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Introduction: In the primary analysis of VISION, tepotinib — a highly selective, potent MET inhibitor — demonstrated durable efficacy, and a tolerable safety profile in patients (pts) with METex14 skipping NSCLC. We report updated outcomes, with interim analyses from a confirmatory cohort. Methods: In the Phase II VISION study, pts with advanced/metastatic METex14 skipping NSCLC, identified by liquid (L+) and/or tissue (T+) biopsy, received 500 mg (450 mg active moiety) tepotinib once daily. All pts were assessed for safety; pts with ≥3 months’ follow-up were assessed for efficacy. Primary endpoint was objective response by independent review (RECIST v1.1). Secondary endpoints were disease control (DCR), duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety. Results: As of Feb 1, 2021, 7,882 pts were pre-screened, and 275 pts were analyzed for efficacy. Patients analyzed for efficacy had a median age of 72.4 years (range, 41-94]), 50.9% were female, 46.5% had smoking history, and median tumor load [target lesions by IRC] was 57.4 mm [range, 10.2-227.8]). Tumor load in L+ pts was 68.0 mm (11.6-227.8) and 52.9 mm (10.2-227.8) in T+ pts. In treatment-naïve pts (n=137), ORR was 54.0% (95% CI: 45.3, 62.6), DCR was 74.5% (66.3, 81.5), and >90% of pts had tumor shrinkage. Median (m) DOR was 32.7 months (9.0, not estimable), mPFS was 10.4 months (8.4, 15.3), and mOS was 17.6 months (13.4, 29.7). In previously treated pts (n=138), ORR was 44.2% (35.8, 52.9), DCR was 75.4% (67.3, 82.3), and >90% of pts had tumor shrinkage. mDOR was 11.1 months (8.4, 18.5), mPFS was 11.0 months (8.2, 12.4), and mOS was 19.9 months (15.8, 22.3). Meaningful clinical activity was observed in L+ and T+ pts (Table). Overall, 14.1% of pts discontinued due to TRAEs; tepotinib was well-tolerated across treatment lines (Table). Conclusions: Tepotinib demonstrated robust and durable clinical activity across treatment lines in pts with METex14 skipping NSCLC, with particularly durable efficacy in first line. TRAEs were manageable with few discontinuations. Table 1. Tepotinib efficacy and safety in patients with METex14 skipping NSCLC (VISION) Tepotinib efficacy (pts with ≥3 months’ follow-up in Cohorts A+C) Overall Treatment-naϊve (n=137) Previously treated (n=138) Combined (N=275) Liquid biopsy* (n=81) Tissue biopsy* (n=86) Liquid biopsy* (n=78) Tissue biopsy* (n=88) Objective response rate, % (95% CI) 49.1 (43.0, 55.2) 54.3 (42.9, 65.4) 54.7 (43.5, 65.4) 43.6 (32.4, 55.3) 47.7 (37.0, 58.6) Disease control rate, % (95% CI) 74.9 (69.4, 79.9) 71.6 (60.5, 81.1) 80.2 (70.2, 88.0) 69.2 (57.8, 79.2) 79.5 (69.6, 87.4) Median duration of response, months (95% CI) 13.8 (9.9, 19.4) 13.8 (7.2, ne) 32.7 (10.8, 32.7) 11.1 (8.4, 19.4) 10.1 (8.3, 15.7) Median progression-free survival, months (95% CI) 10.8 (8.5, 12.4) 8.5 (6.9, 11.3) 15.3 (9.6, ne) 8.3 (5.7, 11.0) 11.1 (8.2, 16.8) Median overall survival, months (95% CI) 19.7 (15.6, 22.1) 15.1 (9.5, 22.1) 29.7 (15.3, ne) 19.9 (12.8, 22.3) 22.3 (17.0, 27.2) Tepotinib safety (all pts who received tepotinib in Cohorts A+C) Overall (N=291) Treatment-naϊve (n=148) Previously treated (n=143) Treatment-related AEs†, n (%) Any grade 264 (90.7) 137 (92.6) 127 (88.8) Grade ≥3 86 (29.6) 49 (33.1) 37 (25.9) Leading to death 2 (0.7) 1 (0.7) 1 (0.7) Leading to a dose reduction 90 (30.9) 51 (34.5) 39 (27.3) Leading to temporary discontinuation 114 (39.2) 63 (42.6) 51 (35.7) Leading to permanent discontinuation 41 (14.1) 24 (16.2) 17 (11.9) All-cause AEs† in ≥20% of patients, n (%) Peripheral edema 191 (65.6) 98 (66.2) 93 (65.0) Nausea 88 (30.2) 51 (34.5) 37 (25.9) Diarrhea 81 (27.8) 42 (28.4) 39 (27.3) Hypoalbuminemia 81 (27.8) 41 (27.7) 40 (28.0) Blood creatinine increase 76 (26.1) 34 (23.0) 42 (29.4) Dyspnea 60 (20.6) 40 (27.0) 20 (14.0) Data cut-off: Feb 1, 2021. *Patients with METex14 skipping detected by both liquid and tissue biopsy are included in both analysis sets (overall, n=59; treatment-naïve, n=30; previously treated, n=29); testing by both methods was not required for enrollment. †AEs were defined as events that started within the day of first dose of trial treatment until 30 days after last dose of treatment or started prior to first dose but worsened during the treatment period. AEs were assessed using the National Cancer Institute Common Terminology Criteria for Adverse Events v4.03. AE, adverse event; CI, confidence interval; MET, mesenchymal-epithelial transition factor; METex14; MET exon 14; ne, not estimable. Citation Format: Marina C. Garassino, Xiuning Le, Wade T. Iam, Enriqueta Felip, Hiroshi Sakai, Remi Veillon, Egbert F. Smit, Julien Mazieres, Jo Raskin, Alexis B. Cortot, Karin Berghoff, Rolf Bruns, Gordon Otto, Paul K. Paik. Tepotinib efficacy and safety in patients with MET exon 14 (METex14) skipping NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT536.

