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Introduction: Molecular profiling of tumors has become the mainstay of diagnostics for metastasized solid malignancies and guides personalized treatment, especially in non-small cell lung cancer (NSCLC). In current practice it is often challenging to obtain sufficient tumor material for reliable molecular analysis. Cell free (cf) DNA in blood or other bio-sources could present an alternative approach to obtain genetic information from the tumor. In a retrospective cohort we analyzed the added value of cfDNA analysis in pleural effusions for molecular profiling. Methods: We retrospectively analyzed both the supernatant and the cell pellet of 44 pleural effusions sampled from 39 patients with KRAS (23) or EGFR (16) positive tumors for the original driver gene mutation as well as for EGFR T790M resistance mutations. Patients were diagnosed with either NSCLC (32), colon carcinoma (4), appendiceal carcinoma (2) or adenocarcinoma of unknown primary (1). Samples collected in the context of routine clinical care were stored in the NKI-AVL biobank. We used Bio-Rad QX200 droplet digital PCR for analysis. Results: The original driver gene mutation could be detected in 37 of the 44 pleural effusions by analysis of both supernatant (35/44 positive) and cell pellet (29/44 positive). In 7 out of 20 pleural effusions from patients with EGFR mutation positive tumors, a T790M mutation was detected. All 7 supernatants were positive as were 5 of the 7 cell pellets. The EGFR T790M mutation was confirmed in all supernatants (4/4) and in 3 of the 4 cell pellets sampled from patients with T790M positive tumors (4). Conclusions: Cell free DNA in pleural effusion proved to be a valuable bio-source and can be used to detect driver gene mutations as well as resistance mechanisms like EGFR T790M in pleural effusion. Citation Format: Karlijn Hummelink, Mirte Muller, Dorothe Linders, Vincent van der Noort, Petra Nederlof, Sjaak Burgers, Gerrit Meijer, Michel van den Heuvel, Daan van den Broek, Kim Monkhorst. Cell free DNA in the supernatant of pleural effusion can detect driver and resistance mutations and can guide TKI treatment decisions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 641.

I: Studies have shown the activity of anti-PD(L)-1 therapies in patients with advanced non-small-cell lung cancer (NSCLC), however response rates are rather low (~20%). Therefore, there is an urgent need to identify biomarkers that predict patient outcome to immunotherapy (IT). Previously we have shown that RNA signatures of tumor-educated platelets (TEPs) may have predictive value for tumor type-specific diagnostics. Platelets are intimately involved in immune responses. We hypothesized that TEP RNA profiles have predictive value for IT response. M: We collected baseline platelet pellets, isolated from whole blood by differential centrifugation, from 389 stage IV NSCLC patients treated with Nivolumab (table 1). Tumor response was evaluated at 3 and 6 months and reported according to RECIST 1.1. Platelet pellets were subjected to total RNA isolation, SMARTer cDNA amplification, and libraries were sequenced on the HiSeq platform. Raw data (~20 M reads per sample) was mapped to the human genome, intron-spanning spliced RNA reads were selected for analysis. Gene panels were calculated by ANOVA statistics. R: Until now 64 samples were sequenced, 30 of patients with clinical benefit (PR or SD at six months; CB) and 34 of patients with no clinical benefit (PD; no CB). 40 randomly selected samples (20 CB, 20 no CB) were used for training of the support vector machine (SVM)-based therapy response classification algorithm. 24 samples were used for independent evaluation of the classifier. Hierarchical clustering of genes with p Table 1.Patient CharacteristicsTotal CohortNumber of patients389Start of treatmentAugust 2015-november 2016Date lock1th of November, 2016Gender (Male)56%Age (med, range)64.5 years (29-83)Treatment lines1st line1%2nd line72%>2nd line27%DiagnosisNon-Squamous68%Squamous26%Other6%ResponseClinical benefit27%Progressive54%Unknown19%PD-L1 expressionResults expected in april Citation Format: Mirte Muller, Myron Best, Nik Sol, Anna-Larissa N. Niemeijer, Adrienne Vancura, Robert D. Schouten, Jeroen J.N. Hiltermann, Sjors G.J.G. In 't Veld, Daan van den Broek, Vincent van der Noort, Adrianus J. de Langen, Ed M.D. Schuuring, Thomas Wurdinger, Michel M. van den Heuvel. RNA-sequencing of tumor-educated platelets enables nivolumab immunotherapy response prediction [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 LB-248. doi:10.1158/1538-7445.AM2017-LB-248