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Summary Tumor-educated platelets (TEPs) are potential biomarkers for cancer diagnostics. We employ TEP-derived RNA panels, determined by swarm intelligence, to detect and monitor glioblastoma. We assessed specificity by comparing the spliced RNA profile of TEPs from glioblastoma patients with multiple sclerosis and brain metastasis patients (validation series, n = 157; accuracy, 80%; AUC, 0.81 [95% CI, 0.74–0.89; p < 0.001]). Second, analysis of patients with glioblastoma versus asymptomatic healthy controls in an independent validation series (n = 347) provided a detection accuracy of 95% and AUC of 0.97 (95% CI, 0.95–0.99; p < 0.001). Finally, we developed the digitalSWARM algorithm to improve monitoring of glioblastoma progression and demonstrate that the TEP tumor scores of individual glioblastoma patients represent tumor behavior and could be used to distinguish false positive progression from true progression (validation series, n = 20; accuracy, 85%; AUC, 0.86 [95% CI, 0.70–1.00; p < 0.012]). In conclusion, TEPs have potential as a minimally invasive biosource for blood-based diagnostics and monitoring of glioblastoma patients., Graphical Abstract, Highlights TEP RNA enables blood-based brain tumor diagnostics TEP RNA is dynamic throughout anti-tumor treatment TEP RNA may be employed for therapy monitoring, Sol et al. show that tumor-educated platelets (TEPs) can be employed for detection of glioblastoma. Glioblastoma TEP RNA profiles can be differentiated from patients with metastatic brain cancer or MS. Furthermore, the tumor signals in TEPs are dynamic, indicating that TEPs can be employed for blood-based therapy monitoring.

BackgroundIn multiple sclerosis (MS), clinical assessment, MRI and cerebrospinal fluid are important in the diagnostic process. However, no blood biomarker has been confirmed as a useful tool in the diagnostic work-up.ObjectivesBlood platelets contain a rich spliced mRNA repertoire that can alter during megakaryocyte development but also during platelet formation and platelet circulation. In this proof of concept study, we evaluate the diagnostic potential of spliced blood platelet RNA for the detection of MS.MethodsWe isolated and sequenced platelet RNA of blood samples obtained from 57 MS patients and 66 age- and gender-matched healthy controls (HCs). 60% was used to develop a particle swarm-optimized (PSO) support vector machine classification algorithm. The remaining 40% served as an independent validation series.ResultsIn total, 1249 RNAs with differential spliced junction expression levels were identified between platelets of MS patients as compared to HCs, including EPSTI1, IFI6, and RPS6KA3, in line with reported inflammatory signatures in the blood of MS patients. The RNAs were subsequently used as input for a MS classifier, capable of detecting MS with 80% accuracy in the independent validation series.ConclusionsSpliced platelet RNA may enable the blood-based diagnosis of MS, warranting large-scale validation.

Diffuse gliomas are the most common malignant primary tumors of the central nervous system. Like other neoplasms, these gliomas release molecular information into the circulation. Tumor-derived biomarkers include proteins, nucleic acids, and tumor-derived extracellular vesicles that accumulate in plasma, serum, blood platelets, urine and/or cerebrospinal fluid. Recently, also circulating tumor cells have been identified in the blood of glioma patients. Circulating molecules, vesicles, platelets, and cells may be useful as easily accessible diagnostic, prognostic and/or predictive biomarkers to guide patient management. Thereby, this approach may help to circumvent problems related to tumor heterogeneity and sampling error at the time of diagnosis. Also, liquid biopsies may allow for serial monitoring of treatment responses and of changes in the molecular characteristics of gliomas over time. In this review, we summarize the literature on blood-based biomarkers and their potential value for improving the management of patients with a diffuse glioma. Incorporation of the study of circulating molecular biomarkers in clinical trials is essential for further assessment of the potential of liquid biopsies in this context. Electronic supplementary material The online version of this article (doi:10.1007/s00401-015-1399-y) contains supplementary material, which is available to authorized users.

Platelets are equipped with RNA processing machineries, such as pre-mRNA splicing, pre-miRNA processing, and mRNA translation. Since platelets are devoid of a nucleus, most RNA transcripts in platelets are derived from megakaryocytes during thrombocytogenesis. However, platelets can also ingest RNA molecules during circulation and/or interaction with other cell types. Since platelets were first described by Bizzozero in 1881, their well-established role in hemostasis and thrombosis has been intensively studied. However, in the past decades, the list of biological processes in which platelets play an important role keeps expanding. In this review, we discuss how platelet RNA biomarker signatures can be altered in the presence of cancer.