Introduction: T-DXd is an antibody-drug conjugate consisting of an anti-HER2 antibody, cleavable tetrapeptide-based linker, and a membrane-permeable topoisomerase I inhibitor payload. In patients (pts) with HER2-expressing or -mutated solid tumors, T-DXd has demonstrated strong antitumor activity with a manageable safety profile and is approved for treatment (tx) of pts with HER2+ metastatic breast cancer (BC) that has progressed on ≥2 anti-HER2 therapies (US) or after chemotherapy (Japan); ILD is an important identified risk. We further characterize ILD in a pooled analysis of 8 single arm phase 1 and 2 T-DXd monotherapy studies. Methods: Pts who received ≥1 dose of T-DXd monotherapy 5.4 mg/kg or higher across 8 single-arm studies were included (between Aug 28, 2015 and the data cutoff June 08, 2020). Potential ILD cases had imaging and clinical data (baseline through the time of ILD case) retrospectively reviewed by an independent adjudication committee to assess whether the reported ILD event was study drug related; events adjudicated as study drug related are reported. Results: 879 pts were included; 510 with BC, 148 with lung cancer, 107 with colorectal cancer, 78 with gastric cancer, and 34 with other cancers (2 had missing tumor type). Median tx duration was 6.9 mo (range, 0.7-50.1 mo); 139 pts (15.8%) experienced an adjudicated ILD event, most were grade (G) 1/2 (G1/2=108 [12.3%]; G3=9 [1.0%]; G4=1 [0.1%]; G5=21 [2.4%]). Among pts treated with T-DXd 5.4 mg/kg (n=315), 48 (15.2%) experienced an ILD event (G1/2=39 [12.4%]; G3=2 [0.6%]; G4=1 [0.3%]; G5=6 [1.9%]). Additional details on ILD events by dose and tumor type will be presented. Among adjudicated ILD events (any grade), the onset date as assessed by investigator was delayed compared with the adjudication committee-assessed date in 71 cases (median difference in onset date, 49 d [range, 1-288 d]). The majority of ILD events occurred in the initial 12 mo of tx. After 12 mo the risk of new onset of ILD decreased with a conditional probability of developing ILD of 7.0% in pts treated to month 12 without ILD and 1.4% in pts treated to month 18 without ILD, which was likely driven by pts with longer tx duration in the BC cohort; however, additional follow-up is required to confirm these observations. Data on the use of steroids to manage ILD will be presented. Conclusion: Pooled analysis showed that most ILD events were low grade (78% of pts with ILD had G1/2 events) and occurred in the first 12 mo of tx, with lowered risk for pts on tx > 12 mo. The adjudication committee identified ILD earlier than investigators in 48% of cases suggesting opportunity for early detection and intervention. Close monitoring, prompt recognition, and proactive management of ILD using current management guidelines may help to improve ILD outcomes. Citation Format: Charles A. Powell, Shanu Modi, Hiroji Iwata, Shunji Takahashi, Egbert F. Smit, Salvatore Siena, Dwan-Ying Chang, Charles S. Fuchs, Kun Nie, Amy Qin, Jasmeet Singh, Corina Taitt, Sunil Verma, D. Ross Camidge. Pooled analysis of drug-related interstitial lung disease (ILD) in 8 single-arm trastuzumab deruxtecan (T-DXd) studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT167.

IntroductionCapmatinib, a highly selective and potent MET inhibitor, was approved for patients with advanced METex14 NSCLC in the US and Japan in 2020, with the FoundationOne® CDx (F1CDx; a next-generation sequencing assay using DNA isolated from FFPE tumor tissue), based on results of the ongoing phase 2 GEOMETRY mono-1 study (NCT02414139). Previously, the F1CDx demonstrated 99% concordance with the METex14 RT-PCR clinical trial assay (CTA) used in the GEOMETRY mono-1 study. Here, we report findings of METex14 detection using the an NGS-based liquid biopsy assay (LDx), which detects MET single nucleotide variants and indels that lead to METex14 in circulating tumor (ct)DNA. MethodsDuring the GEOMETRY mono-1 study, patients were screened for METex14 status using a METex14 RT-PCR CTA on FFPE tissue. Clinical validation of the LDx was performed using plasma samples from patients enrolled in the GEOMETRY mono-1 study, which include METex14-positive samples from Cohort (C)4 (pretreated) and C5b (treatment-naïve), in addition to METex14-negative samples from C1b, C2, and C3, and samples from commercial sources. Concordance of the CTA and LDx were evaluated by positive percent agreement (PPA) and negative percent agreement (NPA). Clinical utility of the LDx in identifying METex14 positive patients for treatment with capmatinib was evaluated by comparing overall response rate (ORR) by BIRC in METex14 positive patients by the CTA and LDx. ResultsOf the 97 patients with METex14 NSCLC in C4 (n=69) and C5b (n=28), 88 patients had plasma volume ≥2.5 mL and cell free DNA ≥20 ng (minimum input). Of the 88 CTA METex14 positive patients, 57 were LDx positive, 26 were LDx negative; 5 had invalid sequencing results. Review of the 26 CTA discordant (METex14-negative) cases on LDx identified 3 with an alternate MET alteration, while the vast majority had evidence of low ctDNA levels. Of the 97 CTA METex14-negative patients who met minimum input requirements, 88 were LDx negative and 9 had invalid sequencing results; the LDx identified no CTA METex14-negative patient as positive. The PPA and NPA for these were 68.7% (95% CI: 57.6%, 78.4%) and 100% (95% CI: 95.9%, 100%), respectively. A post-hoc analysis comparing LDx with F1CDx in patients with METex14 showed similar results. For patients identified as METex14-positive by the LDx, the ORR (95% CI) by BIRC was determined as 48.8% (32.9–64.9%; n=20/41) for C4, and 81.3% (54.4–96.0%; n=13/16) for C5b; corresponding ORRs for patients identified as METex14-positive by the CTA were 40.6% (28.9-53.1%; n=28/69) for C4 and 67.9% (47.6-84.1%; n=19/28) for C5b.ConclusionsCurrent findings from the GEOMETRY mono-1 study support the feasibility and clinical utility of identifying advanced NSCLC patients with METex14 in ctDNA using LDx. For patients identified as METex14-negative by LDx, further testing should be performed on tissue samples, as a negative liquid biopsy result does not preclude a positive result by tissue biopsy. Citation Format: Rebecca S. Heist, Edward B. Garon, Daniel S. Tan, Harry J. Groen, Takashi Seto, Egbert F. Smit, Mike Zou, Yiqun Yang, Ying Li, Andrea Chassot Agostinho, Juergen Wolf. Accurate detection of MET exon 14 skipping using a liquid biopsy assay in NSCLC patients in the GEOMETRY mono-1 study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB056.

