Combination Of Everolimus Research Articles

Abstract OT1-1-02: PI3K-Akt-mTOR pathway analysis to obtain further insight in the efficacy of everolimus in combination with exemestane in metastatic, ER-positive breast cancer: A Dutch breast cancer research group (BOOG) study

Abstract Background In patients with hormone receptor-positive breast cancer, activation of the PI3K-Akt-mTOR pathway is associated with resistance against endocrine therapy. Previous research has shown that genetic aberrations in this pathway occur frequently, including mutation and/or amplification in PI3K subunits or PI3K effectors as well as loss of lipid phosphatases (Fu X, et al. The Breast; 2013). Central review of the BOLERO-2 randomized phase III trial in which patients refractory to a non-steroidal aromatase inhibitor were randomized between exemestane combined with the mTOR inhibitor everolimus versus exemestane and placebo has shown a progression-free survival (PFS) of, respectively, 11.0 and 4.1 months [hazard ratio = 0.38 (95% confidence interval 0.31-0.48; log-rank P <0.0001)] (Yardley DA, et al. Adv Ther; 2013). The combination, however, is known to cause more adverse events and is associated with additional costs as compared to exemestane alone. In the present study we will explore whether there are biomarkers that might indicate which patients most likely benefit from co-targeting PI3K and ER pathways. Trial design/Aims This is a Dutch prospective, open-label, single-arm, investigator-initiated, multicenter trial in which approximately 30 hospitals will participate. A total of 175 patients will be included for baseline blood sampling and archival tumor tissue collection. From 50 patients, a fresh tumor biopsy is required at baseline and from 30 out of 50, another tumor biopsy will be collected upon progressive disease. Exploratory biomarker assessment includes immunohistochemistry (total and phosphorylated PI3K, AKT, mTOR, p70S6K and 4EBP1), tissue phosphoproteomics and circulating tumor DNA (mutations). The results of the biomarker analysis will be compared with clinicopathological characteristics and PFS. Eligibility Postmenopausal patients with hormone receptor-positive, HER2-negative, locally advanced or metastatic breast cancer, refractory to anastrozole or letrozole will be included. No previous treatment with exemestane or mTOR inhibitor for advanced disease is allowed and Informed consent must be signed before enrollment. Statistical methods Since the majority of the tests involve the use of new techniques, the study will be mainly explorative in design. The association between potential biomarkers and clinicopathological characteristics will be tested using Fisher exact test or the Mann-Whitney test. PFS curves will be drawn using the Kaplan-Meier method. PFS in association with potential biomarkers will be tested using Cox proportional hazard regression analysis. Present and target accrual Recently, the study has been opened for inclusion. A period of 2 years is planned for patient enrollment. Up to May 2014, two patients were included. ClinicalTrials.gov identifier NCT02109913 Financial support is received from Novartis, the Netherlands. Citation Format: Dinja T Kruger, Karin Beelen, Connie R Jimenez, Maurice PHM Jansen, Stefan Sleijfer, Sabine C Linn, Epie Boven. PI3K-Akt-mTOR pathway analysis to obtain further insight in the efficacy of everolimus in combination with exemestane in metastatic, ER-positive breast cancer: A Dutch breast cancer research group (BOOG) study [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr OT1-1-02.

Abstract OT1-2-03: The DETECT-study concept: Treatment based on the phenotype of circulating tumor cells in HER2-negative metastatic breast cancer

Abstract Background: The prognostic impact of circulating tumor cells (CTC) in metastatic breast cancer (MBC) is well demonstrated. The role of CTCs in predicting specific treatment response and the importance of CTC phenotypes for therapeutic decisions will be investigated within the DETECT-study concept. Trial Design and eligibility criteria: The DETECT studies are prospective, multicenter, open-label clinical trials designed for patients with HER2-negative MBC and evidence of CTCs in the peripheral blood. DETECT III is a two-arm study for patients with HER2-positve CTCs, randomized to physician’s choice therapy (chemotherapy or endocrine treatment) with or without additional HER2-targeted treatment with lapatinib. DETECT IV combines tow single-arm studies aimed at patients with HER2-negative CTCs. Postmenopausal patients with hormone-receptor-positive MBC will be treated with the mTOR-inhibitor everolimus in combination with an endocrine therapy of physician’s choice (everolimus cohort), whereas patients with triple-negative or hormone-receptor-positive MBC and indication to chemotherapy will receive eribulin (eribulin cohort). Specific aims: The primary objective of the trials is to estimate the clinical efficacy of treatments, assessed by the CTC clearance rate for DETECT III and by progression-free survival (PFS) for DETECT IV. Methods: Prevalence of CTCs at various time points as well as the HER2 status of CTCs are assessed using the FDA-approved CellSearch System (Veridex, USA). After immunomagnetic enrichment with an anti-EpCam-antibody, cells were labelled with anti-CK8/18/19 and anti-CD45 antibodies to distinguish epithelial cells from leucocytes. A fluorescein conjugate antibody with anti-CK-Fluorescein Isothiocyanate (FITC) was used for HER2 phenotyping. The cut-off for CTC-positivity was 1 CTC and for HER2 1 CTC with strong HER2-staining (+++). Survival endpoints will be estimated using the Kaplan-Meier method. Present and target accrual: Overall, about 2000 patients with HER2-negative MBC will have to be screened for CTCs to be able to recruit 228 patients with HER2-positive CTCs for DETECT III (which started in February 2012), 400 patients with HER2-negative CTCs for DETECT IV- everolimus cohort (which started in December 2013) and 120 patients for DETECT IV- eribulin cohort (which will start in the second half of 2014). 907 patients have been recruited for CTC screening until June 2014. Perspectives: One screening for CTCs offers different treatment options for patients with HER2-negative MBC and evidence of CTCs within the DETECT-study concept. DETEC III is the first study to investigate a personalized targeted treatment based on the phenotype of CTCs. The addition of a HER2-targeted therapy in case of HER2-positive CTCs is innovative and in case of success will lead to new treatment strategies in MBC. DETECT IV complements DETECT III with regard to additional therapy indications. Citation Format: Bernadette AS Jaeger, Susanne Albrecht, Fabienne Schochter, Carola A Melcher, Carsten Hagenbeck, Thomas WP Friedl, Brigitte Rack, Volkmar Müller, Peter A Fasching, Wolfgang Janni, Tanja Fehm. The DETECT-study concept: Treatment based on the phenotype of circulating tumor cells in HER2-negative metastatic breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr OT1-2-03.

