Search

The class of circular RNA (circRNA) is characterized by head-to-tail bonds between exons formed by backsplicing. Here, we provide a resource of circRNA expression in a comprehensive panel of 60 lung cancer and non-transformed cell lines (FL3C dataset). RNA sequencing after depletion of ribosomal RNA quantified the expression of circRNA and linear RNA. We detected 148,811 circular RNAs quantified by 2.8 million backsplicing reads originating from 12,251 genes. The number of identified circRNAs was markedly higher using rRNA depletion compared to public polyA-enriched RNA-seq datasets. CircRNAs almost never started in the first exon nor ended in the last exon and started more frequently in earlier exons. Most circRNAs showed high cell line specificity and correlated positively with their linear RNA counterpart. Known cancer genes produced more circRNAs than non-cancer genes. Subsets of circRNAs correlated with cell proliferation, histological subtype or genotype. CircTNFRSF21 was translated crossing the backsplice site in two different reading frames. Overexpression of circPVT1, circERBB2, circHIPK3, circCCNB1, circSMAD2, circTNFRSF21 and circKIF5B significantly increased colony formation. In conclusion, our data provide a comprehensive map of circRNA expression in lung cancer cells and global patterns of circRNA production as a useful resource for future research into lung cancer circRNAs.

Several oncogenic drivers have been identified in non-small cell lung cancer. Targeted therapies for these aberrations have already been successfully developed and implemented in clinical practice. Owing to improved sensitivity in genetic testing, more and more tumors with multiple driver mutations are identified, resulting in dilemmas for treating physicians whether and which targeted therapy to use. In this case series, we provide an overview of patients with intrinsic double mutations in oncogenic drivers and their reported response to targeted therapies, with a focus on epidermal growth factor receptor, anaplastic lymphoma kinase, cMET, and Kirsten rat sarcoma viral oncogene. We also include an unpublished case report on a patient with an epidermal growth factor receptor L858R and cMET exon 14 skipping.

In non‐small cell lung cancer, sensitizing mutations in epidermal growth factor receptor (EGFR) or cMET amplification serve as good biomarkers for targeted therapies against EGFR or cMET, respectively. Here we aimed to determine how this different genetic background would affect the interaction between the EGFR‐inhibitor erlotinib and the cMET‐inhibitor crizotinib. To unravel the mechanism of synergy we investigated the effect of the drugs on various parameters, including cell cycle arrest, migration, protein phosphorylation, kinase activity, the expression of drug efflux pumps, intracellular drug concentrations, and live‐cell microscopy. We observed additive effects in EBC‐1, H1975, and HCC827, and a strong synergism in the HCC827GR5 cell line. This cell line is a clone of the HCC827 cells that harbor an EGFR exon 19 deletion and has been made resistant to the EGFR‐inhibitor gefitinib, resulting in cMET amplification. Remarkably, the intracellular concentration of crizotinib was significantly higher in HCC827GR5 compared to the parental HCC827 cell line. Furthermore, live‐cell microscopy with a pH‐sensitive probe showed a differential reaction of the pH in the cytoplasm and the lysosomes after drug treatment in the HCC827GR5 in comparison with the HCC827 cells. This change in pH could influence the process of lysosomal sequestration of drugs. These results led us to the conclusion that lysosomal sequestration is involved in the strong synergistic reaction of the HCC827GR5 cell line to crizotinib–erlotinib combination. This finding warrants future clinical studies to evaluate whether genetic background and lysosomal sequestration could guide tailored therapeutic interventions., In non‐small cell lung cancer, sensitizing mutations in epidermal growth factor receptor (EGFR) or cMET amplification serve as good biomarkers for targeted therapies against EGFR or cMET, respectively. Here we aimed to determine how this different genetic background would affect the interaction between the EGFR‐inhibitor erlotinib and the cMET‐inhibitor crizotinib. Both genetic characteristics and lysosomal function seem to contribute to the synergistic interaction between crizotinib and erlotinib in the HCC827GR5 cell line, and more research is warranted to determine the exact mechanisms linking these cellular properties.