Background Pulmonary large cell neuroendocrine carcinoma (LCNEC) can be subdivided in two types: the co-mutated TP53 and RB1 subtype, and the TP53 and STK11/KEAP1 mutated subtype (Derks et al. Clin Cancer Res 2018; J Thorac Oncol 2018). DLL3 is a member of the Notch ligand family and a possible treatment target for neuroendocrine carcinoma. We investigated DLL3 and NE marker expression in mutational subtypes of metastatic LCNEC. Methods Immunohistochemical (IHC) analysis for DLL3 (clone SC16.65) was performed on 94 pathological reviewed pretreatment stage IV LCNEC. Samples were scored positive if ≥1% tumor cells showed cytoplasmic or dotlike DLL3 immunostaining. Also an H-score was calculated by multiplying intensity by percentage of positive cells. Targeted next generation sequencing (TP53, RB1, STK11, KEAP1) could be performed in 66 patients. Results DLL3 was expressed in 70/94 (75%) LCNEC, 56 of which showed cytoplasmic immunostaining. 37/70 (53%) samples had an H-score>100. DLL3 staining was more often seen in STK11 and/or KEAP1 mutated LCNEC (13/14;93%) than in tumors with wildtype STK11/KEAP1 (36/52;69%) (trend;p=0.093). In addition, DLL3 positivity was associated with expression of ≥2 NE-markers (64/79 LCNEC (81%) vs 3/8 (37%) tumors with Conclusions A high percentage (75%) of stage IV LCNEC shows cytoplasmatic DLL3 epression in association with NE markers, half of which with H-scores>100. Interestingly, DLL3 positivity was observed in almost all STK11/KEAP1 mutated LCNEC, which is in agreement with recent data (George et al. Nat Commun 2018). It thus might be worthwhile to investigate the efficacy of DLL3 targeted therapy in LCNEC. Citation Format: Ernst-Jan M. Speel, Bregtje C. Hermans, Jules L. Derks, Erik Thunnissen, Robert Jan van Suylen, Michael A. den Bakker, Harry J. Groen, Egbert F. Smit, Ronald A. Damhuis, Esther C. van den Broek, Cecile M. Stallinga, Guido M. Roemen, Anne-Marie C. Dingemans. A high percentage of LCNEC shows DLL3 expression in association with molecular subtypes and neuroendocrine markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 487.

Background: Extracellular vesicles (EVs) are cell-secreted membrane vesicles enclosed by a lipid bilayer derived from endosomes or from the plasma membrane. Since they are released into body fluids, and their cargo includes tissue-specific and disease-related molecules, EVs represent a rich source for disease biomarkers. However, standard ultracentrifugation methods for EV isolation (UC-EV) are laborious, time-consuming, and require high inputs. Recently a novel isolation method was described, which can be performed at small ‘miniprep’ scale, utilizes specific Heat Shock Protein (HSP)-binding peptides to aggregate HSP-decorated EVs (Ghosh et al. (2014), PLoS ONE 9:e110443). Using cancer secretome and biofluid samples, the authors showed enrichment of exosome markers for their method (abbreviated HSP-EV here) but a detailed description of the captured EV proteomes in comparison to the gold-standard ultracentrifugation (UC) method and application to small tumor proximal fluid samples is lacking. Approach: Here we used label-free proteomics of replicate EV isolations from HT-29 cancer cell-conditioned medium to compare EV fractions captured using the new HSP peptide method and UC. Subsequently we applied this novel method to profile EVs released from fresh human colorectal tumors (CRC) (n = 17) and colon adenoma (n = 4) tissue as well as patient-matched normal colon tissue. Results: Despite a 30-fold different input scale (UC-EV: 60 ml versus HSP-EV: 2 ml), both methods yielded comparable numbers of identified proteins (3115 versus 3085), with reproducible identifications (72.5% versus 75.5%) and spectral count-based quantification (average CV 31% versus 27%). EVs obtained by either method contained established EV markers and proteins linked to vesicle-related gene ontologies. In the EV fraction of the tissue secretomes 6390 proteins were identified, of which 471 proteins were significantly 5-fold more present in CRC samples than in normal tissue EVs. Gene ontology analysis revealed enrichment of nuclear proteins involved in DNA damage response, chromosome organization and RNA processing in the CRC EVs. Conclusion: The HSP-EV method provides an advantageous, simple and rapid approach for EV isolation from small amounts of biological samples, enabling high-throughput analysis in a biomarker discovery setting. Citation Format: Connie R. Jimenez, Meike de Wit, Jaco C. Knol, Inge de Reus, Tim Schelfhorst, Logan Bishop-Currey, Valerie Dusseldorp, Nicole van Grieken, Robin Beekhof, Sander R. Piersma, Thang V. Pham, Egbert F. Smit, Remond JA Fijneman, Gerrit Meijer, Henk MW Verheul. Peptide-mediated ‘miniprep’ isolation of extracellular vesicles for high-throughput proteomics; method evaluation and application in colon cancer. [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 3885.

