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PDF file, 47KB, Reproducibility of immunomagnetic separation and ELISA.

PDF file, 80KB, Experiments detailing the validation of immunomagnetic separation and ELISA.

PDF file, 63KB.

PDF file, 66KB, Oncogenes and mutations detected using the OncoCarta Panel v1.0.

PDF file, 63KB, Survival based on signalling output in total population of 88 patients.

PDF file, 71KB, Efficiency of immunomagnetic separation.

PDF file, 44KB, Probes for PIK3CA, AKT2 and PTEN.

Activation of the PI3K/mTOR pathway has been shown to be correlated with resistance to chemotherapy in ovarian cancer. We aimed to investigate the effects of combining inhibition of mTORC1 and 2 using the mTOR kinase inhibitor vistusertib (AZD2014) with paclitaxel in in vitro and in vivo ovarian cancer models. The combination of vistusertib and paclitaxel on cell growth was additive in a majority of cell lines in the panel (n = 12) studied. A cisplatin- resistant model (A2780Cis) was studied in vitro and in vivo. We demonstrated inhibition of mTORC1 and mTORC2 by vistusertib and the combination by showing reduction in p-S6 and p-AKT levels, respectively. In the A2780CisR xenograft model compared to control, there was a significant reduction in tumor volumes (p = 0.03) caused by the combination and not paclitaxel or vistusertib alone. In vivo, we observed a significant increase in apoptosis (cleaved PARP measured by immunohistochemistry; p = 0.0003). Decreases in phospholipid and bioenergetic metabolites were studied using magnetic resonance spectroscopy and significant changes in phosphocholine (p = 0.01), and ATP (p = 0.04) were seen in tumors treated with the combination when compared to vehicle-control. Based on this data, a clinical trial evaluating the combination of paclitaxel and vistusertib has been initiated (NCT02193633). Interestingly, treatment of ovarian cancer patients with paclitaxel caused an increase in p-AKT levels in platelet-rich plasma and it was possible to abrogate this increase with the co-treatment with vistusertib in 4/5 patients: we believe this combination will benefit patients with ovarian cancer.

Background: We aimed to understand the dependence of MEK and m-TOR inhibition in EGFRWT/ALKnon-rearranged NSCLC cell lines. Methods: In a panel of KRASM and KRASWT NSCLC cell lines, we determined growth inhibition (GI) following maximal reduction in p-ERK and p-S6RP caused by trametinib (MEK inhibitor) and AZD2014 (m-TOR inhibitor), respectively. Results: GI caused by maximal m-TOR inhibition was significantly greater than GI caused by maximal MEK inhibition in the cell line panel (52% vs 18%, P

We investigated PD-L1 changes in response to MEK and AKT inhibitors in KRAS mutant lung adenocarcinoma (adeno-NSCLC). PD-L1 expression was quantified using immunofluorescence and co-culture with a jurkat cell-line transfected with NFAT-luciferase was used to study if changes in PD-L1 expression in cancer cell lines were functionally relevant. Five KRAS mutant cell lines with high PD-L1 expression (H441, H2291, H23, H2030 and A549) were exposed to GI50 inhibitor concentrations of a MEK inhibitor (trametinib) and an AKT inhibitor (AZD5363) for 3 weeks. Only 3/5 (H23, H2030 and A549) and 2/5 cell lines (H441 and H23) showed functionally significant increases in PD-L1 expression when exposed to trametinib or AZD5363 respectively. PD-L1 overexpression is not consistent and is unlikely to be an early mechanism of resistance to KRAS mutant adeno-NSCLC treated with MEK or AKT inhibitors.

Evidence that the phosphoinositide 3-kinase (PI3K) pathway is deregulated in ovarian cancer is largely based on the analysis of surgical specimens sampled at diagnosis and may not reflect the biology of advanced ovarian cancer. We aimed to investigate PI3K signaling in cancer cells isolated from patients with advanced ovarian cancer. Ascites samples were analyzed from 88 patients, of whom 61 received further treatment. Cancer cells were immunomagnetically separated from ascites, and the signaling output of the PI3K pathway was studied by quantifying p-AKT, p-p70S6K, and p-GSK3β by ELISA. Relevant oncogenes, such as PIK3CA and AKT, were sequenced by PCR-amplified mass spectroscopy detection methods. In addition, PIK3CA and AKT2 amplifications and PTEN deletions were analyzed by FISH. p-p70S6K levels were significantly higher in cells from 37 of 61 patients who did not respond to subsequent chemotherapy (0.7184 vs. 0.3496; P = 0.0100), and this difference was greater in patients who had not received previous chemotherapy. PIK3CA and AKT mutations were present in 5% and 0% of samples, respectively. Amplification of PIK3CA and AKT2 and deletion of PTEN was seen in 10%, 10%, and 27% of samples, respectively. Mutations of PIK3CA and amplification of PIK3CA/AKT2 or deletion of PTEN did not correlate with levels of p-AKT, p-p70S6K, and p-GSK3β. In patients with advanced ovarian cancer, there is an association between levels of p-p70S6K and response to subsequent chemotherapy. There is no clear evidence that this is driven specifically by PIK3CA or AKT mutations or by amplifications or deletion of PTEN. Mol Cancer Ther; 11(7); 1609–17. ©2012 AACR.