Introduction Lung squamous cell carcinomas (SCC) typically harbor a strong activation of epidermal growth factor receptor (EGFR) pathway. Since one of the most common resistance mechanisms against EGFR inhibition relies on the activation of cMET parallel signaling, we investigated the efficacy of a dual blockade with erlotinib and crizotinib in EGFR and cMET wild-type lung SCC cell lines. Methods Drug sensitivity assays were performed on LUDLU, SKMES-1, H1703, Calu1 and H520 cells. Further studies included analysis of cell cycle, apoptosis, spheroids, migration and Pathscan intracellular signaling array. Expression of emerging proteins was validated by Western blot and evaluated by immunohistochemistry in tissue-microarrays from lung cancer patients. Results Erlotinib and crizotinib showed additive interaction in Calu1, H520 and SKMES-1, and strong synergism in the LUDLU cells (Combination Index: 0.387), associated to G2/M phase arrest, increased apoptosis, spheroid size reduction and inhibition of migration. Remarkably, this combination decreased the phosphorylation of downstream targets of MAPK and PI3K/Akt/mTOR pathways, with the largest decrease observed for PRAS40 Thr246. Moreover, it reduced the expression of both p-Her3 and p-PRAS40 in the synergistic LUDLU cells. Tissue specimens showed a higher expression of both proteins in SCC compared to adenocarcinoma histology. Conclusions Combining erlotinib and crizotinib led to an additive/synergistic interaction in 4 out of 5 SCC cells. By combining both inhibitors, MAPK and PI3K/Akt/mTOR pathways were strongly inhibited, leading to increased cell death. p-Her3 and p-PRAS40 might be used as markers for determining the synergistic effect and for selecting potential candidates for the combination treatment.

The c-Met receptor is a therapeutically actionable target in non-small-cell lung cancer (NSCLC), with one approved drug and several agents in development. Most suitable biomarkers for patient selection include c-Met amplification and exon-14 skipping. Our retrospective study focused on the frequency of different c-Met aberrations (overexpression, amplification and mutations) in 153 primary, therapy-na&iuml, ve resection samples and their paired metastases, from Biobank@UZA. Furthermore, we determined the correlation of c-Met expression with clinicopathological factors, Epidermal Growth Factor Receptor (EGFR)-status and TP53 mutations. Our results showed that c-Met expression levels in primary tumors were comparable to their respective metastases. Five different mutations were detected by deep sequencing: three (E168D, S203T, N375S) previously described and two never reported (I333T, G783E). I333T, a new mutation in the Sema(phorin) domain of c-Met, might influence the binding of antibodies targeting the HGF-binding domain, potentially causing innate resistance. E168D and S203T mutations showed a trend towards a correlation with high c-Met expression (p = 0.058). We found a significant correlation between c-MET expression, EGFR expression (p = 0.010) and EGFR mutations (p = 0.013), as well as a trend (p = 0.057) with regards to TP53 mutant activity. In conclusion this study demonstrated a strong correlation between EGFR mutations, TP53 and c-Met expression in therapy-na&iuml, ve primary resection samples. Moreover, we found two new c-Met mutations that warrant further studies.

The hepatocyte growth factor (HGF) and its tyrosine kinase receptor cMET (MET proto-oncogene) entered into the spotlight mainly as a bypassing pathway for other targeted therapies (e.g., therapies targeting epidermal growth factor receptor). However, the HGF-cMET signaling axis can be oncogenic by itself. Aberrations in HGF-cMET occur in many cancer types, such as non-small-cell lung cancer, pancreatic cancer, and renal carcinoma. This resulted in the development of several inhibitors targeting this signaling axis. Biomarkers for these targeted therapies include cMET amplification and cMET exon 14 skipping. Activation of cMET by HGF results in the activation of several downstream pathways, among which are the mitogen-activated protein kinase cascade and phosphatidylinositol-3-kinase-Akt signaling. During embryogenesis, they control the development of tubules and are involved in the migration and invasion of several cell types. These functions are mirrored in cancer growth, whereby cMET is a known activator of cell migration and metastasis. Therefore, in this chapter, we start by describing the road to discovery, the functions in development, and explain the HGF-cMET signaling in detail. Next we focus on HGF-cMET in cancer, describing the possible aberrations and providing an overview of inhibitors (under development). Finally, we pay attention to the role of HGF-cMET as a resistance mechanism against other (targeted) therapies.

In the last decade, the tyrosine kinase receptor cMET, together with its ligand hepatocyte growth factor (HGF), has become a target in non-small cell lung cancer (NSCLC). Signalization via cMET stimulates several oncological processes amongst which are cell motility, invasion and metastasis. It also confers resistance against several currently used targeted therapies, e.g., epidermal growth factor receptor (EGFR) inhibitors. In this review, we will discuss the basic structure of cMET and the most important signaling pathways. We will also look into aberrations in the signaling and the effects thereof in cancer growth, with the focus on NSCLC. Finally, we will discuss the role of cMET as resistance mechanism.