Background: Novel targeted therapies have been successfully used against subgroups of non-small-cell lung cancer (NSCLC) patients, however, despite initial good response the cancer will eventually relapse. One of those subgroups harbors a rearranged EML4-ALK fusion gene that makes them responsive to crizotinib treatment, but therapy resistance often soon occurs. Real-time monitoring of rearrangement status over the course of treatment will help identify patients showing therapy resistance, but monitoring through serial tumor biopsies has been an obstacle. Therefore, new blood-based ´liquid biopsy´ platforms need to be developed to monitoring biomarkers in the circulation. Here we present one platform using the ability of platelets to sequester RNA released by tumor cells and represent an attractive source for non-invasive biomarker assessment. Methods: EML4-ALK rearrangements were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) in platelets and plasma isolated from blood obtained from 77 NSCLC patients, 38 of whom had EML4-ALK-rearranged tumors. In a subset patients (n = 29) that were treated with crizotinib, progression-free survival (PFS) and overall survival (OS) were correlated with presence of EML4-ALK rearrangements in platelets. Results: The study was designed with three parallel objectives: firstly to determine the sensitivity and specificity of detecting EML4-ALK rearrangements in platelets; secondly, to examine the potential impact of EML4-ALK rearrangement in platelets on outcome to crizotinib; thirdly, to test the feasibility of monitoring patients throughout treatment with EML4-ALK rearrangement assessment in platelets. Detection of EML4-ALK rearrangements in platelets demonstrated 65% sensitivity and 100% specificity. The PFS in patients treated with crizotinib was 3.7 months in patients with a positive EML4-ALK platelet status compared to 16 months for a negative EML4-ALK status (hazard ratio, 3.5; P = 0.02). Furthermore, longitudinal monitoring of EML4-ALK rearrangements in platelets was feasible, as demonstrated in an index patient where crizotinib resistance was observed two months prior to radiographic disease progression. Conclusions: Platelets may provide a useful source for non-invasive assessment of EML4-ALK rearrangements and may prove useful for predicting outcome to crizotinib. Serial analyses of EML4-ALK rearrangements in platelets may help improve clinical decisions based on radiographic imaging alone by detecting resistance to therapy sooner. Citation Format: Jonas A. Nilsson, Niki Karachaliou, Pepijn Schellen, Ana Gimenez-Capitan, Jordi Berenguer, Cristina Teixido, Justine L. Kuiper, Esther Drees, Magda Grabowska, Marte van Keulen, Jihane M. Tannous, Danielle A.M. Heideman, Erik Thunnissen, Anne-Marie C. Dingemans, Santiago Viteri, Bakhos A. Tannous, Ana Drozdowskyj, Rafael Rosell, Egbert F. Smit, Thomas Wurdinger. Monitoring rearrangement of EML4-ALK in blood platelets predicts outcome to crizotinib treatment in non-small-cell lung cancer patients. [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 LB-053. doi:10.1158/1538-7445.AM2015-LB-053

Background: The epidermal growth factor receptor (EGFR) is an established target for the treatment of advanced non-small cell lung cancer (NSCLC). EGFR tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib have been approved for treatment of NSCLC. The Efficacy of those TKIs depends on the presence of activating mutations of EGFR (exon 19 del. or L858R point mutation). Therefore, the EGFR mutational status needs to be ascertained by an invasive biopsy before treatment; however this is often compromised by sampling errors due to tumor heterogeneity or by lack of access to the tumor tissue. We hypothesize that PET-imaging with radiolabeled TKIs (TKI-PET) can provide a tool to determine and predict responsiveness to EGFR TKIs in vivo. Previously, this was demonstrated with the reversible EGFR inhibitor [11C]erlotinib that showed higher uptake in patients harboring exon 19 deletions of EGFR. Therefore, to validate the application of TKI-PET imaging, we developed irreversible second generation TKI afatinib as a PET tracer and extensively evaluated both [18F]afatinib and [11C]erlotinib in a comparative preclinical imaging study to determine the feasibility of TKI-PET as a potential personalized medicine tool. Methods: The synthesis of [11C]erlotinib was already established in our center. The synthesis of [18F]afatinib was developed using an innovative three step radiosynthesis. Both tracers were thoroughly evaluated in a PET imaging study in mice bearing a panel of three clinically relevant EGFR expressing human NSCLC xenografts. The three selected cell lines are: A549 with wild-type EGFR, H1975 with the sensitizing mutation L858R and the acquired resistance related mutation T790M and HCC827 cell line with the sensitizing exon 19 deletion. Other factors influencing uptake were also investigated, such as tracer metabolism and effects of drug efflux transporters. Results: [18F]afatinib was successfully synthesized in an overall decay corrected yield of 17 ± 2.3% with a radiochemical purity of >97% and a specific activity of >200 GBq/µmole at end of synthesis. In a panel of lung cancer xenograft models, [11C]erlotinib and [18F]afatinib both showed an increased uptake in the treatment sensitive cell line, while showing low to no uptake in wild type or acquired resistance cell lines. [18F]afatinib showed a higher tumor-to-background ratio than [11C]erlotinib in the sensitive cell line. In vivo blocking of the P-gp drug efflux transporter with tariquidar revealed an increased uptake of both tracers indicating that they are substrates for efflux pumps. Conclusions: The new TKI-PET tracer [18F]afatinib has been synthesized successfully. [11C]erlotinib and [18F]afatinib show similar uptake patterns across EGFR mutations, while uptake appears to correspond with treatment sensitivity. A treatment study in mice will be performed to directly correlate uptake to sensitivity. Citation Format: Paul Slobbe, Albert Windhorst, Marijke Stigter-van Walsum, Robert Schuit, Egbert Smit, Heiko Niessen, Guus van Dongen, Alex Poot. A comparative preclinical PET imaging study between [11C]erlotinib and [18F]afatinib. [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 113. doi:10.1158/1538-7445.AM2014-113

The five-year survival rate for non-small cell lung cancer (NSCLC) is still less than twenty percent in part due to treatment failure and lack of biomarkers for personalized therapy. Most NSCLC patients are treated by chemotherapy and cisplatin is still the major component of most chemotherapy regimens. However, resistance to cisplatin is a common phenomenon. Aim of this study was to identify protein biomarkers that can be used for cisplatin response prediction. The proteomes and secretomes1 of a series of human NSCLC cell lines with a range of IC50-values for cisplatin (1.5 - 15 μM) were analyzed by label-free proteomics based on GeLC-MS/MS and quantified by spectral counting. Significant differential expression of proteins was determined by the in-house developed beta-binomial statistical test2. Network and gene ontology analysis was performed, with STRING and DAVID respectively, to reveal cellular and molecular functions associated with the differential proteins. In total, 2885 and 2342 proteins were detected in the cell lysates and secretomes, respectively. The proteins with significantly higher expression in the resistant cell lines were associated RNA processing and vesicle transport, while the proteins with higher expression in the sensitive cell lines were known to be involved in RNA splicing, DNA damage repair and extracellular matrix organization. The most promising candidates are being validated in tissues and sputum of patients with known response to cisplatin. 1 Piersma SR et al. J Proteome Res. 2010 Apr 5;9(4):1913-22. 2 Pham TV et al. Bioinformatics. 2010 Feb 1;26(3):363-9. Citation Format: Tieneke B M Schaaij-Visser, Remco Nagel, Inge de Reus, Sander R. Piersma, Thang V. Pham, Egbert F. Smit, Frederik B. Thunnissen, Ruud H. Brakenhoff, Anton Berns, Connie R. Jimenez. Novel candidate protein biomarkers for cisplatin response prediction in non-small cell lung cancer. [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 2488. doi:10.1158/1538-7445.AM2013-2488