Abstract P3-06-04: 4EVER: The impact of mTOR inhibition on bone health in postmenopausal women with hormone receptor positive (HR+) advanced breast cancer treated with everolimus (EVE) in combination with exemestane (EXE)

Abstract Introduction: The BOLERO-2 study showed significant doubling of PFS benefit with the addition of EVE to EXE in postmenopausal women with hormone receptor–positive advanced breast cancer progressing after non-steroidal aromatase inhibitor (NSAI) therapy. The BOLERO-2 bone sub study indicated an immediate positive influence of EVE on bone health after 6 and 12 weeks of treatment. To further investigate the longer term effect of mTOR inhibition on bone health, we included this exploratory objective in the phase IIIb, multi-center, open label study 4EVER for postmenopausal women with hormone receptor positive advanced breast cancer treated with EVE +EXE. Methods: From May 2012 to November 2012 bone biomarker samples of 247 postmenopausal women with metastatic or locally advanced, hormone receptor positive, HER2 negative breast cancer refractory to NSAI were collected within this phase IIIb study. Here, we report the results of the planned exploratory analysis of biomarkers of bone turnover. The objective of the exploratory biomarker analysis of bone resorption and formation was to assess the effect of EVE on changes of biomarker levels of bone metabolism (CTX, P-I-NP, osteocalcin, vitamin D, testosterone, estradiol, DHEAS, SHBG, PTH,TSH and FSH) from day 1 to weeks 4, 12 and 24 to confirm potential protective effects of EVE on bone via inhibition of bone osteoclast resorption. Descriptive statistics were used to summarize the single bone resorption markers by visit and difference to baseline. Results: Trial data base lock will occur in late June 2014, therefore, the final biomarker analysis of bone resorption and formation and their influence on ORR, PFS and safety will be presented at SABCS 2014. The preliminary analysis on the changes of bone marker levels from baseline to week 24 included 247 patients. The measured changes from baseline to week 24 in biomarker levels revealed increasing levels of testosterone, DHEAS and FSH, while SHBG and PTH were significantly decreased from baseline to week 24. The combination of EVE to EXE resulted in stabilization of bone health as documented by a decrease of absolute P-I-NP levels at week 24 compared to baseline as well as a stabilization of bone resorption as measured by CTX. Conclusion: Our first preliminary results (data cut off 15 Nov 2013) on the changes in bone marker levels supported the hypothesis of EVE having direct impact on bone health, suggesting a decrease of bone turnover and a reversal of the increase in bone resorption associated with aromatase inhibitor therapy. The final analysis of the 4EVER sub study on bone marker levels in correlation with the efficacy data will provide more detailed insights into possible benefits of the combination of EVE plus EXE. Citation Format: Peyman Hadji, Hans Tesch, Oliver Stoetzer, Thomas Decker, Christian M Kurbacher, Romy Neumeister, Frederik Marmé, Andreas Schneeweiss, Christoph Mundhenke, Andrea Distelrath, Peter A Fasching, Micheal P Lux, Diana Lüftner, Wolfgang Janni, Mathias Muth, Julia Kreuzeder, Claudia Weiss, Eva-Maria Grischke. 4EVER: The impact of mTOR inhibition on bone health in postmenopausal women with hormone receptor positive (HR+) advanced breast cancer treated with everolimus (EVE) in combination with exemestane (EXE) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-04.

Abstract P4-01-13: CTC enumeration and characterization has predictive and prognostic implications in patients with metastatic breast cancer treated with exemestane plus the mTOR inhibitor everolimus

Abstract Background: The utility of CTC enumeration in predicting patient (pt) outcome has been demonstrated in metastatic breast cancer (MBC) treated with chemotherapy or endocrine therapy. In this study we evaluated the clinical impact of CTC assessment in terms of both enumeration and characterization in breast cancer pts treated with exemestane plus everolimus. Patients and methods: Thirty-nine pts with hormone receptor (HR)-positive, HER2-negative MBC, received exemestane plus everolimus. CTC enumeration in peripheral blood (7.5 ml) was performed before treatment (n=39), post cycles 1 (n=39) and 3 (n=29), on disease re-evaluation and on relapse, whichever occurred first, using the CellSearch System. CTC characteristics were determined at the same time points by immunofluorescence (IF) analysis of PBMC cytospins (106 cells), triple stained with pancytokeratin (CK) antibody along with Ki67 and M30 as proliferation and apoptosis markers, respectively, using the Ariol System. Patients were assessed by CT scans and bone scan, every 3 months or as clinically indicated. Results: At the cut-off of ≥ 1 CTC, 25 of 39 (64%) pts had detectable CTCs at baseline, 12 (31%) of 39 post-1st and 10 (34.5%) of 29 post-3rd cycle. Ten (25.6%) pts remained CTC(+) and 12 (30.8%) CTC(-) both at baseline and post-1st cycle; 15 (38.5%) CTC(+) pts turned to CTC(-) and 2 (5%) CTC(-) turned to (+). CTC positivity after the first cycle was associated with shorter median progression-free survival (PFS) compared to CTC(-) status (3.9 vs 8 mo, p=0.031). Shorter PFS was also recorded for pts that remained CTC(+) at both time points compared to all other (p=0.02). At the cut-offs of ≥ 2 and ≥ 5 CTCs, 16 (41%) and 9 (23%) pts were CTC(+) at baseline, respectively; post-1st cycle, 7 (18%) and 4 (10%) pts were CTC(+) (at ≥ 2 and ≥ 5 CTCs, respectively). Post-3rd cycle the positivity rate was 17% for both cut-offs and these pts had significantly shorter PFS compared to CTC(-) pts (3.7 vs 8.7 months, p=0.048). Efficacy assessment revealed partial response in 3 (7.7%) pts, stable disease in 27 (69.23%) and progressive disease (PD) in 8 (20.5%); 1 pt was non-evaluable for response. Among pts determined CTC(+) post-1st cycle (cut-off ≥ 2 CTCs), 57% progressed compared to 13% of CTC(-) pts (p=0.02). In addition, at the post-3rd cycle evaluation, pts with PD had significantly higher CTC counts compared to non-progressors (mean ± SEM; 10 ± 5.78/pt vs 1.62±0.83/pt, p=0.027). By the use of IF 43%, 44% and 40% of CTC(+) pts had proliferative [Ki67(+)/M30(-)] CTCs at baseline, post -1st and -3rd cycles, respectively (cut-off ≥ 1 CTC); 67%, 50% and 50% of those pts, respectively, experienced PD. Apoptotic [Ki67(-)/M30(+)] CTCs were detected in 14%, 22% and 60% of CTC(+) pts at baseline, post -1st and -3rd cycles, respectively; none of the pts with apoptotic CTCs experienced PD. Conclusions: CTC enumeration and characterization in terms of proliferation and apoptosis during the course of treatment has significant predictive and prognostic implications in patients with MBC receiving the combination of exemestane plus everolimus. Citation Format: Sofia Agelaki, Dimitris Mavroudis, Maria Spiliotaki, Eleni Politaki, Maria A Papadaki, Stella Apostolaki, Christos Nikolaou, Vassilis Georgoulias. CTC enumeration and characterization has predictive and prognostic implications in patients with metastatic breast cancer treated with exemestane plus the mTOR inhibitor everolimus [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-13.