Squamous cell lung carcinoma (SCC) accounts for 30% of patients with NSCLC and to date, no molecular targeted agents are approved for this type of tumor. However, recent studies have revealed several oncogenic mutations in SCC patients, including an alteration of the PI3K/AKT pathway, i.e. PI3K point mutations and amplification, AKT mutations and loss or reduced PTEN expression. Prompted by our observation of a correlation between PTEN loss and FAK phosphorylation in a cohort of patients with stage IV SCC, we evaluated the relevance of PTEN loss in cancer progression as well as the efficacy of a new combined treatment with the pan PI3K inhibitor buparlisip and the FAK inhibitor defactinib. An increase in AKT and FAK phosphorylation, associated with increased proliferation and invasiveness, paralleled by the acquisition of mesenchymal markers, and overexpression of the oncomir miR-21 were observed in SKMES-1-derived cell clones with a stable reduction of PTEN. Notably, the combined treatment induced a synergistic inhibition of cell proliferation, and a significant reduction in cell migration and invasion only in cells with reduced PTEN. The molecular mechanisms underlying these findings were unraveled using a specific RTK array that showed a reduction in phosphorylation of key kinases such as JNK, GSK-3 α/β, and AMPK-α2, due to the concomitant decrease in AKT and FAK activation. In conclusion, the combination of buparlisib and defactinib was effective against cells with reduced PTEN and warrants further studies as a novel therapeutic strategy for stage IV SCC patients with loss of PTEN expression.

The TP53 gene remains the most frequently altered gene in human cancer, of which variants are associated with cancer risk, therapy resistance, and poor prognosis in several tumor types. To determine the true prognostic value of TP53 variants in non–small cell lung cancer, this study conducted further research, particularly focusing on subtype and tumor stage. Therefore, we determined the TP53 status of 97 non–small cell lung cancer adenocarcinoma patients using next generation deep sequencing technology and defined the prognostic value of frequently occurring single nucleotide polymorphisms and mutations in the TP53 gene. Inactivating TP53 mutations acted as a predictor for both worse overall and progression-free survival in stage II–IV patients and patients treated with DNA-damaging (neo)adjuvant therapy. In stage I tumors, the Pro-allele of the TP53 R72P polymorphism acted as a predictor for worse overall survival. In addition, we detected the rare R213R (rs1800372, minor allele frequency: 0.0054) polymorphism in 7.2% of the patients and are the first to show the significant association with TP53 mutations in non–small cell lung cancer adenocarcinoma patients (p = 0.003). In conclusion, Our findings show an important role for TP53 variants as negative predictors for the outcome of non–small cell lung cancer adenocarcinoma patients, especially for TP53 inactivating mutations in advanced stage tumors treated with DNA-damaging agents, and provide the first evidence of the R213R G-allele as possible risk factor for non–small cell lung cancer.

We have read with interest the article by Ortiz-Cuaran and colleagues ([1][1]) on resistance mechanisms to third-generation EGFR tyrosine kinase inhibitors (EGFR-TKI) in lung cancer patients. FISH analysis and targeted sequencing on rebiopsies of these patients were performed, showing that

The development of targeted therapies has led to a revolution in non-small-cell lung cancer, and opened up possibilities for improved personalized medicine. With the constant findings of new targets, a lot of inhibitors are being developed. However, reliable biomarkers are urgently needed. The design of clinical trials needs to become more flexible in order to obtain the best results and gain the US FDA/EMEA approval for the new drugs. A recent example of a failed trial is the Phase III MetLung trial that compared the effects of the c-MET monovalent antibody onartuzumab with erlotinib versus erlotinib alone in late-stage non-small-cell lung cancer. Here, we discuss several points as to why this trial could have failed.

AIM: To investigate the potential benefit of combining the cMET inhibitor crizotinib and cisplatin we performed in vitro combination studies.METHODS: We tested three different treatment schemes in four non-small cell lung cancer (NSCLC) cell lines with a different cMET/epidermal growth factor receptor genetic background by means of the sulforhodamine B assay and performed analysis with Calcusyn.RESULTS: All treatment schemes showed an antagonistic effect in all cell lines, independent of the cMET status. Despite their different genetic backgrounds, all cell lines (EBC-1, HCC827, H1975 and LUDLU-1) showed antagonistic combination indexes ranging from 1.3-2.7. These results were independent of the treatment schedule.CONCLUSION: These results discourage further efforts to combine cMET inhibition with cisplatin chemotherapy in NSCLC.