Purpose Stage IV EGFR mutation positive NSCLC patients treated with reversible tyrosine kinase inhibitors(TKI) (i.e., erlotinib (E) or gefitinib (G)) develop resistance after 6-12 months, in half of the patients mediated by the T790M gatekeeper mutation. Afatinib (BIBW2992, Boehringer Ingelheim) has shown in a phase III study to prolong progression free survival (PFS) in patients progressing on EGFR TKI. The EGFR mutational pattern underlying these responses to afatinib is unknown. The current study was set out to evaluate response rate to afatinib in relation to tumor EGFR genotype. Material/Methods Patients with activating EGFR mutations, or without known EGFR mutations meeting Jackman criteria, progressing on E/G, and who had a re-biopsy before afatinib treatment were included. Mutations in exons 18-21 of the EGFR-gene were tested using high-resolution melting with reflex Sanger sequencing in both centers. We retrospectively studied EGFR mutations, PFS, and overall survival after treatment with afatinib. Statistical analysis was performed using Kaplan-Meier and Chi-square test. Results Initially 21/24 (88%) patients had stage IV NSCLC with a mean performance score of 1 (range 0-2). Before afatinib treatment 17/24 (71%) of patients received a platinum doublet. All patients received E or G. Three patients received both TKI sequentially. Four patients without mutation were treated for more than 6 months with E/G resulting in stable disease. PFS on previous E/G was 16 months (range, 2.0 - 37.9). At rebiopsy, 12/24 (50%) patients had a T790M mutation, no other secondary mutations were detected. There was no difference in duration of E/G administration neither in occurrence of T790M mutation (median 12.4 months (range, 6.8-34.4) with T790M vs. 19.2 months (range, 2.0-37.9) without T790M at rebiopsy; p=0.22). After PD on E/G, patients received afatinib mostly as 2nd or 3rd line therapy.Tumor response rate on afatinib was 12.5% (n=3). Of these, 1 had a T790M mutation. Disease control rate was 71%, median PFS was 3,0 months (range, 1.2-18.7 months). Median OS was 6.8 months (range, 1.3-19.0 months). The presence of T790M mutation did not influence PFS (with T790M, 2.9 vs without T790M, 3.2 months; p=0.684). However, it did influence OS (7.2 months vs. 3.5 months; p=0,039). Conclusion In our cohort of patients, PFS on afatinib was similar to the Lux-lung 1 study and similar for patients with or without T790M mutations. Duration of first generation TKI administration did not influence occurrence of T790M mutation. T790M mutation was in favor of a longer OS on afatinib. Citation Format: Anthonie J. van der Wekken, Justine L. Kuiper, Daniëlle A.m. Heideman, Ed Schuuring, Wim Timens, Erik Thunnissen, Harry J.M. Groen, Egbert F. Smit. Pre-treatment EGFR mutation analysis predicts clinical outcome in a retrospective analysis of 24 non-squamous non-small-cell lung cancer (NSCLC) patients treated with second line afatinib. [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 3526. doi:10.1158/1538-7445.AM2013-3526

Background: EGFR is an established target in advanced NSCLC, and the EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib have been approved for the treatment of patients harbouring activating-EGFR mutations. Unfortunately, their efficacy is limited by acquired resistance, caused in ≈50% of the cases by the T790M secondary point-mutation. Several EGFR inhibitors have been developed with the aim to overcome such resistance. However, emergence of in vitro resistance due to T790M amplification has been reported for second-generation EGFR-TKIs. Therefore we evaluated the efficacy of CNX-2006, a prototype of the novel mutant-selective EGFR-TKI CO-1686, which is currently in a phase I clinical trial in previously treated mutant EGFR NSCLCs. Methods: CNX-2006 was provided by Celgene Avilomics Research. Its antiproliferative activity was tested by sulforhodamine B assay in 12 NSCLC cell lines, previously characterized for EGFR and K-Ras mutational status and gefitinib sensitivity, including PC9GR4 and PC9DR1 (kindly provided by Dr. Jänne, Harvard University, Boston, USA). Novel CNX-2006 resistant clones have been established starting from the EGFR T790M cells H1975 and PC9GR4, and several markers have been characterized by RT-PCR, kinase array and Western blot. Results: CNX-2006 inhibited cell proliferation independently from K-Ras mutations while it was as effective as gefitinib in activating-EGFR mutation positive cells. In the cell lines expressing wild-type EGFR CNX-2006 and gefitinib had limited anti-proliferative activity. CNX-2006 inhibited EGFR-T790M cells growth up to 1000-fold more compared to wild-type EGFR cells. EGFR inhibition was observed in cells harbouring the T790M mutation at IC50 values below 20 nM after 1 hour exposure to the drug. In contrast to gefitinib, CNX-2006 also significantly reduced the volume of tumor spheres derived from H1975 cells. Multiple CNX-2006 resistant clones were generated by exposing H1975 or PC9GR4 cells to increasing drug concentrations, leading to 30-fold resistant clones, which grow in CNX-2006 concentrations 16-20 times the initial IC50s. This resistance was retained for at least 3 months after drug removal. CNX-2006 resistant clones showed differences in expression of several biomarkers associated with EMT, such as a 3-fold reduction of E-cadherin mRNA and a 60-fold increase in MMP9 compared to the parental cells. Conclusions: CNX-2006 is a potent, mutant-selective EGFR inhibitor with excellent in vitro activity in cells with activating EGFR mutations, as well as in cells harbouring the T790M mutation. Future studies in mechanisms underlying EMT are warranted and might be used to prevent CNX-2006 resistance. Citation Format: Elena Galvani, Elisa Giovannetti, Annette O. Walter, Robert Tjin, Henk Dekker, Pier Giorgio Petronini, Egbert F. Smit, Godefridus J. Peters. Role of epithelial-mesenchymal transition (EMT) in sensitivity to CNX-2006, a novel mutant-selective EGFR inhibitor which overcomes in vitro T790M-mediated resistance in NSCLC. [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 3244. doi:10.1158/1538-7445.AM2013-3244