Abstract P2-05-03: Anastrozole and everolimus in hormone receptor-positive metastatic breast cancer: Safety profile, activity and associations of molecular alterations in the PI3K/AKT/mTOR pathway

Abstract Background Combining aromatase inhibitors with PI3K/AKT/mTOR inhibitors in patients with hormone receptor (HR)-positive metastatic breast cancer has demonstrated clinical efficacy. There is limited data on associations between molecular signatures and activity. Patients and Methods We evaluated the combination of anastrozole and everolimus in 56 patients with HR-positive, metastatic breast cancer. The primary objective was to establish safety and maximum tolerated dose (MTD). Dose limiting toxicities (DLTs) were defined as serious grade 3 or 4 toxicities related to treatment that occurred during cycle 1. Dose level 1 was anastrozole 1mg PO QD and everolimus 5 mg PO QD and dose level 2 was anastrozole 1 mg PO QD and everolimus 10 mg PO QD (a dose level -1 included everolimus 2.5 mg PO QD). Secondary endpoints included evaluation of antitumor activity and molecular associations with response. When tissue was available, Next Generation Sequencing (NGS) was performed using genomic libraries selected for all exons of 236 (or 182) cancer-related genes sequenced to average depth of >500× in a CLIA laboratory (Foundation Medicine, Cambridge, MA, USA). An analysis was then performed for all classes of genomic alterations. Results The median age was 59 (range, 37-82) and the median number of prior therapies in the metastatic setting was 3 (range, 0-13). The initial oral daily dose of anastrozole 1 mg oral and everolimus 10 mg PO daily was well tolerated. Five dose-limiting toxicities (DLTs) were seen at full doses, including grade 3 thrombocytopenia (1 patient), grade 3 neutropenia (1 patient), grade 3 increased liver enzymes (1 patient), grade 3 hyperglycemia (1 patient) and, grade 3 mucositis (1 patient). The most common grade 3 or 4 treatment-related toxicities were neutropenia (5%), increased liver enzymes (5%), and hyperbilirubinemia (3%). Of the 56 patients on study, 36 were tested for at least one molecular alteration in the PI3K/AKT/mTOR pathway. Twelve of these 36 patients had NGS analysis of their tumor tissue. Eighteen of 36 patients (50%) tested had at least one alteration in the pathway, including mutations in PIK3CA (n=16), PIK3R1 (n=1), and AKT1 (n=2); PTEN protein loss (n=1); and, AKT3 amplification (n=1). Sixteen of 56 evaluable patients (29%) achieved stable disease (SD) /partial response (PR)/complete response (CR) ≥ 6 months (n = 3 (5%) with PR/CR). Thirteen of the 16 patients who achieved SD/PR/CR ≥ 6 months were tested for a genetic alteration in PI3K/AKT/mTOR pathway and 7 of these patients (54%) had at least one alteration in the pathway, including mutations in PIK3CA (n=6), PIK3R1 (n=1), and AKT1 (n=1); PTEN loss (n=1); and AKT3 amplification (n=1). Conclusions Combination anastrozole 1 mg and everolimus 10 mg is well tolerated and is active in heavily-pretreated patients with HR-positive breast cancer. The presence of a molecular alteration in the PI3K/AKT/mTOR pathway did not predict for clinical activity of this combination. Citation Format: Jennifer J Wheler, Filip Janku, Stacy L Moulder, Aung Naing, Sarina A Piha-Paul, Gerald S Falchook, Ralph G Zinner, Apostolia M Tsimberidou, Siqing Fu, David S Hong, Johnique T Atkins, Roman Yelensky, Vince Miller, Philip J Stephens, Vincente Valero, Funda Meric-Bernstam, Razelle Kurzrock. Anastrozole and everolimus in hormone receptor-positive metastatic breast cancer: Safety profile, activity and associations of molecular alterations in the PI3K/AKT/mTOR pathway [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-05-03.

Abstract P2-03-05: The heterogeneous clinical behavior of luminal breast cancers is associated with different mutational landscapes