Objectives: We hypothesised that it was possible to quantify phosphorylation of important nodes in the phosphatidylinositol 3-kinase (PI3K) pathway in cancer cells isolated from pleural effusions of patients with non-small cell lung cancer (NSCLC) and study their correlation to somatic mutations and clinical outcomes. Materials and Methods: Cells were immunomagnetically separated from samples of pleural effusion in patients with NSCLC. p-AKT, p-S6K and p-GSK3β levels were quantified by ELISA; targeted next-generation sequencing was used to characterise mutations in 26 genes. Results: It was possible to quantify phosphoproteins in cells isolated from 38/43 pleural effusions. There was a significant correlation between p-AKT and p-S6K levels [r = 0.85 (95% confidence interval 0.73-0.92), p < 0.0001], but not p-AKT and p-GSK3β levels [r = 0.19 (95% confidence interval -0.16 to 0.5), p = 0.3]. A wide range of mutations was described and p-S6K was higher in samples that harboured at least one mutation compared to those that did not (p = 0.03). On multivariate analysis, p-S6K levels were significantly associated with poor survival (p < 0.01). Conclusion: Our study has shown a correlation between p-AKT levels and p-S6K, but not GSK3β, suggesting differences in regulation of the distal PI3K pathway by AKT. Higher p-S6K levels were associated with adverse survival, making it a critically important target in NSCLC.

Aim MEK and AKT inhibitors inhibit signaling down steam of KRAS and are being evaluated as single agents or in combination with other anticancer drugs for the treatment of KRAS mutant adenocarcinoma of the lung (adeno-NSCLC). We investigated whether increased PD-L1 expression with functional T cell inhibitory consequences were a common mechanism of resistance to these drugs in this setting. Material and Methods A panel of 10 KRAS mutant adeno-NSCLC cell lines were studied. Cells were exposed to GI50 concentrations of a MEK (trametinib) and AKT (AZD5363) inhibitor for 6hrs, 24hrs and 3 weeks. PD-L1 expression on cell lines was studied by immunofluorescence. Immunofluorescence at various time points were expressed as a ratio of values in drug treated cells compared to untreated control. Functional consequences of PD-L1 expression were studied using a T cell cancer cell line (Jurkat) transfected with a luciferase reporter where co-culture of cancer cells expressing PD-L1 led to reduction in luminescence. Luminescence at various time points were expressed as ratio of luminescence values of drug treated cells compared to untreated controls. Results Following characterization of expression of PD-L1 in 10 KRAS mutant adeno-NSCLC cell lines, 5 cell lines with the highest expression of PD-L1 was chosen for functional assays (H441, H2291, H23, H2030 and A549). When exposed to trametinib for 3 weeks, 3/5 cell lines (H23, H2030 and A549) showed an statistically significant increase in expression of PD-L1 (range 1.1-2.4) however significant reduction of luciferase activity was only observed in H23 and H2030, 0.93 and 0.75, p= 0.018 and p=0.01 respectively. When exposed to AZD5363 for 3 weeks, 3/5 cell lines (H441, H23 and H2030) showed a statistically significant increase in PD-L1 expression (range 1.3-1.9) however significant reduction in luciferase reduction was seen in only H441 and H23, 0.86 and 0.76, p= 0.03 and p= 0.04 respectively. Conclusion Chronic exposure of KRAS mutant cell lines to MEK and AKT inhibitors cause minor increases in PD-L1 expression but this does not result in functional inhibition of T cells in all instances. Consequently, a functional increase in PD-L1 expression is unlikely to be a common mechanism of resistance to MEK and AKT inhibitors in KRAS mutant adeno-NSCLC. 1 Citation Format: Anna R. Minchom, Parames Thavasu, Zai Ahmad, Adam Stewart, Alexandros Georgiou, Mary ER O'Brien, Sanjay Popat, Jaishree Bhosle, Timothy A. Yap, Johann de Bono, Udai Banerji. A study of dynamic changes in PD-L1 expression in KRAS mutant adenocarcinoma of the lung exposed to signal transduction inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2627. doi:10.1158/1538-7445.AM2017-2627

Background: The adenocarcinoma subtype of non-small cell lung cancer (adeno-NSCLC) is routinely treated with chemotherapy if patients do not have molecular aberrations such as epidermal growth factor receptor mutations or anaplastic lymphoma kinase rearrangements. There are currently no validated biomarkers that can predict if patients will gain clinical benefit from chemotherapy, leading to a majority of patients receiving many cycles of unnecessary chemotherapy. We hypothesized that the percentage rise in plasma caspase-cleaved cytokeratin 18 (cCK18) and total cytokeratin 18 (tCK18) assessed before and after chemotherapy correlates with the radiological response to chemotherapy. Methods: Plasma samples from 40 patients with stage IV adeno-NSCLC, treated with first-line chemotherapy with carboplatin (AUC5) plus pemetrexed (500 mg/m2), were collected prior to chemotherapy and 48 h after treatment. ELISA was used to quantify cCK18 and tCK18. Results: The male-to-female ratio was 3:1, and the median age of patients was 63 years. Patients who had a clinical benefit (complete response, partial response or stable disease) at the first radiological assessment following chemotherapy had a significantly higher percentage change in plasma tCK18 levels compared to those who had no clinical benefit, i.e. progressive disease (69.5 ± 75.1 vs. 25.3 ± 30.9%, respectively; p = 0.042). The receiver operating characteristic area was 0.712 (p = 0.039). There was an increase in the percentage change in cCK18 in patients with clinical benefit compared to those without clinical benefit but this was not statistically significant (57.6 ± 112.8 vs. 24.38 ± 45.1%, respectively; p = 0.85). Conclusions: The percentage change in plasma tCK18 levels before and after the first cycle of pemetrexed and carboplatin chemotherapy is associated with clinical benefit. If validated in larger cohorts, this test can be used to identify patients unlikely to respond to treatment who can thus be offered alternative treatments or entry into clinical trials.