Erlotinib is commonly used as a second line treatment in non-small cell lung cancer patients with sensitizing EGFR mutations. In EGFR-wild type patients, however the results are limited. Therefore we evaluated whether the combination of the Protein kinase C-β inhibitor enzastaurin with erlotinib could enhance the effect in the A549 and H1650 cell lines. Cytotoxicity of erlotinib, enzastaurin and their 72-h simultaneous combination was assessed with the MTT assay. The pharmacologic interaction was studied using the method of Chou and Talalay, cell cycle perturbations were assessed by flow cytometry and modulation of ERK1/2 and AKT phosphorylation was determined with ELISA. For protein phosphorylation of GSK3β we performed Western Blot analysis and a Pamgene phosphorylation array, while RT-PCR was used to investigate VEGF and VEGFR-2 expression before and after drug treatments. A synergistic interaction was found in both cell lines with mean CI of 0.58 and 0.63 in A549 and H1650 cells, respectively. Enzastaurin alone and in combination with erlotinib increased the percentage of cells in S and G2M phase, mostly in H1650 cells, while AKT, ERK1/2 and GSK3β phosphorylation were reduced in both cell lines. VEGF expression decreased 5.0 and 6.9 fold in A549 cells after enzastaurin alone and with erlotinib, respectively, while in H1650 only enzastaurin caused a relevant reduction in VEGF expression. The array showed differential phosphorylation of EGFR, GSK3β, EphA1 and MK14. In conclusion, enzastaurin is a protein kinase Cβ inhibitor, working on several cellular signaling pathways that are involved in proliferation, apoptosis and angiogenesis. These features make it a good compound for combination therapy. In the present study the combination of enzastaurin and erlotinib gives synergistic results, warranting further investigation.

Introduction: The Fibroblast Growth Factor Receptor (FGFR) family consists of Tyrosine Kinase Receptors (TKR) involved in several biological functions. Recently, alterations of FGFR have been reported to be important for progression and development of several cancers. In this setting, different studies are trying to evaluate the efficacy of different therapies targeting FGFR. Areas Covered: This review summarizes the current status of treatments targeting FGFR, focusing on the trials that are evaluating the FGFR profile as inclusion criteria: Multi-Target, Pan-FGFR Inhibitors and anti-FGF (Fibroblast Growth Factor)/FGFR Monoclonal Antibodies. Expert opinion: Most of the TKR share intracellular signaling pathways; therefore, cancer cells tend to overcome the inhibition of one tyrosine kinase receptor by activating another. The future of TIC (Tyrosine Kinase Inhibitor) therapy will potentially come from multi-targeted TKIs that target different TKR simultaneously. It is crucial to understand the interaction of the FGF-FGFR axis with other known driver TKRs. Based on this, it is possible to develop therapeutic strategies targeting multiple connected TKRs at once. One correct step in this direction is the reassessment of multi target inhibitors considering the FGFR status of the tumor. Another opportunity arises from assessing the use of FGFR TKI on patients harboring FGFR alterations. (C) 2017 Elsevier B.V. All rights reserved.

The abnormal stimulation of the multiple signal transduction pathways downstream of the receptor tyrosine kinase mesenchymal-epithelial transition factor (cMET) promotes cellular transformation, tumor motility, and invasion. Therefore, cMET has been the focus of prognostic and therapeutic studies in different tumor types, including non-small cell lung cancer. In particular, several cMET inhibitors have been developed as innovative therapeutic candidates and are currently under investigation in clinical trials. However, one of the challenges in establishing effective targeted treatments against cMET remains the accurate identification of biomarkers for the selection of responsive subsets of patients. Recently, splice site mutations have been discovered in cMET that lead to the skipping of exon 14, impairing the breakdown of the receptor. Patients with NSCLC who are carrying this splice variant typically overexpress the cMET receptor and show a response to small molecule inhibitors of cMET. Here, we review the main differences at the structural level between the wild-type and the splice variants of cMET and their influence on cMET signaling. We clarify the reason why this variant responds to small molecule inhibitors and their prognostic/predictive role. (C) 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective tyrosine kinase inhibitors (TKIs). To optimize the use of these TKIs, the discovery of new biomarkers for early detection and disease progression is mandatory. These plasma-isolated exosomes can be used as a non-invasive and repeatable way for the detection and followup of these biomarkers. One ml of plasma from 12 NSCLC patients, with different mutations and treatments (and 6 healthy donors as controls), were used as exosome sources. After RNAse treatment, in order to degrade circulating miRNAs, the exosomes were isolated with a commercial kit and resuspended in specific buffers for further analysis. The exosomes were characterized by western blotting for ALIX and TSG101 and by transmission electron microscopy (TEM) analysis, the standard techniques to obtain biochemical and dimensional data of these nanovesicles. Total RNA extraction was performed with a high yield commercial kit. Due to the limited miRNA-content in exosomes, we decided to perform retro-transcription PCR using an individual assay for each selected miRNA. A panel of miRNAs (30b, 30c, 103, 122, 195, 203, 221, 222), all correlated with NSCLC disease, were analyzed taking advantage of the remarkable sensitivity and specificity of Real-Time PCR analysis; mir-1228-3p was used as endogenous control and data were processed according to the formula 2(-Delta Delta ct 13). Control values were used as baseline and results are shown in logarithmic scale.