Abstract Background: Genomic studies in estrogen-receptor (ER) positive breast cancer showed few commonly mutated genes and suggested that tumors with higher background of mutation rate were enriched in intrinsic aromatase-inhibitor-resistant tumors (Ellis MJ Nature 2012). Different combinations of proliferation (PROL) and estrogen-receptor related genes (ERS) allow the prediction of the risk of recurrence and endocrine treatment sensitivity (Bianchini G Breast Cancer Res 2013). The highPROL/lowERS is enriched in intrinsic endocrine resistant tumors. We explored whether different genomic alterations could contribute to the different clinical behavior of groups defined by PROL and ERS. Method: We defined luminal breast cancers by PAM50 and retrieved gene expression and whole-exome sequencing data of 324 luminal breast cancer cases from the Cancer Genome Atlas (TCGA). PROL and ERS were calculated as previously reported. Low and high expression groups were defined by median cut-points (Bianchini G Breast Cancer Res 2013). As a metric of the background mutation rate of each sample, we used the overall mutation frequency in whole exome (Kandoth C Nature 2013). The most frequently mutated genes (TP53, PIK3CA, MAP2K4, MAP3K1, GATA3, CDH1) were also assessed for their association with PROL and ERS defined groups. Results: The average number of mutations (Nmut) was 44.4 (5-366). High and low proliferation groups had different mutation frequency (average Nmut 53.6 and 35.3, respectively; p=6.1E-06). LowERS group also had a higher Nmut (average Nmut 54.4 vs. 34.5 in high ERS; p=1.2E-06). Notably, neither high or low expression of the ER gene (p=0.89), nor positive and negative progesterone receptor by immunohistochemistry (p=0.69) were associated with Nmut. LowERS group was associated with higher Nmut in luminal A (p=0.005) and B (p=0.006) subtypes. The group with highPROL and lowERS had the highest Nmut (average 62.1), which was not significantly different from basal-like tumors. HighPROL/lowERS was significantly associated with higher frequency of mutTP53 (32.3%) and lower rates of mutPIK3CA (28.1%), mutMAP2K4 and/or mutMAP3K1 (7.2%) and mutCHD1 (3.1%). The lowPROL/highERS had the lowest Nmut (average 29.8), with the higher rate of mutPIK3CA (61.4%) and mutMAP2K4 and/or mutMAP3K1 (29.2%), and lower rate of mutTP53 (6.7%). Discussion: In ER-positive breast cancers, the highPROL/lowERS group, which is characterized by the highest risk of early recurrences despite endocrine treatment and chemotherapy, poor down-regulation of Ki67 after neoadjuvant aromatase-inhibitor (AI), but the highest rate of pCR after neoadjuvant chemotherapy, was also associated with the highest frequency of mutations, of the same order of magnitude of basal-like tumors. This data is in line with the observation that the number of mutations is linked to intrinsic resistance to neoadjuvant AIs (Ellis MJ Nature 2012). These data could be also informative for interpreting results of the combination of everolimus and endocrine therapy (exemestane or tamoxifen) that suggest higher efficacy in tumors with acquired endocrine resistance (lower mutation rate) compared to intrinsic or primary endocrine resistance (higher mutation rate). Citation Format: Giampaolo Bianchini, Eugenia Galeota, Tingting Jiang, Maurizio Callari, Milvia Zambetti, Takayuki Iwamoto, Thomas Karn, Christos Hatzis, Lajos Pusztai, Luca Gianni. The heterogeneous clinical behavior of luminal breast cancers is associated with different mutational landscapes [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-03-05.

Abstract P6-03-07: Targeting multiple pathways in breast cancer: Androgen receptor, HER2, and mTOR

Abstract Background: Androgen receptor (AR) is expressed in ∼60% of breast cancers (BC) and current evidence indicates that some BC become reliant on AR signaling for growth. AR activation affects numerous cellular functions and signaling cascades, including the HER2/PI3K/mTOR pathway. Conversely, the HER2/PI3K/mTOR pathway regulates steroid hormone receptors including AR. Therapeutics targeting this pathway such as the anti-HER2 trastuzumab (TRAS) and the anti-mTOR everolimus (EVE) have shown significant clinical benefit; however the impact that AR inhibition may have on enhancing the efficacy of these agents and a potential mechanism of synergy have not been explored in BC. The anti-androgen enzalutamide (ENZ) inhibits nuclear entry of AR, is clinically effective in castrate-resistant prostate cancer, and is currently being tested in clinical trials for BC. Hypothesis: Given the overlap in these pathways, simultaneous inhibition of AR pathway with agents targeting HER2/PI3K/mTOR may be more effective in BC therapy. Methods: HER2+ and triple-negative BC (TNBC) cell lines were treated with ENZ, TRAS, and EVE, either alone or in combination, at three clinically relevant doses per drug. Cell proliferation was measured either by crystal violet (MDAMB453, SUM225) or on an IncuCyte live cell imager (Essen Bioscience) with nuclear-red labeled cells (BT474, SKBR3, BT549). Drug synergy was calculated using Calcusyn (Biosoft, Inc.) from IncuCyte proliferation data for selected cell lines (BT474, SKBR3, BT549) by comparing combinations of multiple drug concentrations where a combination index (CI)<1 indicated synergy. Protein expression of pathway components was measured by western blot, and gene expression was measured by RT-qPCR. Results: ENZ inhibited proliferation of HER2+ BC cells, and a combination of ENZ plus TRAS inhibited proliferation more effectively than either agent alone. The effect was synergistic in SKBR3 cells (CI<0.1), and additive in BT474 cells. Treatment of a HER2+/ER+ cell line (BT474) as well as an ER-, HER2-non-amplified but overexpressing cell line (MDAMB453) with dihydrotestosterone (DHT) caused increased expression of pHER3 and total HER3 protein, which was attenuated by addition of ENZ. However, in HER2+/ER- cell lines (SKBR3 and SUM225), pHER3 and total HER3 levels did not change, but rather pHER2 levels increased in response to DHT and were attenuated with ENZ treatment. Additionally, ENZ inhibited proliferation of TRAS-resistant SKBR3 cells. However, unlike the parental SKBR3 cells, ENZ did not affect pHER2 levels in the TRAS-resistant cells. ENZ plus EVE treatment resulted in a significant decrease in proliferation compared to single agent; this effect was synergistic in two HER2+ cell lines (SKBR3 CI<0.1, BT474 CI<0.1) as well as one TNBC cell line (BT549 CI<0.5). Treatment with EVE alone caused a compensatory increase in AR protein and downstream AR gene expression in all cell lines. This increase was attenuated with ENZ plus EVE combination treatment. Conclusion: ENZ synergizes in vitro with FDA-approved BC therapies TRAS and EVE through distinct mechanisms. Combination therapies containing ENZ may provide benefit for multiple BC subtypes, including HER2+ and triple negative BC. Funded by DOD BCRP Clinical Translational Award BC120183 to JKR. Citation Format: Michael A Gordon, Nicholas D'Amato, Haihua Gu, Darren Wong, Anthony Elias, Jennifer K Richer. Targeting multiple pathways in breast cancer: Androgen receptor, HER2, and mTOR [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-03-07.

Biomarker signatures correlate with clinical outcome in refractory metastatic colorectal cancer patients receiving bevacizumab and everolimus.

A novel combination of bevacizumab and everolimus was evaluated in refractory colorectal cancer patients in a phase II trial. In this retrospective analysis, plasma samples from 49 patients were tested for over 40 biomarkers at baseline and after one or two cycles of drug administration. Analyte levels at baseline and change on-treatment were correlated with progression-free survival (PFS) and overall survival (OS) using univariate Cox proportional hazard modeling. Multivariable analyses were conducted using Cox modeling. Significant changes in multiple markers were observed following bevacizumab and everolimus treatment. Baseline levels of six markers significantly correlated with PFS and OS, including CRP, Gro-α, IGFBP-1, TF, ICAM-1, and TSP-2 (P < 0.05). At C2D1, changes of IGFBP-3, TGFβ-R3, and IGFBP-2 correlated with PFS and OS. Prognostic models were developed for OS and PFS (P = 0.0002 and 0.004, respectively). The baseline model for OS consisted of CRP, Gro-α, and TF, while the on-treatment model at C2D1 included IGFBP-2, IGFBP-3, and TGFβ-R3. These data demonstrated that multiple biomarkers were significantly modulated in response to bevacizumab and everolimus. Several markers correlated with both PFS and OS. Interestingly, these markers are known to be associated with inflammation and IGF signaling, key modulators of mTOR biology.

