ObjectiveDue to extended application of pharmacogenetic and pharmacogenomic screening (PGx) tests it is important to assess whether they provide good value for money. This review provides an update of the literature.MethodsA literature search was performed in PubMed and papers published between August 2010 and September 2014, investigating the cost-effectiveness of PGx screening tests, were included. Papers from 2000 until July 2010 were included via two previous systematic reviews. Studies’ overall quality was assessed with the Quality of Health Economic Studies (QHES) instrument.ResultsWe found 38 studies, which combined with the previous 42 studies resulted in a total of 80 included studies. An average QHES score of 76 was found. Since 2010, more studies were funded by pharmaceutical companies. Most recent studies performed cost-utility analysis, univariate and probabilistic sensitivity analyses, and discussed limitations of their economic evaluations. Most studies indicated favorable cost-effectiveness. Majority of evaluations did not provide information regarding the intrinsic value of the PGx test. There were considerable differences in the costs for PGx testing. Reporting of the direction and magnitude of bias on the cost-effectiveness estimates as well as motivation for the chosen economic model and perspective were frequently missing.ConclusionsApplication of PGx tests was mostly found to be a cost-effective or cost-saving strategy. We found that only the minority of recent pharmacoeconomic evaluations assessed the intrinsic value of the PGx tests. There was an increase in the number of studies and in the reporting of quality associated characteristics. To improve future evaluations, scenario analysis including a broad range of PGx tests costs and equal costs of comparator drugs to assess the intrinsic value of the PGx tests, are recommended. In addition, robust clinical evidence regarding PGx tests’ efficacy remains of utmost importance.

Tumor necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) signaling is far more complex than initially anticipated and can lead to either anti- or protumorigenic effects, hampering the successful clinical use of therapeutic TRAIL receptor agonists. Cell autonomous resistance mechanisms have been identified in addition to paracrine factors that can modulate apoptosis sensitivity. The tumor microenvironment (TME), consisting of cellular and non-cellular components, is a source for multiple signals that are able to modulate TRAIL signaling in tumor and stromal cells. Particularly immune effector cells, also part of the TME, employ the TRAIL/TRAIL-R system whereby cell surface expressed TRAIL can activate apoptosis via TRAIL receptors on tumor cells, which is part of tumor immune surveillance. In this review we aim to dissect the impact of the TME on signaling induced by endogenous and exogenous/therapeutic TRAIL, thereby distinguishing different components of the TME such as immune effector cells, neutrophils, macrophages, and non-hematopoietic stromal cells. In addition, also non-cellular biochemical and biophysical properties of the TME are considered including mechanical stress, acidity, hypoxia, and glucose deprivation. Available literature thus far indicates that tumor-TME interactions are complex and often bidirectional leading to tumor-enhancing or tumor-reducing effects in a tumor model- and tumor type-dependent fashion. Multiple signals originating from different components of the TME simultaneously affect TRAIL receptor signaling. We conclude that in order to unleash the full clinical potential of TRAIL receptor agonists it will be necessary to increase our understanding of the contribution of different TME components on outcome of therapeutic TRAIL receptor activation in order to identify the most critical mechanism responsible for resistance, allowing the design of effective combination treatments.

Introduction: Non-small cell lung cancer (NSCLC) is the most prevalent form of lung cancer and accounts for most cancer-related deaths worldwide. Despite progress in the treatment of subgroups of patients with targeted therapies, better treatments are required to improve overall prognosis. Agents that target the tumor-necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) receptors (TRAIL-R1 and -R2) are able to trigger selective apoptosis in tumor cells, however showed discouraging activity in clinical studies. Resistance to TRAIL-induced apoptosis and non-canonical pro-tumorigenic signaling hampers therapeutic efficacy. Previously, we found that TRAIL induces migration and invasion via RIPK1/Src/STAT3 pathway in apoptosis resistant NSCLC cells. Here we aim to analyze in more detail the role of Src in TRAIL non-canonical signaling and apoptosis resistance. Methods: Cytotoxicity to rhTRAIL was assessed by MTT assays. Src was chemically inhibited or genetically ablated by short hairpin(sh)RNA or CRISPR/CAS9. Src phosphorylation was studied by western blotting. Protein interactions were examined by co-immunoprecipitation (IP) and subsequent western blotting. Src interacting proteins were examined by co-IP experiments in conjunction with LC-mass spectrometric (MS) analyses. Results: The function of Src in TRAIL signaling was examined in TRAIL resistant A549 and sensitive H460 NSCLC cells. rhTRAIL treatment revealed distinct Src phosphorylation patterns, indicating that Src is differentially activated by TRAIL. Subsequently, co-IP experiments were performed showing that in A549 cells Src interacts with RIPK1 and Caspase-8 upon TRAIL treatment, but not in H460 cells. Next, we explored the possible role of Src in regulating TRAIL-induced apoptosis by chemical modulation of Src or by gene silencing or genetic knockout of Src. We found no role for Src in regulating sensitivity or resistance to TRAIL-induced apoptosis. To further investigate possible biological consequences of TRAIL-dependent Src activation we performed co-IP coupled with LC-MS analysis. Abundant differences were found in the Src interactome of A549 and H460 cells and in absence and presence of TRAIL. Various proteins known to be involved in tumor signaling were identified to be in complex with Src, including components of the RAF/MEK/ERK, Wnt and SMAD3 signaling pathways. Currently, mechanistic and validation studies are in progress to elucidate the role of these proteins. Conclusions: Src has no role in controlling sensitivity or resistance to TRAIL-induced apoptosis in the examined NSCLC cells. On the contrary, the Src interactome showed the activation of various pro-tumorigenic pathways by TRAIL. We anticipate that a deeper knowledge of TRAIL signaling will lead to novel therapeutic strategies to improve TRAIL-receptor targeted therapy. Supported by a grant from the Dutch Cancer Society (KWF 2011-5211). Citation Format: Margot de Looff, Steven de Jong, Frank A.E. Kruyt. The role of Src in TRAIL signaling in non-small cell lung cancer cells [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 4377.