Background Clinical trials have shown that cancers originating from different tissues driven by the same oncogene respond differently to targeted anticancer drugs. We aimed to understand different signaling patterns in KRAS mutant cells derived from non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and pancreatic cancer. Materials and methods We optimized a 50 phosphoprotein antibody-based assay on the Luminex 200 platform. We then exposed a panel of 15 KRAS mutant cell lines (5 cell lines each originating in the lung, pancreas and colon) to a DMSO control (n = 3) and clinically significant concentrations (Cmax achieved in humans adjusted for protein binding in culture medium) of a PI3K (GDC-0941), AKT (AZD5363), m-TOR (everolimus), BRAF (vemurafenib), EGFR (gefitinib), MEK (trametinib) and an HSP90 inhibitor (luminespib) for 1 hr. We quantified the change in phosphorylation of proteins for each drug compared to control. Logistic regression analysis was used to analyse differences between KRAS-driven cell lines originating from different anatomical sites. Results There were changes in phosphorylation related to the pharmacodynamic effects of the drug independent of cell line of origin; however, there were interesting differences between KRAS mutant cells originating from different anatomical sites. In NSCLC cell lines, p-EGFR levels changed significantly less when exposed to PI3K, AKT and m-TOR inhibitors (p = 0.047, 0.022 and 0.047, respectively) when compared to cells originating from CRC and pancreatic cancer. CRC cell lines, when compared to NSCLC and pancreatic cancer cell lines, showed significantly less changes in phosphorylation of key cell cycle regulators such as CHK1 when exposed to PI3K, AKT and m-TOR inhibitors, (p = 0.001, 0.047 and 0.047, respectively) and RB when exposed to an AKT and m-TOR inhibitor (p = 0.047 and 0.047, respectively). Interestingly, pancreatic cell lines showed significantly more changes in p-m-TOR compared to CRC and NSCLC cell lines following exposure to PI3K and AKT inhibitors (p = 0.0095 and 0.022, respectively). Of note, drugs not directly targeting the PI3K pathway differentially regulated different nodes in the PI3K pathway, for example, BRAF inhibitors significantly differentially changed levels of phosphorylation at different nodes in the PI3K pathway such as AKT in NSCLC cell lines, p = 0.047, p70S6K in CRC cell lines, p = 0.0472 and PRAS40 in the pancreatic cancer cell lines, p = 0.022. Conclusion These results suggest that there are significant differences in signaling patterns caused by PI3K pathway inhibitors in KRAS mutant NSCLC, CRC and pancreatic cancer cell lines. Our findings shed light on the putative use of PI3K pathway inhibitors in KRAS mutant cancers. They also question the universal application of solely using genetic mutations to stratify patients in ‘basket’ clinical studies. Citation Format: Adam Stewart, Elizabeth Coker, Anna Minchom, Parames Thavasu, Alexandros Georgiou, Anguraj Sadanandam, Timothy A. Yap, Johann S. de Bono, Bissan Al-Lazikani, Udai Banerji. KRAS and clinical context: Differential dynamic signaling output of KRAS mutant lung, colorectal and pancreatic cancer cell lines when exposed to targeted anticancer drugs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3099.

BACKGROUND: We investigated the association between skin rash and plasma creatine kinase (CK) levels in oncology phase I trials. METHODS: We analysed data from 295 patients treated at our institution within 25 phase I trials which included CK measurements in the protocol. Trials involved drugs targeting EGFR/HER2, m-TOR, VEGFR, SRC/ABL, aurora kinase, BRAF/MEK, PARP, CDK, A5B1 integrin, as well as oncolytic viruses and vascular disrupting agents. RESULTS: Creatine kinase measurements were available for 278 patients. The highest levels of plasma CK during the trial were seen among patients with Grade (G) 2/3 rash (median 249 U l(-1)) compared with G1 (median 81 U l(-1)) and no rash (median 55 U l(-1)) (P