Recently, the development of the third-generation epidermal growth factor receptor-small molecule inhibitor (EGFR-TKI) rociletinib had failed. In this review, the wide-ranging aspects of the evolution of EGFR-TKIs were collected, with a special focus on rociletinib. The influence of different oncogenic mutations on EGFR activity was also discussed. Resistance to the first (erlotinib, gefitinib)- and second (afatinib)-generation EGFR-TKIs provided the rationale behind the development of the third-generation inhibitors (rociletinib, osimertinib). On the basis of these data, a comparison of their efficacy on the different mutated EGFRs and the respective resistance mechanisms is further reported. Moreover, the evolution and results of the clinical trials of rociletinib (TIGER trials) are compared with the trials on osimertinib, another third-generation EGFR-TKI that now has been granted US Food and Drug Administration approval. The reasons behind the arrest in the further development of rociletinib are put in the perspective of future drug development., Video abstract

APR-246 (PRIMA-1(Met)) is able to bind mutant p53 and restore its normal conformation and function. The compound has also been shown to increase intracellular ROS levels. Importantly, the poly-[ADP-ribose] polymerase-1 (PARP-1) enzyme plays an important role in the repair of ROS-induced DNA damage. We hypothesize that by blocking this repair with the PARP-inhibitor AZD2281 (olaparib), DNA damage would accumulate in the cell leading to massive apoptosis. We observed that APR-246 synergistically enhanced the cytotoxic response of olaparib in TP53 mutant non-small cell lung cancer cell lines, resulting in a strong apoptotic response. In the presence of wild type p53 a G2/M cell cycle block was predominantly observed. NOXA expression levels were significantly increased in a TP53 mutant background, and remained unchanged in the wild type cell line. The combined treatment of APR-246 and olaparib induced cell death that was associated with increased ROS production, accumulation of DNA damage and translocation of p53 to the mitochondria. Out data suggest a promising targeted combination strategy in which the response to olaparib is synergistically enhanced by the addition of APR-246, especially in a TP53 mutant background. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

Oncogenic drivers are often overexpressed in adenocarcinoma non-small cell lung cancer (NSCLC) patients, so this tumor type is often sensitive to targeted therapies. Sensitizing mutations in the epidermal growth factor receptor (EGFR) are good predictive biomarkers for response to EGFR-small molecule inhibitors, eg erlotinib. Amplification or exon 14 skipping of cMET are good biomarkers for cMET-small molecule inhibitors, eg crizotinib. In other subtypes of NSCLC, eg squamous, targeted therapy is not considered to be active. We tested the combination of erlotinib and crizotinib on a panel of squamous NSCLC cell lines that were wild-type for both EGFR and cMET. The effect of this drug combination was tested with different assays. The sulforhodamine B-assay was used to determine growth inhibition and combination indexes (CI) were calculated with the method of Chou&Talalay. A CI< 0.8 was synergistic, 0.81.2 was antagonistic. With flow cytometry the cell cycle was studied. With the wound-healing assay we determined the effect on cell migration. A 3D spheroid assay was performed to determine the effect of limited nutrient availability and hypoxia. Protein phosphorylation was studied with a pathscan array and phospho-specific western blotting. The LUDLU cell line gave a synergistic CI of 0.39±0.07, whereas the Calu1 (CI = 0.81±0.04), H520 (CI = 0.87±0.06) and SKMES1 (CI = 0.81±0.02) were additive and the H1703 cell line (CI = 1.20±0.15) was antagonistic. Cell cycle analysis revealed that erlotinib monotherapy caused a G1 phase arrest (38 to 76%) and crizotinib caused a G2/M (13 to 64%) phase arrest. In cells treated with the combination, the effect of crizotinib was dominant (13 to 52%). In the wound-healing assay the LUDLU cells were not migratory. The pathscan revealed a decrease in the phosphorylation of pERK1/2 (Thr202/204), pPRAS40 (Thr246) and pGSK3β (Ser9). Western blotting showed that the levels of pPRAS40 were decreased in the LUDLU cells after treatment with erlotinib or crizotinib, and almost gone when treated with the combination. In the SKMES1 cells only the combination decreased pPRAS40. H1703 cells showed no change in pPRAS40. These changes in phosphorylation make us hypothesize that Her3 and PI3K/Akt signaling may be important in the interaction of both drugs. Her3 is a promiscuous receptor that is able to heterodimerize with EGFR and cMET. When blocking one of these receptors, Her3 is still able to continue downstream PI3K/Akt signaling. When both EGFR and cMET are blocked, Her3 and PI3K/Akt signaling is inhibited, leading to synergy. The expression of total-Her3 was lowest in the antagonistic cell line. The combination of both inhibitors lead to a complete inhibition of pHer3 (Tyr1289). In conclusion: Blocking both EGFR and cMET signaling causes inhibition of Her3 downstream signaling through PI3K/Akt, leading to synergy in wild-type squamous NSCLC cells. Citation Format: Nele Van Der Steen, Christian Rolfo, Patrick Pauwels, Godefridus J. Peters, Elisa Giovannetti. Combining forces: Synergy of erlotinib and crizotinib in a wild-type squamous non-small cell lung cancer cell line [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 1074. doi:10.1158/1538-7445.AM2017-1074