Rationale. In observational and preclinical studies, metformin is found to have anticancer properties and to prolong survival in various cancer types. It is unknown whether metformin use confers a survival benefit in Small Call Lung Cancer (SCLC) patients. We studied the influence of metformin usage on the overall survival (OS) in SCLC patients. Methods. SCLC patients in the period 1998 to 2009 were selected from the population-based Eindhoven Cancer Registry. Those included were linked to the PHARMO Record Linkage System. The following data were recorded: date of diagnosis, age, gender, stage of disease, comorbidity (Charlson comorbidity index), drug usage, administration of chemotherapy and date of death. Kaplan Meier curves were used to estimate OS. Cox regression analyses were used to estimate the hazard ratio (HR), this was corrected for age, gender and stage of disease in multivariate analyses. Due to the small group of patients using metformin, a multivariate analysis to correct the HR estimation was not performed. Results. In this study, a total of 1,136 SCLC patients were included. Median age was 67 years (range 37-95), 63%/37% male/female, 63%/37% extended/limited disease. Patients were treated with chemotherapy (75%), radiotherapy (28%) or surgery (2%). A total of 256 patients (23%) did not receive active anticancer treatment. Most common comorbidities were cardiovascular diseases (35%), COPD (24%), and hypertension (19%). A total of 151 patients (12%) were diagnosed with diabetes mellitus (DM), of which 92 (68%) received antidiabetics at time of diagnosis of SCLC. Fifty patients received metformin. DM was not significantly associated with a worse prognosis, with a median OS of 6.0 vs. 7.5 months in patients without DM (HR= 1.2 (95% CI 0.9-1.5)). Patients receiving metformin had no survival benefit comparing to all other SCLC patients, with a median OS of 8.4 and 7.0, respectively (HR= 0.9 (95% CI. 0.7-1.2)). Compared to patients receiving insulin monotherapy or sulfanylureas (n= 42), outcome was non-significantly improved in patients receiving metformin monotherapy (n= 20): median OS 11.0 vs. 7.0 months (HR= 0.9 (95% CI. 0.5-1.6)). Conclusions. In our series, having diabetes was not associated with worse prognosis in SCLC patients. However, within the SCLC patients with diabetes, those receiving metformin had a clinically relevant survival benefit compared to those receiving other antidiabetics. Due to a relative small cohort we could not demonstrate significance. Larger (retro- and) prospective studies and preclinical research must be performed to elucidate the role of metformin, if any, in the clinical management of SCLC. Citation Format: Wouter W. Mellema, Mieke J. Aarts, Myrthe P.P. Van Herk-Sukel, Egbert F. Smit. A retrospective evaluation of metformin usage in patients with small cell lung cancer (SCLC). [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 3635. doi:10.1158/1538-7445.AM2013-3635

Background: Non Small Cell Lung Cancer (NSCLC) remains the number one cause of cancer death worldwide. For a number of cancers, restoration of tumor suppressor p53 activity is being studied as a possible treatment strategy. Approximately 50% of NSCLCs harbor wild type p53. The p53 inhibitor RCHY1 was previously shown to be highly expressed in lung cancer and suggested to play a critical role in lung tumor progression by degrading p53 (Duan et al., J Natl Cancer Inst 96: 1718-1721). We hypothesized that more molecules could be inhibiting p53 in NSCLC. Here we set out to identify these inhibitors as potential therapeutic targets in NSCLC. Methods: Three siRNA screens were done with the Dharmacon siGENOME whole human genome siRNA library on a derivative of the A549 NSCLC cell line harboring the p53 dependent firefly luciferase reporter PG13Luc. Luciferase activity was measured three days after siRNA transfection using an in-house developed assay (Siebring-van Olst et al, J Biomol Screen, PMID: 23112084). Results: The screens showed excellent assay metrics (Z’ factors >0.5 and Spearman Rank Correlations >0.89). Hit selection threshold for stratification was set at a robust Z-score ≥ 3, which selected 54 genes. Silencing these candidates increased luciferase activity more than 5-fold in the primary screen, which exceeded commonly known p53 inhibitors such as MDM2 and MDM4 (2.5 and 3.2-fold, respectively). For 37 hits, the phenotype was confirmed with two or more individual siRNAs targeting that gene, as well as in a different A549-PG13Luc cell clone. In A549 cells carrying a shRNA construct suppressing p53, a reduction in p53 activity was seen for all 37 candidates, thus showing that the effects of gene silencing on luciferase activity were indeed p53 dependent. A second reporter system with a different p53 response element, the Cignal assay, confirmed p53 dependency for 14 hits. Silencing these 14 inhibitors in a second NSCLC cell line (NCI-H292), also resulted in p53 activation. Western blot analysis on siRNA-transfected A549 cells confirmed p53 activation by showing increased levels of p53 and/or increased levels of p21 (12 and 8 candidate genes, respectively). Interestingly, silencing 9 putative p53 inhibitors was accompanied with a subsequent loss of cell viability. Protein interaction analysis using STRING showed that six of these genes, i.e., CWC22, SF3A3, SF3B1, SF3B14, HNRPL and SNRPD3, cluster together in a network involved in RNA processing, while the other candidates appeared unrelated. Conclusion: We identified a number of potent inhibitors of p53 in NSCLC cells that have not previously been associated with p53 function. Some of these proteins could be potential therapeutic targets in cancers harboring wild type p53. Citation Format: Ellen Siebring-van Olst, Renee X. de Menezes, Ida H. van der Meulen, Egbert F. Smit, Victor W. van Beusechem. Genome-wide siRNA screens identify potent p53 inhibitors in NSCLC cells. [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 4388. doi:10.1158/1538-7445.AM2013-4388