The stability and recovery of six human recombinant cytokines (tumour necrosis factor (TNF), interferon-alpha (IFN-alpha), IFN-gamma, interleukin-1 alpha (IL-1 alpha), IL-1 beta, and IL-6) from whole blood was investigated with a view to optimizing blood collection and storage procedures prior to performing immunoassays. Blood from healthy volunteers was subjected to various processing and storage procedures. Blood samples were treated with either: ethylenediamine tetraacetic acid (EDTA) (1.5 mg/ml blood) (E); EDTA/Trasylol (1.5 mg and 1000 KIU/ml blood) (ET); heparin (30 IU/ml) (H) or allowed to clot (serum). The bloods were spiked with individual cytokines, split into aliquots and kept at 4 degrees C or RT. In the first instance spiked bloods from healthy volunteers (n = 5 per cytokine) were processed using sterile and non-pyrogenic materials and procedures. At regular time intervals, samples were cold spun, separated, flash frozen and assayed for the appropriate cytokine using RIA/IRMA methods. In a further study, timed separation was repeated with spiked blood from healthy volunteers (n = 5 per cytokine) using normal commercially available blood collection materials and procedures. In a third study, spiked blood from healthy volunteers (n = 3 per cytokine) was processed under sterile and non-pyrogenic conditions, and the blood samples separated, aliquoted and flash frozen within half hour of collection. These were then subjected to repeated cycles of freeze thawing at 4 degrees C or RT before assaying. In general, the stability of cytokines in whole blood was improved by storage at 4 degrees C and/or rapid separation. There was no significant difference between samples handled under sterile, non-pyrogenic conditions and those collected using normal blood collection procedures. The blood collection procedures described in this paper did not induce any of the six cytokines in the unspiked blood. Overall, EDTA-treated samples performed most consistently. The addition of trasylol did not significantly affect the results. Most of the cytokines appeared unaffected by up to three freeze thaw cycles. The stability and recovery of the spiked cytokines varied from least stable to most stable spiked cytokine as follows; TNF-alpha less than IL-6 less than IFN-gamma less than IL-1 alpha less than IFN-alpha less than IL-1 beta. The recovery of spiked IFN-gamma from heparinized plasma samples was considerably higher than any other plasma or serum samples. The recovery of spiked TNF-alpha and IL-6 from serum samples was consistently lower than amounts recovered from plasma samples (anticoagulant treated).(ABSTRACT TRUNCATED AT 400 WORDS)

Background: We have previously immunomagnetically separated cancer cells from ascites samples of patients with ovarian cancer who went on to receive chemotherapy. We reported that a raised p-S6K in cancer cells was associated with chemoresistance (Carden C, et al, Mol Cancer Ther 2012;11:1609-17). We hypothesized that combining chemotherapy with a novel dual m-TORC1/2 inhibitor would be effective in this setting. Methods: We studied apoptosis induced by paclitaxel (P), AZD2014 (A) and the combination (C) on 2 ovarian (A2780Cis, SKOV3), 2 lung (PC9, H520), 2 breast (MCF7, SKBR3) and 1 endothelial cell line (HUVEC) by quantifying cleaved-PARP using ELISA. We further investigated the effects of each drug and the combination on tumor growth and signal transduction (pSer473-AKT, p-S6) with a combination of P (20 mg/kg/week) and A (50 mg/kg/3 days/week) in 2 xenograft models (A2780Cis and H520). We also designed an investigator-initiated clinical trial of the combination of P 80 mg/m2/week in combination with an escalating dose of A administered twice a day in 2 schedules (2/7 and 3/7 started concomitantly with the P infusions). Radiological response was measured by RECIST and clinical benefit (CB) defined as progression at 14 weeks or later. Results: There was a major increase in apoptosis induction, as determined by c-PARP levels quantitation, in 3/6 cancer cell lines and the endothelial cell line HUVEC when A was added to P when compared to P alone utilizing concentrations of both drugs at GI50 and 5 x GI50 for 24 hrs. Additive growth inhibition was observed in both xenograft models after 2 weeks of combination treatment with tumor volume increased by 281 ± 98% with the combination (p Conclusions: The combination of P+A is effective in preclinical ovarian and lung cancer models, with additive growth inhibition and apoptosis seen. Promising drug combination antitumor activity has also been reported in heavily pre-treated patients with ovarian cancer and squamous NSCLC in an ongoing clinical trial. Citation Format: Parames Thavasu, Anne-Christine LF Wong Te Fong, Begona Jimenez Rodriguez, Bristi Basu, Alison Turner, Emma Hall, Timothy A. Yap, Susana Banerjee, Martin Leach, Johann S. de Bono, Yuen-Li Chung, Udai Banerji. Translating preclinical observations to the clinic: Combination of the dual m-TORC1/2 inhibitor AZD2014 and paclitaxel in ovarian and lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT138. doi:10.1158/1538-7445.AM2015-CT138