e20043 Background: Targeted therapies caused a revolution in treatment of non-small cell lung cancer (NSCLC). Response to Epidermal Growth Factor Receptor such as erlotinib, can be predicted by the presence of sensitizing mutations in EGFR. In case of cMET, amplification or exon 14 skipping serve as biomarkers for response to cMET-TKIs, crizotinib. Methods: In this study, we investigated the combination of erlotinib and crizotinib on several squamous NSCLC cell lines wild-type for EGFR and cMET. Growth inhibition was determined using the sulforhodamine B-assay. Combination indexes (CI) were calculated based on the method of Chou and Talalay. A CI below 0.8 was considered synergistic, between 0.8 and 1.2 was considered additive and above 1.2 was considered antagonistic. The influence of treatment on the cell cycle, cell migration, 3D cell cultures and phosphorylation was studied. Results: The H1703 cell line gave an antagonistic (CI = 1.20±0.15), the Calu1 (CI = 0.81±0.04), H520 (CI = 0.87±0.06) and SKMES1 (CI = 0.81±0.02) were additive and LUDLU (CI = 0.39±0.07) was synergistic. A G1 phase arrest occurred after treatment with erlotinib (38 to 76%), whereas crizotinib caused a G2/M phase arrest (13 to 64%). In the combination, the effect of crizotinib was dominant (13 to 52%). A decrease in p-ERK1/2 (Thr202/204), p-PRAS40 (Thr246) and p-GSK3 β (Ser9) was observed. Phospho-PRAS40 showed a decrease after treatment with erlotinib or crizotinib, whereas combination treatment almost abolished p-PRAS40. In the SKMES1 cell line, p-PRAS40 only decreased after the combination, whereas H1703 cells showed no change in p-PRAS40 levels. Phospho-PRAS40 activity is strongly linked to PI3K/Akt signaling. Her3 is able to heterodimerize both with EGFR and cMET, thus activating downstream PI3K/Akt signaling. No expression of Her3 in the antagonistic cell line was detected. In the LUDLU, levels of p-Her3 were not detectable after erlotinib treatment and in the combination, whereas in the SKMES1 levels of p-Her3 slightly decreased only after combination treatment. Conclusions: To conclude, the simultaneous inhibition of both EGFR and cMET leads to a strong inhibition of PI3K/Akt signaling, leading to synergy in the LUDLU wild-type squamous NSCLC cells.

The advent of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) represented the most important innovation in NSCLC treatment over the last years. However, despite a great initial activity, secondary mutations in the same target, or different alterations in other molecular pathways, inevitably occur, leading to the emergence of acquired resistance, in median within the first year of treatment. In this scenario, the mesenchymal-epidermal transition (cMET) tyrosine kinase receptor and its natural ligand, the hepatocyte growth factor (HGF), seem to play an important role. Indeed either the overexpression or the amplification of cMET, as well as the overexpression of the HGF, have been reported in a substantial subgroup of NSCLC patients resistant to EGFR-TKIs. Several cMET-inhibitors have been developed as potential therapeutic candidates, and are currently under investigation in clinical trials. These compounds include both monoclonal antibodies and TKIs, and most of them have been investigated as dual combinations including an anti-EGFR TKI, to improve the efficacy of the available treatments, and ultimately overcome acquired resistance to EGFR-inhibitors.

