Francisco Javier Ramos, Heidi A. Lane, José Baselga

Data analysis and interpretation: Josep Tabernero, Federico Rojo, Emiliano Calvo, Howard Burris, Ian Judson, Katharine Hazell, Erika Martinelli, Santiago Ramon y Cajal, Suzanne Jones, Laura Vidal, Nicholas Shand, Teresa Macarulla, Francisco Javier Ramos, Sasa Dimitrijevic, Ulrike Zoellner, Pui Tang, Michael Stumm, Heidi A. Lane, David Lebwohl, José Baselga

Manuscript writing: Josep Tabernero, Federico Rojo, Emiliano Calvo, Ian Judson, Katharine Hazell, Nicholas Shand, Sasa Dimitrijevic, Ulrike Zoellner, Heidi A. Lane, David Lebwohl, José Baselga

Final approval of manuscript: Josep Tabernero, Federico Rojo, Emiliano Calvo, Howard Burris, Ian Judson, Katharine Hazell, Erika Martinelli, Santiago Ramon y Cajal, Suzanne Jones, Laura Vidal, Nicholas Shand, Teresa Macarulla, Francisco Javier Ramos, Sasa Dimitrijevic, Ulrike Zoellner, Pui Tang, Michael Stumm, Heidi A. Lane, David Lebwohl, José Baselga

	Appendix

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Pharmacodynamic assessments.
Tissue samples were immediately placed into a 4°C precooled 4% neutral buffered formalin solution and fixed for 8 to 16 hours (and no longer than 24 hours). Fixed specimens were further processed through routine specimen dehydration using graded ethanols to xylene. Specimens were embedded in paraffin wax under vacuum at 60°C and stored until analysis at the Molecular Oncology Laboratory at Vall d’Hebron University Hospital.

Immunohistochemical analysis of total AKT, phosphorylated Akt at Ser473 (pAkt), total 4E-BP1, phosphorylated 4E-BP1 at Thr70 (p4E-BP1), phosphorylated eIF-4G at Ser1108 (peIF-4G), total S6, phosphorylated S6 at Ser235/236 and at Ser240/244 (pS6), and proliferation marker Ki-67 were performed in formalin-fixed paraffin-embedded sections from tumor and skin samples. Rabbit polyclonal antitotal Akt #9272 and antipAkt #9277, antitotal 4E-BP1 #9452 and antip4E-BP1 #9455, antipeIF-4G #2441 and antipS6 #2211 (Ser235/236) and #2215 (Ser240/244) from Cell Signaling Technology, Beverly, MA; mouse monoclonal anti-S6 from Novartis Pharma AG, Basle, Switzerland, and anti-Ki67 clone MIB1 #M7240 from Dako, Carpinteria, CA were used. We validated these primary antibodies in breast cancer samples from the Vall d’Hebron University Hospital tumor bank, comparing the expression between western blot and immunohistochemistry studies (Rojo F, Najera L, Lirola J, et al: Clin Cancer Res 13:81-89, 2007). Immunostaining was performed on 4 µm tissue sections mounted onto positively charged glass slides. After removal of paraffin in xylene and graded alcohols, sections were hydrated. Epitope retrieval was performed in a water bath at 95°C in 10 mmol/L citrate buffer, pH6 for 40 minutes. After target retrieval, endogenous peroxidase was blocked by immersing the sections in 0.03% hydrogen peroxide for 5 minutes. Incubation with primary antibodies was performed at room temperature for 1 hour using the following dilutions: Akt 1:50, pAkt 1:50, 4E-BP1 1:50, p4E-BP1 1:50, p eIF-4G 1:50, S6 1:500, p S6 Ser235/6 1:80, p S6 Ser 240/4 1:50 and Ki67 1:100. Appropriate antirabbit or antimouse peroxidase-conjugated goat polymers were used to detect antigen-antibody reaction (En Vision+ System; DAKO) for 30 minutes at room temperature. Sections were visualized with 3,3'-diaminobenzidine as a chromogen for 5 minutes and counterstained with Mayer's hematoxylin. Two negative control rabbit polyclonal immunoglobulins (Biogenex, San Ramon, CA; and Santa Cruz Biotech) and a negative control mouse monoclonal immunoglobulin (Biogenex) were also used.

Qualitative changes in the expression of markers were assessed in a blinded fashion. For quantitative analysis, the Histo score (Hscore) was calculated to evaluate complete tumor sections and epidermis on skin samples at high magnification using a light microscopy.

The Hscore was determined by estimation of the percentage of tumor or skin cells positively stained with low, medium, or high staining intensity. The final score was determined after applying a weighting factor to each estimate. The following formula was used: Hscore = (low %) x 1 + (medium %) x 2 + (high %) x 3. Scoring of the proliferation marker Ki-67 was assessed by estimation of a ratio of tumor cells positively stained for Ki-67 versus the total number of tumor cells. This result was expressed as a percent of tumor cells stained. For a positive score, cytoplasmic staining was required for total AKT, pAKT at Ser473, total 4E-BP1, p4E-BP1 at Thr70, peIF-4G at Ser1108, total S6 and pS6 at Ser235/236 and at Ser240/244, and nuclear staining for Ki-67. Paired pretherapy and on-therapy samples were analyzed using the Wilcoxon rank test by SPSS Data Analysis Program version 10.0 (SPSS Inc, Chicago, IL). Statistical tests were conducted at the two-sided .05 level of significance. Pearson linear correlation was employed to examine the potential relationships between trough concentration values of everolimus and PD effects in tumor and skin samples.

	ACKNOWLEDGMENTS

 
We thank Jurgen Mestan, PhD, Novartis Institutes for BioMedical Research Oncology Basel for the provision of the S6 protein monoclonal.

	NOTES

 
Submitted September 20, 2007; accepted November 16, 2007.

Presented in part at the 41st Annual Meeting of the American Society of Clinical Oncology, Orlando, FL, May 13-17, 2005; and at the 13th European Cancer Conference, Paris, France, October 30 to November 3, 2005.

Authors’ disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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