Impact of Previous Somatostatin Analogue Use on the Activity of Everolimus in Patients with Advanced Neuroendocrine Tumors: Analysis from the Phase III RADIANT-2 Trial

Background/Aims: The phase III placebo-controlled RADIANT-2 trial investigated the efficacy of everolimus plus octreotide long-acting repeatable (LAR) in patients with advanced neuroendocrine tumors (NET) associated with carcinoid syndrome. Here we report a secondary analysis based on the previous somatostatin analogue (SSA) exposure status of patients enrolled in RADIANT-2. Methods: Patients were randomly assigned to receive oral everolimus 10 mg/day plus octreotide LAR 30 mg intramuscularly (i.m.) or to receive matching placebo plus octreotide LAR 30 mg i.m. every 28 days. SSA treatment before study enrollment was permitted. Patient characteristics and progression-free survival (PFS) were analyzed by treatment arm and previous SSA exposure status. Results: Of the 429 patients enrolled in RADIANT-2, 339 were previously exposed to SSA (95% received octreotide); 173 of 339 patients were in the everolimus plus octreotide LAR arm. All patients had a protocol-specified history of secretory symptoms, but analysis by type showed that more patients who previously received SSA therapy had a history of flushing symptoms (77%), diarrhea (86%), or both (63%) compared with SSA-naive patients (62, 62, and 24%, respectively). Patients who received everolimus plus octreotide LAR had longer median PFS regardless of previous SSA exposure (with: PFS 14.3 months, 95% confidence interval, CI, 12.0-20.1; without: 25.2 months, 95% CI, 12.0-not reached) compared with patients who received placebo plus octreotide LAR (with: 11.1 months, 95% CI, 8.4-14.6; without: 13.6 months, 95% CI, 8.2-22.7). Conclusion: Everolimus in combination with octreotide improves PFS in patients with advanced NET associated with carcinoid syndrome, regardless of previous SSA exposure.

Second-Line Treatment of Advanced Urothelial Cancer with Paclitaxel and Everolimus in a German Phase II Trial (AUO Trial AB 35/09)

Objective: The efficacy of second-line treatment after failure of platinum-based chemotherapy in patients with advanced urothelial cancer is limited. Based on encouraging preclinical and clinical phase I data, we evaluated the safety and efficacy of the combination of paclitaxel and everolimus in these patients. Methods: In this trial, patients having failed to respond to prior platinum-based combination treatment of urothelial cancer were treated with paclitaxel (175 mg/m² i.v., 3-weekly) and the mTOR-inhibitor everolimus (10 mg p.o., once daily). The patients were treated until tumor progression or until a maximum of 6 cycles was completed. A one-stage design was used to evaluate the objective response rate (ORR) as the primary endpoint. Results: A total of 27 patients (67% male; median age 63 years) were enrolled. The most frequent grade III/IV toxicities were anemia (28%), peripheral neuropathy (28%), and fatigue (24%). No treatment-related deaths were reported. Complete and partial remissions were observed in 0/24 and 3/24 patients eligible for efficacy analysis, respectively (ORR 13%). Progression-free survival was 2.9 months [95% confidence interval (95% CI) 1.9-4.2], and the median overall survival was 5.6 months (95% CI 4.8-10.2). Conclusion: The combination of paclitaxel and everolimus has not achieved the expected efficacy in second-line treatment of urothelial cancer and should not be further explored.

P1.14 - A Dose Escalation Single Arm Phase Ib Combination Study of BEZ235 with Everolimus in Patients with Advanced Solid Malignancies

ABSTRACT Background/Purpose: The dual phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitor, BEZ235, was shown to be efficacious as an inhibitor of cancer cell proliferation in combination with the mTOR-allosteric inhibitor, RAD001 or everolimus, in a preclinical model over everolimus alone. We hypothesized that the combinatorial effect on the mTOR and PI3K pathway would result in a synergistic anti-tumor effect in humans. Therefore, we designed a first in human, phase 1b trial, in order to study the safety and pharmacokinetics of the combination of BEZ235 and everolimus in patients with solid tumor malignancies. Methods: Nineteen patients with solid tumor malignancies that had progressed on standard of care treatment were enrolled. The experimental medication, BEZ235, was provided to us by Novartis. BEZ235 was administered orally once a day at escalating doses of 200, 400, and 800mg and everolimus was administered orally once a day at 2.5mg in 28 day cycles. Results: The most common toxicities observed included fatigue (68%), diarrhea (74%), nausea (63%), mucositis (42%) and elevated liver enzymes (63%). The maximum tolerated dose (MTD) was not reached due to toxicity resulting in early discontinuation of the trial. BEZ235 serum levels were inconsistent suggesting poor absorption. Everolimus exposure was increased over what would be expected for administration alone, likely due to the previously postulated drug interaction between BEZ235 and everolimus. Of the twelve patients evaluated for response, one had SD at 5 weeks and no PR or CR was observed. Conclusion: The combination therapy of BEZ235 and everolimus was shown to be poorly tolerated in our patient population likely due to poor absorption, drug interaction, and inter-patient variability leading to inconsistent drug levels. Although, there were no confirmed responses, one patient did respond clinically for a brief period. We expect that the combination of everolimus with new dual inhibitors with improved toxicity profiles and increased absorption will be more promising.

Phase I trial of gemcitabine and split-dose cisplatin plus everolimus (RAD001) in patients with advanced urothelial cancer.

