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Purpose: Deregulated phosphatidylinositol 3-kinase pathway signaling through AGC kinases including AKT, p70S6 kinase, PKA, SGK and Rho kinase is a key driver of multiple cancers. The simultaneous inhibition of multiple AGC kinases may increase antitumor activity and minimize clinical resistance compared with a single pathway component. Experimental Design: We investigated the detailed pharmacology and antitumor activity of the novel clinical drug candidate AT13148, an oral ATP-competitive multi-AGC kinase inhibitor. Gene expression microarray studies were undertaken to characterize the molecular mechanisms of action of AT13148. Results: AT13148 caused substantial blockade of AKT, p70S6K, PKA, ROCK, and SGK substrate phosphorylation and induced apoptosis in a concentration and time-dependent manner in cancer cells with clinically relevant genetic defects in vitro and in vivo. Antitumor efficacy in HER2-positive, PIK3CA-mutant BT474 breast, PTEN-deficient PC3 human prostate cancer, and PTEN-deficient MES-SA uterine tumor xenografts was shown. We show for the first time that induction of AKT phosphorylation at serine 473 by AT13148, as reported for other ATP-competitive inhibitors of AKT, is not a therapeutically relevant reactivation step. Gene expression studies showed that AT13148 has a predominant effect on apoptosis genes, whereas the selective AKT inhibitor CCT128930 modulates cell-cycle genes. Induction of upstream regulators including IRS2 and PIK3IP1 as a result of compensatory feedback loops was observed. Conclusions: The clinical candidate AT13148 is a novel oral multi-AGC kinase inhibitor with potent pharmacodynamic and antitumor activity, which shows a distinct mechanism of action from other AKT inhibitors. AT13148 will now be assessed in a first-in-human phase I trial. Clin Cancer Res; 18(14); 3912–23. ©2012 AACR.

Biomarkers have many applications along the care process in rare cancers and have potential to optimize treatment decisions. However, many diagnostic tests struggle to gain market access and appropriate value in terms of payer coverage because of a lack of evidence on their health economic benefit to patients. CMS reimbursement guidelines, of cost-plus pricing for diagnostics does not always work for niche small rare disease subpopulations. Nor does it reflect the lifelong value of effective patient care. To address this, we have developed a cost benefit model for early diagnostic intervention, based on retrospective data to determine mutation status and monitor for disease progression in people diagnosed with neurofibromatosis type 1 (NF1) and soft tissue tumors. NF1 is an autosomal dominant hereditary cancer syndrome that affects around 1:2,500 people and is caused by mutations in the NF1 gene. 50% of patients can have few to many neurofibroma which vary in clinical behavior from benign to malignant. About 10% of people can develop malignant peripheral nerve sheath tumors (MPNSTs) thought to develop from pre-existing benign neurofibroma. Currently, there is no reliable way of knowing which individual and which tumors are malignant. MPNSTs are often difficult to diagnose in the early phases with current imaging methods like CT or MRI. Overall, these patients face a lifetime risk of cancer up to 16-26% higher than the normal population. Once metastasizes MPNSTs are associated with poor prognosis, less than 25% survival rate at 5 years. We built a model used retrospective anonymized patient treatment records to demonstrate cost and risk based benefits to patients, payers and clinical practitioners, on the effect of early intervention versus late on treatment cost, and patient outcome in the long-term care process. For NF1 patients. In conclusion, our model proposes a new framework to engage healthcare insurers and managed care physician groups in the US to quantify the health economic benefits of prognostic biomarker tests in patient populations with a lifelong genetic predisposition to cancer. Citation Format: Lydia Turkson, Hannah Mamuszka, Kyla Grimshaw, Erica Marie Marshall. MPNST treatment and diagnosis in NF1: A health economic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5288.