Introduction and Aims Efficacy of targeted anticancer drugs is limited by de novo resistances related to cross talk within and between complex signal transduction networks. Rational combinatorial targeted therapies are one of the main solutions to increase activity or overcome resistance. We aimed to investigate the effects of inhibiting different nodes within the MAPK pathway (RAF and MEK) and PI3K-AKT pathway (PI3K, AKT and m-TORC1/2) either alone or in different combinations, on cell growth in a panel of KRAS mutant and KRAS wt NSCLC cell lines. Material & Methods We used dabrafenib, trametinib, GDC-0941, MK2206 and AZD2014 to inhibit RAF, MEK, PI3K, AKT and m-TORC1/2 respectively. Inhibition of signalling output in MEK, PI3K, AKT and m-TORC1/2 was assessed by maximal reduction of p-ERK, p-AKT (Thr308), p-AKT (Ser473) and p-S6 by trametinib, GDC-0941, MK2206 and AZD2014 respectively. In keeping with its mechanism of action, maximal induction of p-MEK was considered as inhibition of RAF by dabrafenib in the cell line panel (none had mutations in BRAF). Quantification of phosphoproteins was done using MesoScale Discovery ELISA. Using concentrations of drugs shown to inhibiting signalling though these nodes, the effects of inhibiting the nodes alone or in combination on cell growth was studied using WST-1 assays. The NSCLC cell line panel included KRAS wild-type (H522; H1838; H1651) or KRAS mutant (A-549; Calu-6; H23) cells. Results The concentration range of dabrafenib, trametinib, GDC-0941, MK2206 and AZD2014 to inhibit RAF, MEK, PI3K, AKT and m-TORC1/2 related to the different cell lines was [40-1000 nM], [40-200 nM], [1000 - 10 000 nM], >50 000 nM, and [500-1500 nM] respectively. The degree of growth inhibition following inhibition of an individual node varied between 5% and 75%. Highest inhibitions of cell proliferation by drugs alone were obtained with PI3K-AKT pathways compared to the MAPK pathway inhibitors for 6/6 cell lines. Drug combinations improved inhibition of cell proliferation in all cell lines. In 3/3 KRAS mutant cells, best combinations included MEK inhibitor and any other of the PI3K-AKT pathway as compared to inhibition of different nodes within the PI3K-AKT or MAPK pathways. In 3/3 KRAS wt cell lines, vertical combinations within the PI3K-AKT pathway induced highest inhibitions of cell proliferation compared to vertical combinations within the MAPK pathway or horizontal combinations between the PI3K-AKT and MAPK pathways. Discussion/Conclusions In the cell line panel tested, KRAS mutant cell lines were more sensitive to horizontal combinations than vertical combinations with the MAPK and PI3K-AKT pathways. KRAS wt cells were more sensitive to vertical combinations within the PI3K-AKT pathway. This preclinical study has clinical implications while planning combinations of targeted agents to treat NSCLC. Citation Format: Sophie Broutin, Adam Stewart, Parames Thavasu, Angelo Paci, Jean-Michel Bidart, Udai Banerji. The effect of horizontal and vertical inhibition of nodes within the MAPK and PI3K-AKT pathways in NSCLC models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5343. doi:10.1158/1538-7445.AM2015-5343

Specific antitumor immune responses require expression of MHC class I or II molecules on tumor cells, and MHC antigen down-regulation is a presumed tumor growth promoting mechanism. Because IFN-gamma up-regulates tumor MHC antigen expression in vitro, in this Phase II trial of an immunologically active dose and schedule we evaluated whether this was the case in vivo. Twenty-three patients with metastatic melanoma were treated with IFN-gamma 100 microg/m(2) s.c. once weekly for a maximum of 6 months. There were three complete responses, now maintained for 53, 36, and 25 months. The remainder had progressive disease. The treatment was well tolerated, with no toxicity exceeding National Cancer Institute Common Toxicity Criteria grade II. Immunohistochemical analysis of tumor biopsies during treatment was performed using monoclonal antibodies to HLA class I (W/632) and class II (CR3/43) monomorphic determinants. HLA class I was down-regulated in 2 of 19 patients pretreatment and up-regulated by IFN-gamma in both. HLA class II was down-regulated pretreatment in 14 of 18 patients and up-regulated by IFN-gamma in 6 (43%). The HLA up-regulation persisted throughout the study. IFN-gamma induced significant but short-lived up-regulation of surrogate markers of monocyte activation (serum neopterin) and class I up-regulation (serum beta-2-microglobulin) in most patients. There was no consistent relationship between surrogate marker up-regulation, tumor antigen up-regulation, and responses. The study shows that the significant immune modulation induced by IFN-gamma does not correlate with tumor responses and that the serum surrogate marker changes do not reflect tumor events. The durable and long-lived responses, clear demonstration of tumor MHC up-regulation, and low toxicity suggest that weekly IFN-gamma 100 microg/m(2) would be a useful addition to chemoimmunotherapeutic regimens.