INTRODUCTION: Blood-based liquid biopsies are a potential approach for leveraging clinical cancer diagnostics and patients' treatment. However, so far, diagnostic, prognostic, predictive, and monitoring biomarkers for patients with diffuse glioma are only limitedly available, and require further validation. Tumor-educated blood platelets (TEPs) sequester several biomolecules, including EGFRvIII mRNA, during tumor growth, thereby altering their RNA profile. Therefore, we reasoned that TEPs may serve as an potential biosource for blood-based biomarkers. METHODS: Blood platelets of patients with glioblastoma and healthy individuals were isolated and subjected to total RNA isolation. Platelet mRNA was amplified using full-length SMARTer mRNA amplification, and processed for sequencing on the Illumina platform. Raw sequencing data was aligned and summarized to the human reference genome using STAR and HTseq, and subjected to differential expression analyses, hierarchical clustering and support vector machine (SVM) classification. RESULTS: We characterized the platelet RNA of 39 glioblastoma patients, and 52 healthy individuals by high-throughput RNA-sequencing. Glioblastoma patient-derived TEPs contained 457 RNAs (FDR < 0.001) that were differentially expressed as compared to healthy individuals. Gene ontology showed that these RNAs are implicated in several biological processes such as RNA translation, immune response, and DNA repair. Using SVM classification we readily separated both groups from each other (accuracy: 94%, AUC: 0.982). CONCLUSIONS AND FUTURE EXPERIMENTS: Our results suggest that TEPs harbour potential as a diagnostic biosource. To further evaluate the biomarker potential, follow-up studies using additional samples, including samples of lower grade glioma and brain metastases patients are under way. Furthermore, we have initiated the collection of clinical follow-up platelet samples to identify the potential of TEPs as disease monitoring biomarkers in glioma patients. Eventually, we believe TEPs have the potential to guide clinical decision making in every day practice.

Summary Tumor-educated blood platelets (TEPs) are implicated as central players in the systemic and local responses to tumor growth, thereby altering their RNA profile. We determined the diagnostic potential of TEPs by mRNA sequencing of 283 platelet samples. We distinguished 228 patients with localized and metastasized tumors from 55 healthy individuals with 96% accuracy. Across six different tumor types, the location of the primary tumor was correctly identified with 71% accuracy. Also, MET or HER2-positive, and mutant KRAS, EGFR, or PIK3CA tumors were accurately distinguished using surrogate TEP mRNA profiles. Our results indicate that blood platelets provide a valuable platform for pan-cancer, multiclass cancer, and companion diagnostics, possibly enabling clinical advances in blood-based “liquid biopsies”., Graphical Abstract, Highlights • Tumors “educate” platelets (TEPs) by altering the platelet RNA profile • TEPs provide a RNA biosource for pan-cancer, multiclass, and companion diagnostics • TEP-based liquid biopsies may guide clinical diagnostics and therapy selection • A total of 100–500 pg of total platelet RNA is sufficient for TEP-based diagnostics, Best et al. show that mRNA sequencing of tumor-educated blood platelets distinguishes cancer patients from healthy individuals with 96% accuracy, differentiates between six primary tumor types of patients with 71% accuracy, and identifies several genetic alterations found in tumors.

Summary Blood-based liquid biopsies, including tumor-educated blood platelets (TEPs), have emerged as promising biomarker sources for non-invasive detection of cancer. Here we demonstrate that particle-swarm optimization (PSO)-enhanced algorithms enable efficient selection of RNA biomarker panels from platelet RNA-sequencing libraries (n = 779). This resulted in accurate TEP-based detection of early- and late-stage non-small-cell lung cancer (n = 518 late-stage validation cohort, accuracy, 88%; AUC, 0.94; 95% CI, 0.92–0.96; p < 0.001; n = 106 early-stage validation cohort, accuracy, 81%; AUC, 0.89; 95% CI, 0.83–0.95; p, Graphical Abstract, Highlights • Tumor-educated platelet (TEP) RNA profiles allow for blood-based cancer diagnostics • Inflammatory conditions only minimally confound TEP-based cancer detection • Swarm intelligence algorithms enable efficient selection of biomarker gene panels • TEP gene panels enable support vector machine-based classification of lung cancer, Best et al. use particle-swarm optimization algorithms and RNA-seq of tumor-educated platelets from patients to generate RNA sets capable of identifying patients with non-small-cell lung cancer, including those having early stage, from individuals without cancer, including those having inflammatory conditions.

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

Allowance of tumor-educated platelets for multiclass liquid biopsy-based diagnosis of cancer

Tumor-educated Platelets (TEPs) have emerged as rich biosources of cancer-related RNA profiles in liquid biopsies applicable for cancer detection. Although human blood platelets have been found to be enriched in circular RNA (circRNA), no studies have investigated the potential of circRNA as platelet-derived biomarkers for cancer. In this proof-of-concept study, we examine whether the circRNA signature of blood platelets can be used as a liquid biopsy biomarker for the detection of non-small cell lung cancer (NSCLC). We analyzed the total RNA, extracted from the platelet samples collected from NSCLC patients and asymptomatic individuals, using RNA sequencing (RNA-Seq). Identification and quantification of known and novel circRNAs were performed using the accurate CircRNA finder suite (ACFS), followed by the differential transcript expression analysis using a modified version of our thromboSeq software. Out of 4732 detected circRNAs, we identified 411 circRNAs that are significantly (p-value < 0.05) differentially expressed between asymptomatic individuals and NSCLC patients. Using the false discovery rate (FDR) of 0.05 as cutoff, we selected the nuclear receptor-interacting protein 1 (NRIP1) circRNA (circNRIP1) as a potential biomarker candidate for further validation by reverse transcription–quantitative PCR (RT-qPCR). This analysis was performed on an independent cohort of platelet samples. The RT-qPCR results confirmed the RNA-Seq data analysis, with significant downregulation of circNRIP1 in platelets derived from NSCLC patients. Our findings suggest that circRNAs found in blood platelets may hold diagnostic biomarkers potential for the detection of NSCLC using liquid biopsies., Marie SkłodowskaCurie Grant Agreement No. 765492., Cancer Center Amsterdam (CCA) Foundation (Grant #CCA2017-2-16).