Background Extensive-stage disease, small-cell lung cancer (ED-SCLC) is a lethal disease with poor clinical outcomes given current systemic therapy options. The objective of this study was to examine potential prognostic factors identifiable through genome-wide characterization methodologies to enhance our current understanding of ED-SCLC and gene networks involved in ED-SCLC. Methods Patient samples obtained within the framework of a randomized Phase III trial (n=908) were successfully assayed for copy number variations (CNVs, n=92), exon array gene expression (n=73), and miRNA array (n=92). The CNV data were adjusted to account for degraded nucleic acids from formalin-fixed, paraffin-embedded samples, and copy number was estimated using the Partek® genomics suite. Unsupervised clustering analysis of exon array data was performed to detect groups of samples with similar expression profiles. Cluster stability was assessed by using bootstrapping procedures, and its association with survival was studied. For miRNAs, which were expressed in >75% of samples, a rank-based correlation analysis was performed between the miRNA expression and mRNA expression. A Cox-regression model was built to calculate miRNAs’ association with survival time. Results A SNP array analysis revealed CNVs at chromosomes 3p, 4q, 5q, 10q, 13q, and 17p, which was in accordance with previous reports. Novel changes at other chromosomal sites are being analyzed. Cluster analysis of expression data suggests the presence of 2 distinct molecular subtypes within the ED-SCLC patient samples examined in this analysis. A significant difference in progression-free survival (PFS) was observed (log rank, p ≤ 0.009); the median PFS for patients belonging to cluster 1 was 5.2 months versus 3.5 months for patients assigned to cluster 2. Cluster 1 showed high expression of genes associated with antiapoptotic pathways through NFKB, I-KB, IRAK1 and IRAK2; Gn-Rh signaling and downstream MAPK induction; and EGR1 factor signaling cascades, including c-Jun and c-Fos. Cluster 2 showed high expression of genes associated with DNA damage repair including ATM, MSH2, BRIP1, Rad50, and MRN complex; and G2/M checkpoint arrest through ATM phosphorylation and cyclin-B induction. Several miRNAs were associated with improved survival, including miRNAs from the miR7 and miR320 families. Conclusions This analysis of ED-SCLC patient samples, which used multiple genomics platforms, confirmed several known aberrations and identified novel markers not previously described with ED-SCLC outcomes. Two clusters of patients with distinct molecular profiles and outcomes were revealed, and miRNAs associated with improved survival outcomes were identified. While these data are exploratory and ongoing, confirmation of these observations may lead to advances in profiling and targeting important genes integral to SCLC. 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 LB-396. doi:1538-7445.AM2012-LB-396

Introduction Exosomes are nanometer-sized vesicles secreted by tumor cells after fusion of multivesicular bodies with the plasma membrane that reflect tumor cell biology. Exosomes contain distinct classes of molecules including mRNA, miRNA and proteins. Because they can be isolated from blood they are promising candidate biomarkers. Diagnostic biomarkers for selection of patients for treatment with tyrosine kinase inhibitors are urgently warranted. We hypothesize that exosomes contain tumor derived kinases that may serve as biomarkers for treatment with kinase inhibitors. Methods The U87 glioma cell line with or without EGFRvIII (U87ΔEGFR) was used as an in vitro model in this study. Cell viability was studied with the MTT-assay. Exosomes were isolated from 32h conditioned media either serum-free or containing 10% exosome-deprived serum and from 5-9mL of fresh human serum from 3 cancer patients. The isolation procedure consisted of subsequent centrifugation at 500G, 2000G, 10.000G and 100.000G. The exosome pellet was treated with proteases for 1h at 37°C to digest soluble proteins, washed in PBS and lysed. Western blots for phospho-EGFR (y1068), EGFR, phospho-AKT, AKT, phospho-ERK, ERK, Alpha-Tubulin and exosome markers CD63, ALIX and CD81 were performed. Results The sensitivity of U87ΔEGFR to erlotinib (ERL) was significantly higher compared to parental U87 cells with IC50s of 1µM and 5.4µM (p 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 176. doi:1538-7445.AM2012-176

TKI represent the largest group of novel anticancer drugs. Loosely based on a core pyrimidine structure TKI selectively target the ATP activity of a TK. Their poor solubility represents a challenge in administration. TKI are administered orally but available PK indicates that in most cases bioavailability is relatively low (∼60% or lower). Despite a molecular weight in a similar range, doses vary significantly: Sunitinib (Sun) - 50 mg/d, Dasatinib (Das) - 150 mg/d, Erlotinib (Erl) - 150 mg/d, Gefitinib (Gef) - 250 mg/d and Sorafenib (Sor) - 400 mg BID. Steady state plasma levels vary from 0.13 (Sun), 0.21 (Das), 0.29 (Gef), 2.54 (Erl) to 12.1 µM (Sor). In combination Erl increased systemic plasma levels of Sor. Variations in intestinal absorption may seriously affect plasma concentrations, tumour exposure and anti tumour effect. To investigate the mechanisms behind these differences a well established model for intestinal transport was used: the human colon cell line, CaCo2, when grown in special coated Transwell plates forms a confluent differentiated polarised monolayer resembling gut epithelium. This model was used to determine the permeability of Gef, Erl, Sun, Sor, Das and combined Erl/Sor. Absorption from the gut given as the transfer rates from Apical to Basolateral (A/B) sides using 20 µM TKI at the apical side was determined over a 3 hour period. Transfer was linear in this period. Transfer rates varied from 43 for Sun, 209 for Das, 180 for Gef, 223 for Sor, to 479 pmol/hr for Erl. At 10 µM Erl increased transfer rate of Sor from 145 to 185.7 pmol/hr in line with observed plasma levels. In order to determine the role of ABC pumps, we depleted ATP with azide, which partially reduced transfer of Gef, Sun and Sor, but did not affect Erl. Ko143, a specific ABCG2 inhibitor decreased transfer of Gef and Sor but unexpectedly increased Sun. Remarkably, permeability transfer rates from the basolateral (blood) side to gut (B/A) were much higher (252 for Gef, 621 for Sor, 685 for Sun, 1623 for Erl and 4630 pmol/hr for Das) than A/B transfer. In order to explain this difference we determined cellular accumulation in the monolayer cells at the end of the experiments. For the A/B experiments this was undetectable for Erl, 0.79 for Gef, 1.65 for Sor, 11.9 for Sun and 1.1 for Das (pmol/µg protein). For the B/A experiments a proportional decrease in accumulation relative to the observed increased transfer rate was seen with Gef as the exception, with values from undetectable for Erl, 2.1 for Gef, 0.15 for Sor, 4.72 for Sun and 0.26 pmol/µg protein for Das. In conclusion, absorption of TKI from the apical side (gut epithelium) is relatively poor, while there was a relatively high negative flow (B/A); this is in line with the low bioavailability of most TKI. Some ABC transporters play a role in the absorption, in line with their substrate specificity. This may also affect retention of TKI in tumour cells 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 3785. doi:1538-7445.AM2012-3785