Background: Lapatinib is a tyrosine kinase inhibitor that inhibits EGFR and HER2 and is licensed to treat HER2-amplified breast cancer. Understanding mechanisms of acquired resistance is an unmet clinical need. Methods: SKBR3 cells were continuously exposed to increasing concentrations of lapatinib over a 12-month period. The parent cell line (SKBR3) and resulting lapatinib-resistant cell line (SKBR3LR) then underwent a set of studies including: a) western blot analysis for PGP, b) whole exome sequencing, c) whole genome expression microarray, d) quantification of 587 phosphoproteins. All data were integrated using Metacore software. Sensitivity of SKBR3 and SKBR3LR to a panel of drugs based on the findings was carried out by 96-hr sulforhodamine assays. Results: The GI50 values for lapatinib in SKBR3LR and SKBR3 cells were 3582 nM (SD 241.6) and 10.3 nM (SD 1.1). There was no induction of PGP protein or mRNA expression levels in SKBR3LR compared to SKBR3. Somatic mutations in KIT and MEK3 which were not present in SKBR3 were detected in SKBR3LR. There was a reduction in the levels of p-HER2 and p-EGFR in SKBR3LR; however, no differences in the levels of p-HER3, p-HER4, and p-KIT between the cell lines were observed. Analysis of proteins downstream in the RAS-RAF-MEK and PI3K-AKT-m-TOR showed increased levels of p-MEK but no significant changes in p38MAPK and no differences in p-AKT levels, but an increase in p-P70S6K in SKBR3LR compared to SKBR3. The phosphorylation network changes were integrated with transcription factors that showed significant enrichment for binding sites in the genes that were differentially expressed in SKBR3LR. Deregulation of the MAPK signalling network was one of the proposed mechanisms of resistance of SKBR3LR cells to lapatinib. Differential sensitivity to small-molecule agents was used to interrogate the mechanism of lapatinib resistance further. There was no significant differential sensitivity of SKBR3 and SKBR3LR to imatinib, GI50 values 16.2 uM (SD 1.2) and 13.64 uM (SD1.1), respectively. SKBR3LR was more resistant than SKBR3 to an AKT inhibitor AKT1/2i; GI50 values 8 uM (SD 0.45) vs 17.3 uM (SD 1.1) respectively. Interestingly, SKBR3LR was more sensitive to the HSP90 inhibitor, AUY922; GI50 values 3.2 nM (SD 1.1) and 11.2 nM (SD 0.42) in SKBR3LR and SKBR3 cells, respectively. Experiments are ongoing using targeted agents to understand the mechanism of lapatinib resistance and suggest potential therapeutic strategies to overcome these, including novel combinations, particularly the use of HSP90 inhibitors. Conclusions: Integrating genomic-transcriptomic and phosphoproteomic data provides useful insights into the mechanism of resistance to lapatinib. The lapatinib-resistant cell line model was sensitive to the HSP90 inhibitor, AUY922, suggesting HSP90 inhibitors could be clinically effective in this setting. Citation Format: Parames Thavasu, Robert Te Poele, Mark Hughes, Adam Stewart, Paul Workman, Udai Banerji. A genomic-transcriptomic-phosphoproteomic study to interrogate lapatinib resistance in an HER2 over expressing SKBR3 human breast cancer cell line model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1828. doi:10.1158/1538-7445.AM2014-1828

e13559 Background: Current development is focused on agents targeting the RAF-MEK-ERK and PI3K-AKT-mTOR pathways. Combinations of these agents may address causes of de novo resistance. Identifying patients (pts) most likely to benefit from these combinations is important. Methods: A panel of 20 cell lines (5 BRAFM, 5 KRASM, 5 PIK3CAM and 5 BRAF/PIK3CA/KRASWT) were exposed to MEK and/or AKT inhibitors and evaluated for inhibition of pathway signaling and cell growth. Inhibition of signaling output of the RAF-MEK-ERK (p-ERK) and PI3K-AKT-mTOR (p-S6) pathways was quantified by ELISA. Growth inhibition was determined using sulforhodamine B assay. Based on the results of preclinical experiments, at our center we prioritized enrolling pts with RAS mutant tumors (including ovarian cancer and non-small cell lung cancer [NSCLC]) into an ongoing phase 1b clinical trial (CMEK162X2109) evaluating the combination of MEK inhibitor binimetinib (MEK162) and the PI3K-α inhibitor BYL719 in pts with RAS and RAF mutant tumor...

Background: Epidermal growth factor receptor (EGFR) inhibition with tyrosine kinase inhibitors (TKIs) has proven successful in the management of patients with non small cell lung cancer (NSCLC). However, clinical benefit is predominantly limited to the subgroup of patients with an activating EGFR mutation (10%). Treating the remaining 90% of patients with wild type (wt) EGFR is an unmet clinical need. Aim: To evaluate the effect of an allosteric AKT inhibitor (AKTi 1/2) in combination with the EGFR inhibitor gefitinib on cell proliferation, apoptosis and signalling output in EGFR mutant/wt NSCLC cell lines. Methods: Four NSCLC cell lines were selected: NCI-H522 and NCI-H1651 (EGFR wt and K-RAS wt), PC9 and HCC827 (EGFR mutant). The 96hr sulphorhodamine assays was used to assess the effect of gefitinib and AKTi 1/2 on growth inhibition, and median effect analysis to calculate combination indices (CIs) to assess the effect of both drugs in combination. The expression of p-EGFR, p-AKT, p-S6, p-ERK and cleaved PARP were studied by Western blotting in the EGFR wt NCI-H522 and the EGFR mutant PC9 cell lines. Results: EGFR wt cell lines NCI-H522 and NCI-H1651 were relatively resistant to gefitinib (IC50 values of 7.0 ± 2.5 uM and 8.8 ± 0.9 uM, respectively) compared with the EGFR mutant cell lines PC9 and HCC827 (IC50 values of 0.07 ± 0.03 uM and 0.004 ± 0.0005 uM, respectively). The combination of gefitinib and AKTi 1/2 produced synergistic inhibition of growth in both EGFR wild type and mutant cell lines. Synergism was more marked in EGFR wt cell lines with CI values (ED50) of 0.53 ± 0.28 and 0.49 ± 0.17 for NCI-H522 and NCI-H1651 respectively, compared with 0.74 ± 0.11 and 0.76 ± 0.14 for the EGFR mutant cell lines PC9 and HCC827, respectively. Concomitant exposure of cells to gefitinib and AKTi 1/2 for 24 hrs caused incremental inhibition of p-AKT, p-S6 and p-ERK in both the EGFR wt (NCI-H522) and EGFR mutant (PC9) cell lines. Gefitinib alone resulted in partial inhibition of AKT phosphorylation in PC9 cells and up-regulation in NCI-H522 cells. Incremental induction of cleaved PARP, a marker of apoptosis, was seen in PC9 cells treated with the combination of gefitinib and AKTi 1/2. Conclusions: These results demonstrate synergistic growth inhibition of EGFR wt and mutant NSCLC cell lines by the combination of gefitinib with AKTi 1/2. Additional pre-clinical studies are investigating this further. Clinical studies of the combination doublet are warranted to expand the utility of EGFR TKI in EGFR wt NSCLC. Citation Format: Martina Puglisi, Parames Thavasu, Adam Stewart, Jaishree Bhosle, Sanjay Popat, Mary E R O'Brien, Udai Banerji. Evaluation of combination of an EGFR and AKT inhibitor in EGFR mutant and EGFR wild type non-small cell lung cancer cells lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2441. doi:10.1158/1538-7445.AM2013-2441