The serine/threonine kinase AKT plays a pivotal role in signal transduction events involved in malignant transformation and chemoresistance and is an attractive target for the development of cancer therapeutics. Fragment-based lead discovery, combined with structure-based drug design, has recently identified AT7867 as a novel and potent inhibitor of both AKT and the downstream kinase p70 S6 kinase (p70S6K) and also of protein kinase A. This ATP-competitive small molecule potently inhibits both AKT and p70S6K activity at the cellular level, as measured by inhibition of GSK3β and S6 ribosomal protein phosphorylation, and also causes growth inhibition in a range of human cancer cell lines as a single agent. Induction of apoptosis was detected by multiple methods in tumor cells following AT7867 treatment. Administration of AT7867 (90 mg/kg p.o. or 20 mg/kg i.p.) to athymic mice implanted with the PTEN-deficient U87MG human glioblastoma xenograft model caused inhibition of phosphorylation of downstream substrates of both AKT and p70S6K and induction of apoptosis, confirming the observations made in vitro. These doses of AT7867 also resulted in inhibition of human tumor growth in PTEN-deficient xenograft models. These data suggest that the novel strategy of AKT and p70S6K blockade may have therapeutic value and supports further evaluation of AT7867 as a single-agent anticancer strategy. Mol Cancer Ther; 9(5); 1100–10. ©2010 AACR.

Successful drug development in oncology requires a deeper understanding of the functional consequences of the diverse genetic changes observed in human cancers. For example, responses to epidermal growth factor receptor (EGFR) inhibitors are observed in patients whose tumors express EGFR alleles with activating mutations, rather than in tumors overexpressing EGFR. Furthermore, antibodies against EGFR are ineffective in tumors bearing certain activating alleles of KRAS. Horizon Discovery has used its proprietary rAAV gene engineering technology to generate isogenic cell lines covering a range of mutations commonly found in cancer patients. Use of a non-tumorigenic ‘clean’ cell line background such as MCF10A allows specific evaluation of the mutations without any confounding factors due to the presence of other genetic alterations. Mutations introduced into cancer cell line backgrounds allow the contextual evaluation of a cancer related gene. Here we describe the use of isogenic cell line panels as powerful tools for investigating sensitivity and resistance markers to cancer therapeutics. Some 50% of human tumors exhibit p53 loss or inactivation. To investigate how p53 loss in combination with other common cancer-driving mutations may influence therapeutic responses, we have generated a suite of MCF10A isogenic cell lines covering some of the major cancer genotypes, either in isolation or on a TP53 (-/-) background. These genotypes include EGFR (delE746-A750/+), EGFR (L858R/+), KRAS (G12V/+), BRAF (V600E/+), BRAF (V600K/+) and PIK3CA (H1047R/+). Thus, we have been able to investigate the interaction effects of discrete mutations in molecularly defined, but more tumor-like cell models. One data highlight arose from the profiling of the EGFR mutant panel using small molecule EGFR inhibitors; in isolation, the introduction of common activating EGFR mutations L858R or deletion of E746-A750 led to increased sensitivity, recapitulating clinical findings. However, combining EGFR mutation with loss of p53 further enhanced the cell response. Through systematic profiling of this panel to targeted therapeutic agents such as gefinitib, selumetinib, vemurafenib, and pictilisib, we have identified interesting differential sensitivities, which can be directly attributable to introduction of a given mutation. Results such as these can enable better patient stratification for anticancer agents, and allow incorporation of molecular markers into clinical trial design for personalised therapeutic regimens. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A148. Citation Format: Annette S. Little, Jessica Hunt, David Hughes, Ruth Feltell, Daniel Gitterman, Rachel Leah, Holly Astley, Ramu Mangena, Kyla Grimshaw, Christopher Torrance. Modeling patient responses to targeted therapy with rAAV mediated gene editing. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A148.