Tumour-necrosis factor related apoptosis-inducing ligand (TRAIL) receptors (TRAIL-R1 and -R2) are appealing therapeutic targets to eradicate tumours specifically via caspase-dependent apoptosis. However, resistance is often observed and TRAIL-R activation can even activate pro-tumorigenic non-canonical signalling pathways. Previously, we found that TRAIL-induced RIPK1-Src-STAT3 signalling was mediating cell migration and invasion in resistant non-small cell lung cancer (NSCLC). Here, the contribution of Src in TRAIL signalling in NSCLC cell lines was further examined. TRAIL sensitive H460 and resistant A549 NSCLC cells showed distinct time-dependent rhTRAIL-induced Src phosphorylation patterns with early activation in A549 cells. Pharmacological Src inhibition as well as shRNA knockdown or CRISPR/CAS9-dependent knockout of Src expression did not alter sensitivity to rhTRAIL-induced apoptosis in both cell lines. Silencing of secondary complex proteins showed that TRADD, but not TRAF2, FADD nor caspase-8, was required for Src activation in A549 cells. Possible mediators of Src-dependent rhTRAIL signalling were identified by Src co-IP-LC-mass spectrometric analyses. In A549 cells the number of Src-interacting proteins increased after rhTRAIL treatment, whereas protein numbers decreased in H460 cells. In rhTRAIL treated A549 cells, Src biding proteins included components of the RAF-MEK1/2-ERK, Wnt and SMAD3 signalling pathways. Functional analyses showed that Src mediated phosphorylation of MEK1/2 and ERK, prevented phosphorylation of SMAD3 and was required for nuclear translocation of ERK and β-catenin in A549 cells. Clonogenic growth of both Src proficient and deficient A549 cells was not affected by rhTRAIL exposure, although Src depletion and MEK1/2 inhibition reduced colony size and numbers significantly. In conclusion, rhTRAIL-induced and Src dependent MEK/ERK, SMAD3 and β-catenin signalling may contribute to the known pro-tumorigenic effects of rhTRAIL in resistant NSCLC cells. However, this needs to be further examined, as well as the potential therapeutic implications of targeting these pathways when combined with TRAIL receptor agonists.

General introductionNon-small cell lung cancer Cancer is currently the second cause of death after cardiovascular diseases worldwide. However, it is predicted to become the primary cause of death in the near future 1 . It is expected that globally the burden of cancer rises to 18.1 million new cases and 9.6 million deaths 1 . Lung cancer is the major cause of total cancer related deaths (18.4%) and has a 5 year survival rate of only 18.3% 1,2 . Non-small cell lung cancer (NSCLC) is the most prevalent type, accounting for 85% of lung cancer cases, of which lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LSCC) are the most prevailing subtypes 3 . The remaining 15% of the lung cancers consist of small cell lung cancer (SCLC).Current treatment options depend on the stage and genetic profile of the cancer and include surgery, radiotherapy, chemotherapy, targeted therapies and/or immune checkpoint inhibitors. Intensive research to biological drivers of NSCLC and the technological developments during the past decade, including next generation sequencing and the different -omics platforms, have resulted in the discovery and application of targeted therapies. Numerous activating mutations in oncogenic drivers have been found in NSCLC, of which KRAS (25%), epidermal growth factor receptor (EGFR) (17%) and anaplastic lymphoma kinase (ALK) (7%) are the three most frequent occurring aberrations 4 . Targeted therapies for patient with mutant EGFR and ALK are successfully applied [5][6][7] . For other genetic abnormalities new strategies are being developed, of which some are currently under investigation in clinical trials 4 . In spite of the successful application of targeted therapies the overall survival rate of lung cancer remains low compared to other cancer types 2 . Apoptotic TRAIL signalling An interesting therapeutic biological agent for various tumours, including NSCLC, is the tumour-necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL). TRAIL induces selective apoptosis in tumour cells by pro-apoptotic/canonical signalling that is induced via TRAIL-receptor 1 (-R1/DR4) and/or TRAIL-R2 (DR5), but leaves normal cells unharmed [8][9][10] . Three other TRAIL receptors have been identified: the TRAIL-R3 (DcR1), TRAIL-R4 (DcR2) and the soluble Osteoprotegerin (OPG). These three receptors fail to trigger apoptotic responses and are believed to function as decoy receptors that sequester TRAIL, although TRAIL-R4 was reported to modulate T cell cytotoxicity towards cancer cells 11,12 . However, the exact function(s) of the decoy receptors need to be further explored 13 . Activation of TRAIL-R1 and TRAIL-R2 by TRAIL induces homo-or heterotrimerization and subsequent recruitment of Fas-Associated protein with Death Domain (FADD) and pro-caspase 8 at the intracellular death effector domains (DED) of the receptors (Fig. 1). At that instant Caspase 8 is cleaved and activated, and triggers the so-called death receptor, or extrinsic apoptotic pathway, by cleaving downstream substrates including 16 Chapter 1 ...