Purpose We hypothesized that understanding the relationships between combinatorial inhibition of signalling output and growth in cell line models will help design clinical trials of combinations of MEK and AKT inhibitors which are currently challenged by overlapping toxicity. Methods We exposed two BRAF mutant cell lines (WM266.4 and SKMEL-28), two PIK3CA mutant cell lines (MCF-7 and SKOV-3) and a cell line with no BRAF or PIK3CA mutations (Caco2) to increasing concentrations of a MEK inhibitor (MEKi) and an AKT inhibitor (AKTi), PD0325901 and AKT11/2 kinase inhibitor respectively, for 24hrs. ELISAs were used to quantify signalling output downstream of MEK and AKT i.e. p-ERK and p-S6 respectively. Concentrations of MEKi and AKTi that inhibited phosphorylation of 0, 25, 50, 75 and 100% of ERK and S6 respectively were calculated in these models. The cell line panel was then exposed to different functionally inhibitory concentrations of both MEKi and AKTi in combination and effects on growth inhibition was quantified by 96hr sulforhoamine B assays. Results In BRAF mutant lines WM266.4 and SKMEL-28, the growth inhibition (GI) by maximally inhibiting p-ERK by MEKi was 83.2 % and 64 % respectively. There was no statistical difference on GI by additionally maximally inhibiting p-S6 in addition to maximal p-ERK inhibition in these cell lines (86.9 vs 83.2 % p=0.34 and 74% vs 64%, p= 0.27 respectively). In PIK3CA mutant cell lines MCF-7 and SKOV3 the GI by maximally inhibiting p-S6 by AKTi was 93 % and 69.4 % respectively. There was no statistical difference on growth inhibition by additionally maximally inhibiting p-ERK in addition to maximal p-S6 inhibition in these cell lines (94.6 vs 93 % p=0.34 and 79.6% vs 69.4%, p= 0.05 respectively). In Caco2 which does not harbour either a BRAF or PIK3CA mutation, the GI by maximally inhibiting p-ERK was 48.9 % and additionally maximally inhibiting p-S6 caused a significantly improved growth inhibition (78.1 vs 48.9%, p= 0.0007). GI by maximally inhibiting p-S6 was 64.1% and additionally maximally inhibiting p-ERK caused a statistically significant improved growth inhibition (78.1 vs 64.1%, p=0.0203) in this cell line model. In Caco2 cells the incremental significant GI caused by the addition of AKTi and MEKi started at concentrations that inhibited 50% of p-S6 and 75% of p-ERK respectively. Conclusions In the cell line panel studied, significant additional growth inhibition by maximally inhibiting p-ERK and p-S6 in combination occurred in BRAF and PIK3CA wt cells and not in those that harboured activating mutations in these genes. Titration of the degree of p-ERK and p-S6 inhibition needed to maximally inhibit growth in combination may obviate the necessity to completely inhibit both p-ERK and p-S6. These findings have important implications in the way we design phase I/II trials of combinations of MEK and AKT inhibitors. Citation Format: Adam Stewart, Parames Thavasu, Johann S. de Bono, Udai Banerji. A study of relationships between signalling output and growth inhibition to personalize combinations of MEK and AKT inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2341. doi:10.1158/1538-7445.AM2013-2341

Plasma cytokines and immune markers were assessed during the clinical management of 42 patients with multiple myeloma, MM. Of the patients 22/42 (all with progressive disease) were studied from the time of diagnosis, through various treatment regimes, to remission, progression or death. 5/42 patients had monoclonal gammopathy of undetermined significance (MGUS), 8/42 others had either indolent MM or stable MM, and a further 7/42 with progressive disease were also studied. IL-6, TNF-alpha, IL-1 alpha, IL-1 beta, beta 2 microglobulin (beta 2M), and neopterin were estimated in bloods taken under optimal conditions for cytokine detection. The levels were compared with a panel of samples from healthy volunteers. Both immunoreactive and biologically active plasma IL-6 levels were measured. Pretreatment IL-6 levels (both immunoreactive and biologically active) were found to correlate with severity of disease. In 13/22 patients with progressive disease who had been followed from the time of diagnosis over a 12-month period or until death, pretreatment IL-6 levels were predictive of response to therapy. Elevated plasma levels of TNF-alpha, beta 2M and neopterin were found in patients with progressive multiple myeloma, and this correlated with renal impairment. The analytes measured during the course of chemotherapy did not show correlation with disease progression or response to therapy.