Cancer is a genetic disease, and as such, genetic variation is almost exclusively responsible for onset and progression, and critically is also a key component in an individual's response to therapeutics. Understanding the complex of genetics of cancer and identifying the key mutations driving phenotype requires patient-relevant disease models. Horizon Discovery's expertise in translational genomics has led to the development of GENESIS™ a proprietary genome-editing platform based on the unique ability of rAAV to stimulate homologous recombination. By exploiting a cell's natural means of repairing its own DNA in an error-free manner, rAAV GENESIS™ enables the precise insertion, deletion or substitution of a desired DNA sequence into any endogenous gene loci in mammalian cells. Horizon has generated over 500 X-MAN™ (gene-X mutant and normal) isogenic cell lines, the world's first source of genetically-defined and patient-relevant human cellular models. We are currently using multiple panels of these disease models in combination with high-throughput RNAi screens to create a large scale synthetic lethality target identification platform. Our platform consists of lines that are identical except for the targeted modification, enabling us to identify gene target hits that have tumor selective properties. Here we have selected isogenically matched HCT116 parental, p53 null (-/-) and p53 mutant (R248W/+) lines; by use of reverse transfection methodologies we achieved >80% knockdown efficiency for a library of over 2,200 druggable targets. This approach yielded targets that selectively induce lethality in the presence of a p53 mutation for both the null (-/-) and mutant lines (R248W/+), interestingly two of the top hits inducing synthetic lethality in the p53 null lines are p21 and the p21 binding partner CDKN1A. The full power of these experiments comes from comparisons of the isogenically matched lines. Our results demonstrate that the p53 null lines (-/-) have a marked resistance to siRNA mediated synthetic lethality when compared to the mutant (R248W/+) lines, hit rates being 0.6% and 13% respectively. The number of p53 R248W/+ mutant hits are also of great interest. It is well reported that p53 transgenic mice rapidly succumb to certain types of cancers not commonly observed in p53 null mice (Song et. al. Nat Cell Biol. 2007). It is thought this mutation promotes tumorigenesis by a novel mechanism involving active disruption of critical DNA damage-response pathways. This mechanism of action clearly renders these cells more susceptible to therapeutic intervention. Overall these results highlight the challenge of identifying therapeutics that are able to target p53 null tumors. The mutant (R248W/+) hits, however may offer a therapeutic opportunity, especially when considering that mutations at this locus are the most commonly occurring p53 mutations reported in human tumors. Citation Format: Jo Francis, Jessica Hunt, Kyla Grimshaw, Agustin Chicas, Lihua Yu, Ping Zhu, Markus Warmuth, Chris Torrance. X-MANTM isogenic disease models: Precision tools for synthetic lethality screening. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B40.

The AGC kinase AKT is a key component of the phosphatidylinositol 3-kinase (PI3K) pathway, which is frequently deregulated in cancer, making AKT a target of major therapeutic interest. However, PI3K signaling through both AKT-dependent and AKT-independent mechanisms involving other AGC kinases, such as p70S6K, PKA, SGK and ROCK, is important in a range of cancers. Hence, the pharmacological inhibition of these multiple AGC kinases may increase response rates and minimize clinical resistance compared with targeting AKT alone. The clinical drug candidate AT13148 is a multi-AGC kinase, ATP-competitive inhibitor, identified utilizing high-throughput X-ray crystallography and fragment-based lead discovery techniques. Screening of this oral small molecule against a panel of kinases at 10μM revealed >80% inhibition of the structurally related AGC kinases AKT, PKA, ROCK2, p70S6K, MSK, RSK1/2, and SGK. We demonstrate that AT13148 has antiproliferative activity in a range of in vitro models harboring relevant genetic abnormalities, including PTEN, KRAS, PIK3CA and HER2 aberrations. AT13148 caused substantial blockade of AKT, p70S6K, PKA, ROCK and SGK substrate phosphorylation and induction of apoptosis in both a concentration and time-dependent manner in cancer cells with clinically relevant genetic defects both in vitro and in vivo. Antitumor efficacy in HER2-positive, PIK3CA-mutant BT474 breast, PTEN-deficient PC3 