322 Background: Gemcitabine and split-dose cisplatin (GC) is standard first-line therapy for patients with advanced urothelial cancer (UC). Everolimus is an inhibitor of the PI3K/AKT/mTOR pathway, dysregulated in ~35% of UCs. The drug has been shown to have single-agent activity in phase II trials of advanced UC. The safety of combining everolimus with GC in the first-line setting for UC is unknown. Methods: Previously untreated cisplatin-eligible patients with advanced UC were enrolled. Patients received gemcitabine 800 mg/m2 and cisplatin 35mg/m2 on days 1 and 8 with everolimus at dose levels (DL1: 5 mg QOD, DL2: 5 mg daily or DL3: 10 mg daily) for six 21-day cycles. Patients with at least SD continued maintenance everolimus until progression. Restaging evaluations were performed every 2 cycles. The primary objective was to establish the dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) of the three-drug combination. Results: 12 patients were enrolled, 7 with bladder and 5 with upper tract primaries. 4/12 patients completed at least 6 cycles. 0/3 patients at DL1 had DLTs. 3/3 patients at DL2 had DLTs (pancytopenia, hypersensitivity reaction to everolimus and anemia). Following de-escalation to DL1, 2/6 patients in a new DL1 cohort had DLTs (neutropenia and diarrhea, 1/6 patients inevaluable). Grade 3/4 events included anemia (67%), neutropenia (42%), lymphopenia (42%), thrombocytopenia (25%), urinary tract infection (25%), hypomagnesemia (17%), and hypophosphatemia (17%). Tumor responses included: 4 PR (33%), 5 SD (42%), 1 PD (8%), and 2 inevaluable (17%). 2 patients with PR underwent consolidative surgery but eventually progressed. Responses by MSKCC Risk Scores of 0, 1, and 2 were seen in 3/5 (60%), 1/6 (17%), and 0/1 patients, respectively. Median PFS was 3.9 months (95% CI, 3.7-no reached) and median OS was 10.8 months (95% CI, 6.9-not reached). Conclusions: MTD was reached at DL1 for combination gemcitabine, split-dose cisplatin and everolimus, with 2/8 evaluable patients exhibiting DLTs at this dose level. Clinical trial information: NCT01182168.

Everolimus plus bicalutamide for castration resistant prostate cancer (CRPC): Bench to bedside and back.

189 Background: We previously reported that the mammalian target of rapamycin (mTOR) pathway was upregulated in CRPC, and that simultaneous blockade of both androgen receptor (AR) and mTOR pathways synergized in killing CRPC cells. Methods: Eligible patients (pts) must have progressive CRPC with serum testosterone &lt;50 ng/dl. No prior bicalutamide (except to prevent flare) or everolimus was allowed. Treatment regimen included daily bicalutamide 50 mg and everolimus 10 mg orally. Primary endpoint was prostate specific antigen (PSA) response (defined as ≥30% reduction). Sample size of 23 pts would have the power of 0.8 and alpha error of 0.05 (one-sided) if the combination had a PSA response rate of 50% vs. a historical rate of 25% with bicalutamide alone. Molecular correlative studies including serum ERBB3 levels were performed by ELISA. LNCaP and C4-2 cells, modeling bicalutamide-sensitivity and resistance, respectively, were transfected with ErbB3 or an empty vector and subjected to western blotting. Results: Of 24 pts enrolled, the mean number of treatment cycles was 7.9 (1.0-23.0). Eighteen pts had a PSA response (75%, 95% CI: 0.53-0.90). Median overall survival was 28 months (95% CI 14.1-42.7). Fourteen pts (54%, 95% CI 0.37, 0.78) developed Grade 3 (13 pts) or Grade 4 (1 pt with sepsis) adverse events attributed to study treatment. Pts with partial response showed a greater decline in ErbB3 (P=0.037) compared to those with stable disease or disease progression. Consistent with these findings, everolimus induced a decline in ErbB3 in hormone-sensitive LNCaP cells but failed to affect ErbB3 in CRPC C4-2 cells, which expressed 8-fold higher levels of ErbB3. Increased expression of ErbB3 in LNCaP cells resulted in resistance to everolimus, whereas bicalutamide initially prevented this effect. Prolonged exposure to bicalutamide also resulted in an increase in ErbB3 which correlated with increased growth rates and cell invasion. Conclusions: The combination of bicalutamide and everolimus has very encouraging efficacy in men with bicalutamide-naïve CRPC, but with substantial everolimus-related toxicity. ERBB3 may contribute to resistance and deserves further study. Clinical trial information: NCT00814788.

Toxicity evaluation of convection-enhanced delivery of small-molecule kinase inhibitors in naïve mouse brainstem.

Diffuse intrinsic pontine gliomas (DIPGs) are inoperable and lethal high-grade gliomas lacking definitive therapy. Platelet-derived growth factor receptor (PDGFR) and its downstream signaling molecules are the most commonly overexpressed oncogenes in DIPG. This study tested the effective concentration of PDGFR pathway inhibitors in cell culture and then toxicity of these small-molecule kinase inhibitors delivered to the mouse brainstem via convection-enhanced delivery (CED) for potential clinical application. Effective concentrations of small-molecule kinase inhibitors were first established in cell culture from a mouse brainstem glioma model. Sixteen mice underwent CED, a local drug delivery technique, of saline or of single and multidrug combinations of dasatinib (2 M), everolimus (20 M), and perifosine (0.63 mM) in the pons. Animals were kept alive for 3 days following the completion of infusion. No animals displayed any immediate or delayed neurological deficits postoperatively. Histological analysis revealed edema, microgliosis, acute inflammation, and/or axonal injury in the experimental animals consistent with mild acute drug toxicity. Brainstem CED of small-molecule kinase inhibitors in the mouse did not cause serious acute toxicities. Future studies will be necessary to evaluate longer-term safety to prepare for potential clinical application.

Everolimus in diffuse large B-cell lymphomas.

Satisfactory treatment of relapsed/refractory diffuse large B-cell lymphoma (DLBCL) is not currently available and novel therapies are needed. mTOR is an intracellular kinase that is part of an aberrantly activated pathway in DLBCL. Preclinical studies in DLBCL cell lines demonstrated that everolimus, an oral selective mTOR inhibitor, induces cell cycle arrest and is synergistic with rituximab. Phase I studies indicated 10 mg daily to be the best dosing of everolimus in DLBCL. A large Phase II study in relapsed/refractory DLBCL confirmed the substantial activity (overall response rate: 30%) and good tolerability of everolimus in DLBCL, with thrombocytopenia being the main toxicity. The combination of everolimus and rituximab showed encouraging results (objective response rate: 38%; complete response: 13%), without increasing toxicity. Combination studies of everolimus with novel agents or with immunochemotherapy are underway.