Introduction: KRAS mutation is thought to be related with poor outcome of NSCLC pts. The predictive value of KRAS mutation for response to chemotherapy (CT) has not been established Patients and methods: From a retrospective database from 2 university hospitals all pts with advanced NSCLC treated with palliative platinum containing chemotherapy were selected. Pts were included when archive tumor tissue was available for DNA extraction. High resolution melting followed by PCR sequencing was performed for KRAS exon1 and 2. Results: KRAS mutation analysis was evaluable for 122 pts. All pts had advanced NSCLC and were no candidate for treatment with curative intent. Mean age 61 years (range 24-80 years), 81 males and 41 females. All pts received platinum-containing CT. Response was evaluable for 110 pts: 1 complete response, 21 partial response, 54 stable disease and 54 progressive disease. Median progression free survival (PFS) was 5.7 months (m) (95% CI 4.2-7.2 m), median overall survival (OS) was 7.5 m (94% CI 5.2-9.9 m). 44 pts (36%) had a KRAS mutation. 38% of pts with a KRAS mutation had as best response PD on CT, and 27% of pts without a KRAS mutation had PD, this difference was not significant. No difference in PFS and OS was observed for pts with a KRAS mutation (median PFS 5.3 m, 95% CI 3.2-7.3 m and median OS 6.2 m, 95% CI 4.8-7.3 m) and without a KRAS mutation (median PFS 6.1, 95% CI 5.0-7.2m and median OS 8.9 m, 95% CI 6.4-11.4). Conclusion: In our series KRAS mutation was not predictive for response to platinum containing CT in pts with advanced NSCLC. Furthermore, KRAS mutation was not prognostic. Additional data on specific KRAS mutations will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 340. doi:10.1158/1538-7445.AM2011-340

Objectives Thymidylate synthase (TS) is a key enzyme for de novo synthesis of DNA and as such a target for anticancer drugs. In a mouse model, effective TS inhibition resulted in a 1.8 fold increase of 3′-deoxy-3′-[(18)F]fluorothymidine (18F-FLT) cellular uptake (Cancer Res 2006;66:8558-64). Here we investigated changes in 18F-FLT uptake 4 hours after the first dose of pemetrexed in patients with non-small cell lung cancer (NSCLC). Methods Twelve NSCLC patients (mean age 64 yrs, 4 male) treated with pemetrexed (in combination with cisplatin, carboplatin or monotherapy) were included. Patients underwent dynamic 18F-FLT PET at baseline and 4 hours after the first therapeutic dose of pemetrexed. For primary tumors, regions of interest (ROI) were defined with a 41, 50 and 70% threshold of the maximum pixel, and the maximum standardized uptake value (SUVmax) was determined (as well as full kinetic analysis). One, 2, 3, 4 and 6 hours after pemetrexed administration, blood samples were collected to measure deoxyuridine levels using LCMSMS. After six weeks, RECIST was used to evaluate tumor response. Association between 18F-FLT uptake and RECIST was tested with a chi-square test. Results According to RECIST 3 patients had PR, 7 SD and 2 PD. Four hours after administration of pemetrexed, two patients had increased 18F-FLT uptake (beyond test-retest borders, 22% for SUVmax), while three patients had a significantly decreased uptake (see Table). In 7 out of 12 patients, the change in 18F-FLT uptake was within the test-retest variability. In all patients deoxyuridine levels raised after administration of pemetrexed with a average maximum at 4 hours. There was no significant association between 18F-FLT uptake and RECIST. Conclusions Measuring TS inhibition in a clinical setting 4 hours after pemetrexed revealed a non-systematic change in 18F-FLT uptake. The association with response measurement on CT was not significant.18F-FLT PET and RECIST per patientPt No.Baseline 18F-FLT uptake (SUVmax)18F-FLT uptake 4 hours after pemetrexed (SUVmax)Change 18F-FLT in uptake (%)RECIST after 6 weeks14.74.94.1SD24.44.75.0SD34.12.3−44.7SD44.04.411.0PD53.54.528.6PR63.42.2−33.3SD74.85.05.7SD812.57.9−36.6PR911.312.17.1PD105.67.228.3SD115.15.35.7PR124.73.7−21.5SD 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 5225.

An ongoing effort in the clinical application of anti-angiogenic cancer therapy is to identify and validate molecular and/or cellular biomarkers that could predict response as well as enhance our insight in the pharmacodynamics of these agents. Here we study therapy-induced changes in the frequency of a novel rare circulating cell population, characterized by a CD45neg / CD34bright / CD133neg marker profile, which was recently shown to be the source of highly proliferative endothelial outgrowth cells. These candidate cEPCs (ccEPCs) were measured during treatment of 23 renal cell cancer patients with the receptor tyrosine kinase inihibitor, sunitinib and 19 non-small cell lung cancer patients with the monoclonal antibody bevacizumab together with the EGFR inhibitor erlotinib. In addition plasma VEGF was monitored. Tumor response was assessed with Computed Tomography (CT)-scans according to RECIST criteria. In patients treated with sunitinib an opposite change in the frequency of CD34bright/ CD45dim/CD133pos (hematopoetic progenitor cells, HPCs) and CD34bright/CD45neg /CD133neg (ccEPCs) was seen. The median of ccEPCs increased from 69 before treatment (C1Day1) to 180 cells/ml on C1Day14 (P= 0.001) and to 229 (n=14, P=0.013) on C1Day28, while the HPCs decreased from 1350 on C1Day1 to 372 cells/ml on C1Day14 (n= 23, P=0.000) to 409 on C1Day28 (n=14, P= 0.001). Plasma VEGF levels showed an increase during treatment, parallel to the ccEPC numbers. These effects were treatment-related as both cell populations and plasma VEGF levels returned to pretreatment levels after the 2 weeks rest period (C2Day1). The responders group (PR+SD) showed an increase of ccEPCs ( median 82 to 188, P=0.001), while no significant increase was found in the non-responders group (PD) but this group (n=5) was limited in size (median 69 to 145, P=0.686). In patients treated with bevacizumab and erlotinib, but not with erlotinib alone (controls) ccEPCs also increased, whereas the HPCs remained stable during treatment and the free plasma VEGF levels decreased. The relation to tumor response is being analyzed. In conclusion, two CD34bright populations, ccEPCs and HPCs, which both are partly VEGFR2pos, showed a different response to anti-VEGF(R) treatment. This study therefore implicates that in future analysis of CD34bright/VEGFR2pos cEPCs, a distinction has to be made between CD45neg and CD45dim cells. Since, recent studies in mice have shown that human highly proliferative EPC outgrowth cells are functionally important in neovascularization, the potential role of ccEPCs in human tumor angiogenesis and their increased numbers upon anti-VEGF therapy warrants further study. Larger studies are needed to define the predictive potential of these cells for the outcome of anti-angiogenic therapies.