human prostate cancer and PTEN-deficient MES-SA uterine tumor xenografts was demonstrated. We show for the first time that induction of AKT phosphorylation at serine 473 by AT13148, as reported for other ATP competitive inhibitors of AKT, is not a therapeutically relevant reactivation step for this compound. We used gene expression microarray studies to characterize the underlying molecular mechanisms of action of AT13148 and the selective AKT inhibitor CCT128930, and observed the induction of upstream regulators including insulin receptor substrate-2 and PIK3IP1 due to compensatory feedback loops, consistent with blockade of AKT signaling. These studies also showed that AT13148 and CCT128930 have distinct molecular effects in cancer cells: AT13148 had a predominant effect on apoptosis genes and caused a greater apoptotic phenotype, while CCT128930 modulated genes in the network regulating cell cycle. This finding emphasizes the functional differences of AT13148 as a multi-AGC kinase inhibitor in contrast to a more AKT-selective inhibitor. In view of the potential mechanistic advantages detailed above, and the potent antitumor activity observed at well tolerated doses against established human tumor xenografts with clinically relevant genetic drivers, the clinical utility of such an AGC kinase inhibitor strategy will now be assessed in a first-in-human Phase I trial of AT13148. 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 928. doi:1538-7445.AM2012-928

Ras mutations are present in approximately 49% of human colorectal cancers, with mutations in K-Ras G12 and G13 being the most common. Given their high prevalence, understanding how Ras mutations impact response to anticancer agents is crucial to the successful development of novel agents. Recent evidence suggests that not all variants of K-Ras respond in the same way to targeted agents, where some patients with G13D mutations respond better to Cetuximab compared to patients expressing G12V (De Roock, JAMA 2011). Therefore there is a need for better in vitro models to facilitate the characterisation of inhibitor selectivity and efficacy in K-Ras mutant cell systems. We created suites of isogenic X-MAN SW48 and LIM1215 colorectal cancer cells, each of which harbour one of 7 different K-Ras G12 or G13 mutations at their endogenous loci introduced via AAV-mediated homologous recombination. This system allows use of endogenous promoters and has enabled us to create a panel of human cell lines in which the only difference between the cells is the point mutation of interest. This more accurately recapitulates the genomic architecture in human tumors and provides an excellent platform for addressing the impact of K-Ras mutations on responses to therapeutic agents. We selected over 30 targeted agents and cytotoxic drugs based on their clinical relevance to colorectal cancer. We profiled the anti-proliferative activity of each of these across the suite of K-Ras variants in SW48 cells. Strikingly, we found a consistent pattern of resistance towards 5 chemically distinct MEK1/2 inhibitors across the 7 K-Ras variants compared to wild-type cells. These results were confirmed in a second suite of K-Ras mutant lines generated in LIM1215 cells, where clear resistance to MEK inhibitors was observed. To investigate the mechanism of this resistance to MEK inhibitors, we combined the anti-EGFR monoclonal antibody Cetuximab with the MEK inhibitor AZD6244, both compounds being ineffective as single agents in the K-Ras G12D and G12C mutant SW48 cells. Concentrations of Cetuximab as low as 0.01μg/ml re-sensitised previously resistant K-Ras mutant cells to the effects of AZD6244. Given that this dose of Cetuximab was ineffective alone, this result suggests that upregulation of the EGFR axis may be a potential mechanism in compensating for MEK inhibition, and these studies are on-going. In conclusion, we have created two suites of isogenic colorectal cancer cell lines each expressing a different K-Ras G12 or G13 mutation and demonstrated that K-Ras mutations impart resistance to clinical MEK inhibitors. Despite screening over 30 chemotherapeutics covering an array of targets, we did not identify any other significant differences in sensitivity between K-Ras wild-type and mutant cells, demonstrating the mechanistic relevance of this model to MEK inhibitors. Our data highlight the advantages of using this system in profiling molecularly targeted agents in order to identify potential mechanisms of resistance and in rationally identifying combinations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A70.