Everolimus and pasireotide for advanced and metastatic hepatocellular carcinoma

Few treatment options are available for patients with advanced or metastatic hepatocellular carcinoma (HCC). Based on preclinical and early clinical efficacy signals and lack of overlapping toxicity, we undertook this multicenter phase II trial to estimate efficacy and safety of everolimus and pasireotide in advanced HCC. Patients with advanced HCC not amenable to locoregional therapy and Child-Pugh A cirrhosis received everolimus 7.5 mg PO daily and pasireotide LAR 60 mg IM every 28 days. The primary endpoint was time to progression (TTP), with 26 events needed to evaluate if everolimus + pasireotide improved TTP from 2.8 to 4.4 months, with 80% power and an alpha of 0.05. Secondary endpoints included response as measured by RECIST modified for HCC, treatment-emergent adverse events, and overall survival. After 24 patients were enrolled, results of a randomized trial showing no benefit of everolimus in HCC were released prompting an unplanned interim analysis that found the conditional probability of rejecting the null hypothesis based on events in those patients was 0.08. Therefore accrual was halted. Patients had a median age of 59 years, 21 (88%) had BCLC stage C cancer, and 11 (46%) metastatic disease. Median TTP was 3.5 months (95% CI 2-5.8) and median survival 6.7 months (95% CI 6-infinity). Best response was stable disease in ten patients. Grade 3 hyperglycemia occurred in 6 (25%). There were no grade 4 treatment-emergent events. Despite promising early efficacy signals, we found no benefit for the combination of everolimus and pasireotide in HCC.

Treatment of neuroendocrine tumors (NETs) with combination of everolimus and lu-177 radiolabeled somatostatin analogue.

TPS502 Background: Treatment of Grade 1 and 2 NET is complex and involves surgical, Locoregional and systemic modalities depending on the manifestations as well as extent of disease. Of the emerging new systemic modalities are everolimus and radiolabelled somatostatin analogue. everolimus is FDA approved for advanced pancreatic NET whereas radiolabelled somatostatin analogue is still not standard therapy in the USA. However, it is gaining popularity especially in Europe as effective systemic therapy for somatostatin receptor positive tumors. Subclinical animal model research showed synergistic effect of the combination of everolimus and the Lu-177 radiolabeled somatostatin analogue. The goal of this study is to assess the safety and efficacy of the combination of Everolimus and the intravenous radiolabeled Lu-177 DOTATOC Therapy in the treatment of unresectable G1-2 neuroendocrine tumors of all gastrointestinal, lung and pancreatic origins. Methods: This is a phase 1 – 2 study. The phase 1 part involves finding the maximum tolerating dose (DLT) of everolimus and accordingly to recommend dose for the phase II part. The second part is a standard phase II trial assessing the efficacy and toxicity of the regimen at the recommended dose. A total of 30 patients will be enrolled, based on the expected response of the combination to be 20% with power of 80 and type 1 of.05, compared to the known everolimus alone overall all response of 5%. In phase 1, three patients will be treated with 5mg daily with expansion to six patients if one of the three patients experienced DLT. If DLT occurred in none of three or no more than one of six patients, everolimus dose will be escalated to 10 mg and will be considered the recommended dose for phase 2 part. Otherwise, everolimus dose will be kept at 5 mg daily and will be considered the recommended dose for phase II part. DLT in more than two of six patients will be considered unacceptable, and the trial will be closed because of high toxicity.

Sorafenib and everolimus for patients with unresectable high-grade osteosarcoma progressing after standard treatment: a non-randomised phase 2 clinical trial

Results of previous study showed promising but short-lived activity of sorafenib in the treatment of patients with unresectable advanced and metastatic osteosarcoma. This treatment failure has been attributed to the mTOR pathway and might therefore be overcome with the addition of mTOR inhibitors. We aimed to investigate the activity of sorafenib in combination with everolimus in patients with inoperable high-grade osteosarcoma progressing after standard treatment. We did this non-randomised phase 2 trial in three Italian Sarcoma Group centres. We enrolled adults (≥18 years) with relapsed or unresectable osteosarcoma progressing after standard treatment (methotrexate, cisplatin, and doxorubicin, with or without ifosfamide). Patients received 800 mg sorafenib plus 5 mg everolimus once a day until disease progression or unacceptable toxic effects. The primary endpoint was 6 month progression-free survival (PFS). All analyses were intention-to-treat. This trial is registered with ClinicalTrials.gov, number NCT01804374. We enrolled 38 patients between June 16, 2011, and June 4, 2013. 17 (45%; 95% CI 28-61) of 38 patients were progression free at 6 months. Toxic effects led to dose reductions, or short interruptions, or both in 25 (66%) of 38 patients and permanent discontinuation for two (5%) patients. The most common grade 3-4 adverse events were lymphopenia and hypophosphataemia each in six (16%) patients, hand and foot syndrome in five (13%), thrombocytopenia in four (11%), and fatigue, oral mucositis, diarrhoea, and anaemia each in two (5%). One patient (3%) had a grade 3 pneumothorax that required trans-thoracic drainage, and that recurred at the time of disease progression. This was reported as a serious adverse event related to the study drugs in both instances. No other serious adverse events were reported during the trial. There were no treatment-related deaths. Although the combination of sorafenib and everolimus showed activity as a further-line treatment for patients with advanced or unresectable osteosarcoma, it did not attain the prespecified target of 6 month PFS of 50% or greater. Italian Sarcoma Group.

A Novel Combination of the mTORC1 Inhibitor Everolimus and the Immunomodulatory (IMiD) Drug Lenalidomide Produces Durable Responses in Patients with Heavily Pretreated Relapsed Lymphoma