A rapidly growing domain in present-day oncology is the development of targeted therapies, including several cMET inhibitors against non-small cell lung cancer (NSCLC). Nowadays, cMET amplification is used as a standard biomarker for patient selection, however there is no clear cut-off value to determine amplification, nor is there a real consensus about the way to test this. More recently, splicing variants of cMET, which skip exon 14, are gaining importance since it has been shown that patients harboring this mutation can respond to cMET directed targeted therapy. In this study, we explored the occurrence of cMET aberrations and their correlation with cMET expression in a population of 155 primary EGFR-TKI naive NSCLC tumors. Since cMET is considered as important in metastatic behavior, we also investigated the correlation of cMET expression and amplification between the primary tumor and metastases. Finally, given the link between the EGFR and cMET pathways, the EGFR status was also studied. Samples were collected at the Antwerp University Hospital and the Onze-Lieve-Vrouw Hospital Aalst. The expression of EGFR and cMET was determined by immunohistochemistry (Ventana, clones 3C6 and SP66 respectively), while cMET amplification was examined by in situ hybridization (Ventana, MET DNP and CEN7 DIG probes). EGFR mutations and cMET splice site mutations were detected by Sanger sequencing. Significant correlations were tested using the Chi2 and kappa test (SPSS 23). In 146/155 tumors cMET expression could be determined; 73/146 samples showed high (2+ or 3+ score) cMET expression and 4/118 samples showed amplification (ratio ≥ 2). No significant correlations could be determined between cMET expression and histological subtype of NSCLC (p = 0.065), differentiation degree (p = 0.468) and cMET amplification (p = 0.214). However, a significant correlation was found between cMET expression, EGFR expression (p = 0.015) and EGFR mutations (p = 0.029). Splice site mutation regions were sequenced in 87/155 samples, all of which were wild-type. When comparing cMET amplification between the primary tumor and the corresponding metastases (n = 40), only one sample showed amplification in the metastases and not in the primary tumor. Hence, cMET expression showed a significant correlation between primary tumors and their metastases (kappa = 0.003). The overall results of our study are in agreement with earlier data. Moreover, we showed that overall expression levels of cMET in primary tumors and metastases are very similar despite large intratumor heterogeneity. In conclusion, this study shows that high cMET expression in most NSCLC samples does not originate from presently known genetic cMET aberrations. High cMET expression is likely to be caused by temporary changes in transcription and translation, influenced by EGFR-signaling, miRNAs or other regulatory mechanisms. Citation Format: Nele Van Der Steen, Vanessa Deschoolmeester, Filip Lardon, Christian Rolfo, Elisa Giovannetti, Godefridus J. Peters, Patrick Pauwels. cMET in non-small cell lung cancer: pieces of the puzzle. [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 2242.