Background: Human derived cell lines represent an important model system for fundamental research and translational medicine. We sought to gather patient-relevant information by examining a previously published metastasis score (MS) (proliferation index) for operable breast cancer in well-characterized isogenic cell lines. We chose to examine the impact on MS of specific genetic alterations in known oncogenes and consequence of treatment with a class I PI3K inhibitor. Investigation of the proliferation index of cells without the complexity of cell mixtures found in tumor microenvironment may shed light on tumor cell-specific transcriptional dysregulation. Methods: Nontumorigenic MCF10A and isogenic cell lines harboring (G12V), PI3Kα (H1047R) and p53 (null) mutations were cultured in T75cm tissue culture flasks overnight. MCF10A and isogenic PI3Kα (H1047R) cell lines were seeded and allowed to adhere overnight before addition of 0.3 µM, 1 µM and 3 µM GDC-0941 or DMSO control and incubated for 24 hours. Total RNA was prepared using RNeasy Kit (Qiagen) from cells. Expression profiling was carried out using RT-PCR for the previously described metastasis score (Tutt et al, 2009) that includes 14 genes and 3 reference genes. Results: MCF10A and the isogenic mutant cell lines had equivalent levels of proliferation as measured by MS and similar phenotypes under these monolayer culture conditions with high levels of growth factors. Modest dose-dependent decreases in the expression level of the constituent MS genes (1.5-3 fold) were observed with GDC-0191 exposure that translated into a marked decrease in overall MS (14 to 4) due to their equally weighted additive impact. The four genes that demonstrated the greatest decrease were CCNB1, BUB1, MYBL2, and UBE2S. These genes are highly associated with cyclin-dependent kinase (CDK1), a key player in cell cycle regulation. The MS score for these isogenic lines are also being defined under in vitro assay conditions that more accurately mimic the tumor microenvironment; in particular a 3-dimentional Matrigel assay that unleashes a marked differential metastatic/invasive phenotype in mutant vs. WT cells. Conclusion: MCF10A and isogenic cell lines harboring KRAS (G12V), PI3Kα (H1047R) and p53 (null) mutations had equivalent levels of proliferation as measured by MS. These results suggest that a combination rather than a single oncogenic mutant are required to alter the MS, although in vitro conditions likely play an important role in fully unmasking genotype-specific phenotypes in these clean gain-of-function models. Exposure of parental and PI3K (H1047R) cell lines to PI3K inhibitor GDC-0941 led to a dose-dependent decrease in constituent genes and composite MS. These results suggest that even though genes in the PI3K/PTEN pathway are not included in the molecular score, perturbation of this signaling pathway leads to alteration of the MS, by affecting cell cycle progression. Future studies are required to determine if alteration of MS in cell lines treated with different targeted oncogenic pathway inhibitors will recapitulate patient tumor response. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A33.