IntroductionNew treatment strategies are improving outcomes for patients with Hodgkin (HL) and non-Hodgkin lymphoma (NHL) but achieving durable responses and cure in the relapsed patient remain a challenge. Both the mTORC1 inhibitor everolimus (RAD001) and the IMiD, lenalidomide (LEN) have efficacy as single agents in patients with relapsed and refractory (R/R) HL and NHL. Individually, LEN and RAD present a low toxicity profile and in combination may offer synergistic therapeutic efficacy by unique mechanisms of action. A phase I/II clinical trial of RAD and LEN was designed to explore the tolerability, toxicity and to determine the maximum tolerated dose (MTD) of this combination for the Phase II study. The results of phase I/II data are reported here.MethodsMC0981 was activated on January 10, 2011. In phase I, the study enrolled a total of 25 patients and RAD 5 mg daily + LEN 10 mg/d X 21d in a 28-day cycle was defined as the MTD. The phase II opened on May 14, 2013, and closed on Feb 19, 2014, during which 33 patients were enrolled and were treated until progression of disease. Eligibility required age ≥18, biopsy proven relapsed or refractory NHL or HL, ECOG PS 0- 2, measurable disease, Hb >9g/dl, ANC ≥1200/uL, platelet ≥ 50,000/uL, creatinine ≤1.5xULN and willingness to sign informed consent. Women of child bearing potential had pregnancy testing and all patients followed recommendations for contraception. Follow up data was analyzed as of July 2014.ResultsA total of 58 patients were enrolled to this study and fifty five are evaluable for analysis (2 never started therapy, 1 deemed ineligible). The median age was 62 years (21-82) and 38 (69.1%) were male. Sixty-five percent had stage IV disease and 42% had 4 or more prior treatments including stem cell transplantation in 49%. Histologies included HL (n=10) and NHL (n=45 with 23 DLBCL, 2 FL-III, 5 LPL, 3 MCL, 2 MF, and 3 other). At least 1 Gr≥3 adverse event was seen in 74.5% of patients. The most common Gr≥3 toxicities were hematologic - neutropenia (30.9%), and thrombocytopenia (21.8%), and anemia (10.9%). Nine of 55 patients (16%) had Gr≥3 infection; 2 with skin infection, 1 with cholecystitis, 1 with lung infection, 1 with pharyngitis 1 with otitis externa, 1 with otitis media, and 1 with pleural infection and 1 with upper respiratory tract infection. Seven patients discontinued treatment due to adverse events. At this time, 44% (25/55) remain progression-free. A documented tumor response, as overall response rate (ORR) was seen in 20% (11/55) with 1CR, 8PR, and 2MR; 17 remain on therapy; and 22 deaths have occurred with a median survival of 15.0 months (95% CI 7.5-NR). Median follow up time was 5.5 (range, 0.7- 41.0) months. The median duration of response (DOR) in those responding is 19.4 months.ConclusionsIn a heavily pretreated population with baseline compromised bone marrow function, the combination of everolimus and lenalidomide is feasible with a modest ORR and a surprisingly long DOR. Future studies in more defined groups of NHL or HL are necessary to better predict which patients will be long-term responders.This trial is sponsored by Novartis and Celgene DisclosuresOff Label Use: mTORC1 inhibitor (Everolimus) and the Immunomodulatory (IMiD) drug Lenalidomide for patients with Heavily Pretreated Relapsed Lymphoma . Mikhael:Onyx: Research Funding; Celgene: Research Funding; Sanofi: Research Funding; Novartis: Research Funding. Nowakowski:Celgene, Morphosis: Consultancy. Witzig:Celgene: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Novartis: Research Funding. Reeder:Millennium, Celgene, Novartis: Research Funding.

Everolimus in Combination with Crizotinib Synergistically Inhibits the Growth of ALK-Positive Anaplastic Large Cell Lymphoma Cells

More than a half of anaplastic large cell lymphoma (ALCL) harbors an aberrant NPM-ALK fusion gene, which activates a number of down-stream signaling pathways such as Ras/ERK, PI3K/AKT, and JAK3/STAT3. Through this mechanism, mTOR pathway is also activated in ALK-positive ALCL (Vega F, et al. Cancer Res 2006). Everolimus, an mTOR inhibitor, has shown promising anti-tumor activity in a variety of lymphomas (Jundt F, et al. Blood 2005; Wanner K, et al. Br J Haematol 2006; Haritunians T, et al. Leukemia 2007), although the clinical efficacy of everolimus monotherapy was not satisfactory, possibly due to activation of several pro-surviving signaling pathways. The combined effect of everolimus and crizotinib, an ALK inhibitor, has not yet been investigated in ALK-positive tumors so far. The aim of this study was to evaluate the effect of everolimus in combination with crizotinib in ALK-positive ALCL cell lines, K-299 and SU-DHL-1.We treated K-299 and SU-DHL-1 cells with various concentrations of everolimus and crizotinib at a fixed ratio of 1:40 (Figure 1). After 72 hours, the combination index (CI) values calculated by the Chou-Talalay method were less than 1 (range, 0.583-0.763 in K-299 cells and 0.271-0.616 in SU-DHL-1 cells) in all tested combinations, suggesting synergistic cytotoxicity of everolimus and crizotinib. The Western blot analysis (Figure 2) demonstrated that everolimus treatment up-regulated the phosphorylation of ERK Thr202/Tyr204 and AKT Thr308 and Ser473 in K-299 cells. However, this aberrant activation of ERK and AKT was attenuated by the addition of crizotinib. In addition, while everolimus selectively inhibited phosphorylation of mTOR Ser2448, a marker for mTORC1 activity, the combination treatment more potently inhibited mTOR Ser2448 phosphorylation and decreased phosphorylated mTOR at Ser2481, a marker for mTORC2, as well. In the cell-cycle analysis, the combination treatment induced G1 arrest. Everolimus treatment alone did not increase the fraction of cells in the sub-G1 region compared to the control (2.16% vs. 4.03% in K-299 and 1.34% vs. 1.68% in SU-DHL-1), while crizotinib monotherapy increased the sub-G1 population (11.88% vs. 4.03% in K-299 and 28.68% vs. 1.68% in SU-DHL-1). The combination of crizotinib and everolimus markedly increased the sub-G1 population in both ALK-positive ALCL cell lines (22.25% in K-299 and 46.40% in SU-DHL-1). PARP cleavage was also increased after the combination treatment. To test the hypothesis that our findings could be applyed to other ALK-positive malignancies, we treated NCI-H2228, a lung adenocarcinoma cell line that harbors an EML4-ALK fusion gene, with everolimus and crizotinib for 72 hours. The CI values were less than 1 in all tested combinations: 0.228 in 1 nM everolimus plus 80 nM crizotinib, 0.216 in 2 nM everolimus plus 160 nM crizotinib, and 0.349 in 4 nM everolimus and 320 nM crizotinib.In summary, everolimus combined with crizotinib synergistically inhibited the growth of ALK-positive ALCL cells. Crizotinib abrogated aberrant ERK and AKT signaling activation induced by everolimus and more potently inhibited both mTORC1 and mTORC2 activity when combined with everolimus, resulting in increased G1 cell-cycle arrest and apoptosis (Figure 3). Our findings may provide an evidence for future research using everolimus and crizotinib combination in ALK-positive ALCL and could be used to improve the therapeutic outcome in patients with ALK-positive ALCL. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.