Intro: The use of targeted therapies confers great responses in non-small cell lung cancer patients, but ultimately results in acquired resistance, e.g. against epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) due to cMET amplification. Therefore, combination therapy with EGFR-TKI and cMET TKIs in cell lines with a different EGFR and cMET background was investigated. Materials & methods: All experiments were performed in 5 cell lines with a different EGFR and cMET background: the cMET amplified squamous lung cancer cell line EBC-1, the HGF-secreting myeloma cell line JJN3, the HCC827 and H1975 lung adenocarcinoma cell lines with an activating EGFR exon 19 deletion and an L858 as well as a T790M resistance mutation, respectively. Finally LUDLU-1 is a squamous lung cancer cell line, wild-type for both EGFR and cMET. The cytotoxicity of combination therapy with erlotinib (EGFR-TKI) and crizotinib (cMET-ALK-ROS1-TKI) or PHA665752 (cMET-TKI) was determined by the sulforhodamine B assay after 72h of simultaneous/sequential treatment (Table 1). Results: For all treatments, the JJN3, EBC-1 and HCC827 cell lines showed no synergistic effect. For the H1975, a decrease in resistance could be observed, albeit not significant. For LUDLU-1, a trend towards synergism was observed when erlotinib was administered simultaneously with crizotinib (p = 0.074) and PHA665752 (p = 0.055). Also a significantly synergistic effect was observed when treatment with erlotinib was followed by PHA665752 (p = 0.014). Discussion: Synergism between erlotinib and crizotinib/PHA665752 depends on the genetic background of the cells and the treatment schedule. This was most pronounced in the EGFR and cMET wild-type cell line. These results open up the discussion to include EGFR and cMET wild-type patients into future clinical trials. Cell lineErlotinib (μM)Crizotinib (μM)PHA665752 (μM)Erlo+CrizErlo = >CrizCriz = >ErloErlo+PHAErlo = >PHAPHA = >ErloEBC-10-100-0.025-0.050-0.1-0.150-0.025-0.050-0.1-0.20-0.3-0.40-0.1-0.2JJN30-100-2-30-2-30-2-30-5-70-5-70-5-6HCC8270-50-3-50-3-50-1-20-7-90-7-90-4-5H19750-100-2-30-2-30-1-20-6-70-6-70-2-3LUDLU-10-100-3-50-3-50-2-30-7-80-7-80-3.5-4 Table 1: Therapeutic concentrations per cell line & treatment schedule: control-IC20-IC40. Citation Format: Nele Van Der Steen, Vanessa Deschoolmeester, An Wouters, Filip Lardon, Christian Rolfo, Patrick Pauwels. Combination therapy with EGFR-TKI and cMET-TKIs in non-small cell lung cancer: the golden duo. [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 2593. doi:10.1158/1538-7445.AM2015-2593

BACKGROUND Cancer cells produce a heterogeneous mixture of vesicular, organelle-like structures (microvesicles or MVs) into their surroundings including blood and body fluid. In particular exosomes are biological nanovescicles (40-100 nm) that are formed by the inward budding of multivescicular bodies (MVB), as a component of the endocytic pathway. They are released from different cell types under both normal and pathological conditions. Exosomal content is composed by proteins, DNA, mRNA and microRNA (miRNA) that are transferred to distant site and mediate inter-cellular communication. PURPOSE OF THE STUDY The aim of this pilot study is to investigate whether exosomes isolation from clinical samples (plasma from NSCLC patients) is feasible. Furthermore we have analyzed a panel of 7 miRNA related to NSCLC. EXPERIMENTAL DESIGN AND METHODS The study population was selected among lung cancer patients diagnosed with NSCLC, histotype adenocarcinoma, in the Oncology department of Antwerp University Hospital. A total of 12 patients participated in the project. Six blood samples from healthy volunteers were also collected. After obtaining the informed consent, blood samples (10 ml) were stored in the UZA-tumor biobank. Clinical data were also collected from patients’ medical records. Exosomes were isolated by means of both Density Gradient (DG) centrifugation and the Total Exosome Isolation kit (from plasma) (Invitrogen) according to manufacturers’ instructions. The Total exosome RNA and protein isolation kit (Invitrogen) was used for proteins recovery from exosome. Extracted proteins are used for western blot analysis aimed at the identification of specific exosome protein markers: CD63, ALIX and TSG101. Nanoparticle Tracking Analysis (NTA) was performed by means of the NanoSight NS500 instrument. In order to visualize the isolated exosomes a TEM (Transmission Electron Microscopy) analysis was performed. The total exosome RNA and protein isolation kit (Invitrogen) was used to extract small-RNA from exosomal samples. The analysis of 7 miRNAs (miR-30b, -30c, -103, -122, -195, -221, -222) was performed on the LightCycler 480 (Roche) and the fold change was calculated according to the formula 2ααCq. miR-1228 was used has endogenous control to normalize the reaction. RESULTS • ALIX protein is a reliable protein marker for exosome identification • TEM analysis have shown that exosomes can be isolated in NSCLC plasma samples • 7 selected miRNAs are strongly deregulated in our clinical samples and seem related to staging • miR30b and -30c might be related to squamous cell carcinoma histotype CONCLUSION The analysis of exosomes in NSCLC could represent a noninvasive test for patients’ management. With this pilot study we have demonstrated that exosome analysis and exosomal miRNA profiling is feasible. Due to the small sample size we cannot make significant statistic conclusion. Further analyses in a larger series are needed. Citation Format: Christian D. Rolfo, Marta Castiglia, Marco Giallombardo, Jorge Chacartegui, Nele Van Der Steen, Inge Mertens, Marc Peeters, Antonio Russo, Patrick Pauwels. Exosomes analysis in non-small cell lung cancer: looking for a clinical application. [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 3382. doi:10.1158/1538-7445.AM2015-3382