The tumour microenvironment promotes selective outgrowth of cells carrying mutations in oncogenes or tumour suppressors, enabling the activated pathways to drive tumour growth. Two such pathways are the PI3-K/AKT and RAS/RAF/MEK signaling pathways, which demonstrate a high frequency of mutations in human cancer. These pathways represent important therapeutic targets, and a number of targeted agents are now in clinical trials. Despite being pertinent to the clinical situation, the role that the tumour microenvironment plays in modifying responsiveness to therapeutic agents targeted against these pathways is poorly understood. A major challenge faced during the development of PI3-K/AKT and RAS/RAF/MEK targeted agents is to demonstrate inhibitor selectivity and efficacy in a patient-relevant cell based system. Isogenic ‘X-MAN’ paired cell lines help address this by providing a system in which the endogenous wild-type or mutant alleles of the parental cell line are selectively activated or silenced via targeted homologous integration, providing a pair of human cell-lines in which the only difference between the cells is the mutation of interest. This approach therefore overcomes the issues that may occur when over-expression methods are employed. We therefore set out to investigate how changes in the tumour microenvironment altered cell responses to PI3-K and MEK inhibitors. We employed isogenic HCT116 human colon cancer cells where the parental cells harbor pre-existing H1047R PIK3CA and G13D KRAS alleles and compared these to isogenic clones that retain either the PIK3CA or KRAS wild-type alleles. Inhibitors were profiled under a range of defined conditions chosen to model the tumour microenvironment, including low glucose, low serum and hypoxia, and the activity of the compounds investigated. Under standard culture conditions, we found that PI3-K and MEK inhibitors showed minimal selectivity for cells with activating mutations compared to wild-type cells. When grown under defined culture conditions which more closely mimic the tumour microenvironment, the effects of the driving mutations became dominant and a differential between wild-type and mutant cell responses to inhibitors was observed. The selective PI3-K inhibitors GDC-0941 and PI-103 showed a >10-fold increase in potency when tested in anti-proliferative assays in mutant vs, wild-type cell lines. However, the dual PI3-K/mTOR inhibitor BEZ-235 did not show selectivity in this system. We show that by using isogenic cell lines under conditions which model the tumour microenvironment, differences in activity profiles between specific inhibitors can be quickly and definitively identified, highlighting the advantages of using this system in profiling molecularly targeted agents. 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 1651.

Deregulated AKT signalling is implicated in cancer. The preclinical characterization of AKT inhibitors and development of PD biomarkers are vital prior to clinical trials. Invasive techniques for clinical PD analyses pose ethical and logistical issues, hence hair follicles may represent a non-invasive option. The pyrazole AT7867 and pyrrolopyrimidine CCT128930 are novel ATP-competitive AKT inhibitors from different chemical series developed at The Institute of Cancer Research in collaboration with Astex Therapeutics (Cambridge, UK). AT7867 and CCT128930 have IC50 values against AKT2 of 17nM and 6nM respectively. Growth inhibitory IC50 (GI50) values were 2.4μM and 6.3μM in PTEN-null U87MG glioblastoma cells as measured by SRB assay. Cellular studies of both compounds in U87MG cells were carried out by Meso Scale Discovery (MSD) ELISA, western blot (WB) and immunofluorescence. These showed an initial induction of phosphorylated (p) Ser473 AKT as expected with ATP-competitive AKT inhibitors, and inhibition of downstream AKT targets, including pSer9 GSK-3β, pThr246 PRAS40, pSer127 YAP and pSer235/236 S6RP, indicating AKT pathway blockade. Interestingly, CCT128930 inhibited phosphorylation of AKT targets GSK-3β, S6RP and PRAS40 at lower concentrations (0.5-1µM), compared to AT7867 (5-10µM) in U87MG cells. Phosphorylation was also inhibited at earlier time points with equipotent doses (3XGI50) of CCT128930 versus AT7867. CCT128930 also caused a predominant G0/G1 phase arrest, while AT7867 resulted in a predominant G2 arrest in U87MG cells at equipotent doses (3XGI50), using BrdU and PI staining and flow cytometry. We report for the first time in vivo efficacy with intraperitoneally (ip) or orally administered AT7867 in PTEN-null MES-SA uterine sarcoma and U87MG mouse xenograft models; and ip administered CCT128930 in HER2-overexpressing BT474 breast cancer and U87MG xenografts, which correlated with in vivo pharmacokinetics (PK) and PD biomarker modulation of pSer9 GSK-3β and pSer235/236 S6RP using WB and MSD. Hair follicles were developed as a robust surrogate PD biomarker, with pThr246 PRAS40 as a PD readout. This assay was validated with healthy volunteer hair follicles, which were treated ex vivo with CCT128930. A significant decrease in pThr246 PRAS40 (p In conclusion, by employing an integrated PK and PD biomarker-driven drug discovery strategy, we have developed 2 novel and potent AKT inhibitors with antitumor activity, and have validated a robust surrogate biomarker assay for AKT inhibition for use in clinical trials. 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 4481.