Background Weekly administration of paclitaxel is increasingly used to treat patients with platinum-resistant ovarian cancer. We have previously reported that activation of the phosphatidylinositide-3-kinase pathway as measured by p-p70S6K is associated with resistance to chemotherapy in studies using ovarian cancer cells isolated from ascites (Carden et al, 101st AACR Annual Meeting, 2010; Abstract no. 788). We thus set out to explore the combination of paclitaxel and an inhibitor of the mTORC1/2 complex, AZD2014, in ovarian cancer cell line models. Purpose We aimed to study the effects of combining paclitaxel and the mTORC1/2 inhibitor AZD2014 on growth inhibition in a panel of human ovarian cancer cell lines. We also studied the effects of drug administration schedule on growth inhibition. Methods A panel of 5 human ovarian cell lines was studied: A2780, CH1, SKOV3, IGROV and OVCAR-3. The effects of paclitaxel and AZD2014 administered concomitantly on cell growth were studied using 96hr sulforhodamine B assays. Combination indices (CI) were calculated using median effect analysis and a one-sided t-test was performed to calculate significant differences from a hypothetical value of 1 (indicating additivity). All experiments were carried out in triplicate. Results The combination was additive when drugs were administered concomitantly. In addition, in A2780 and SKOV3 cell lines exposed to paclitaxel followed by AZD2014 the combination indices were 1.112 and 1.099 respectively. Conclusion AZD2014, a novel mTORC1/2 inhibitor, and paclitaxel have additive growth inhibitory effects in a panel of ovarian cancer cell lines when administered concomitantly. Further experiments to understand the effects of this combination on apoptosis and signal transduction are ongoing. The combination of AZD2014 and paclitaxel has potential applications in the treatment of ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 931. doi:1538-7445.AM2012-931

Background: AKT pathway activation is commonly described in ovarian cancer, and implicated in preclinical models of chemoresistance. Ascites occurs in a high proportion of patients at some stage in their illness. Purpose: To study the relationship between activation of the AKT pathway in human ovarian cancer ascites specimens (measured by the ratio of phosphorylated (P) to total (T) AKT pathway proteins) to patient survival and response to chemotherapy. In addition we studied the correlation of P/T AKT ratio to carboplatin sensitivity in a human ovarian cancer cell line panel. Method. 99 samples of ascites from 75 patients with ovarian cancer and a known treatment history were purified using anti-EpCam antibody coated magnetic beads (Dynal Biotech), stored at −80 degrees C, and then analysed with Mesoscale Discovery (MSD) ELISA for P/T AKT, GSK3β and p70S6K, and Pathscan ELISA for P/T 4EBP1 . Differences in survival in patient subgroups were analysed by logrank tests. Five cell lines (A2780, CH1, IGROV1, OVCAR3, SKOV3) were analysed in triplicate by sulphurhodamine B colorimetric assay to establish half maximal inhibitory concentration (IC50) for carboplatin and PI3K-AKT-mTOR pathway inhibitors (rapamycin, LY 294002, PI103, and Merck AKT inhibitor VIII), and the results correlated with baseline AKT pathway protein activation ratios. Results: Samples from patients who responded to chemotherapy (n=32) subsequent to sample collection had a lower median P/T S6K ratio (0.43) than those (n=28) who progressed (0.71, p=0.026, Mann-Whitney test; 39 samples were from patients who received no subsequent chemotherapy). Analysing the first sample for each patient, (n=75), those with a P/T AKT ratio above median (0.140) had a statistically significantly longer survival than those patients below median (28.0 months (m) vs 49.7 m, HR 1.74 (95% CI 1.01-3.01), p=0.048). A similar trend for P/T S6K ratio was also found, however this was not statistically significant (22.0 m vs 47.5 m, HR 1.56 (95%CI 0.90-2.71), p=0.112). In the ovarian cell line panel studied, a high P/T AKT ratio was found in the cells most resistant to carboplatin, whereas there was no relationship between AKT pathway activation and resistance to the AKT pathway inhibitors. Potential reasons for activation of the AKT pathway include amplification and mutation of PIK3CA and AKT2. Fluorescence in-situ hybridisation and sequencing of somatic DNA from these samples are ongoing. Conclusion: Patients who responded to subsequent chemotherapy had a significantly lower P/T S6K ratio than those who progressed. A P/T AKT ratio greater than 0.144 in malignant ascites predicts a statistically significant poorer prognosis. In vitro cell line models suggest a trend of P/T AKT ratio to correlate with carboplatin resistance. P/T AKT ratio should be studied prospectively as a prognostic and predictive biomarker in ovarian cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 788.