Platelet Diagnostics:A novel liquid biomarker

The aim of this thesis is to find a novel liquid biomarker for the detection of cancer and to optimize treatment. The first chapter gives an introduction to the oncology biomarker field and focuses on platelets and their role in cancer. In part 1, we evaluate extracellular vesicles (EVs). EVs are small vesicles released by all types of cells, including tumor cells, into the circulation. They carry protein kinases and can be isolated from plasma. We demonstrate that AKT and ERK kinase protein levels in EVs reflect the cellular expression levels and treatment with kinase inhibitors alters their concentration, depending on the clinical response to the drug. Therefore, EVs may provide a promising biomarker biosource for monitoring of treatment responses. Part 2 starts with reviews describing the function and role of platelets in greater depth. Chapter 3 focusses on thrombocytogenesis and several biological processes in which platelets play a role. Furthermore, the RNA processing machineries harboured by platelets are discussed. Both chapter 3 and 4 evaluate the change platelets undergo after being exposed to tumor and its environment. The exchange of biomolecules with tumor cells results in educated platelets, so-called tumor educated platelets (TEPs). TEPs play a role in several hallmarks of cancer and have the ability to respond to systemic alterations making them an interesting biomarker. In chapter 5 the diagnostic potential of platelets is first discussed. We determine their potential by sequencing the RNA of 283 platelet samples, of which 228 are patients with cancer, and 55 are healthy controls. We reach an accuracy of 96%. Furthermore, we are able to pinpoint the location of the primary tumor with an accuracy of 71%. In part 3, our developed thromboSeq platform is taken to the next level. Several potential confounding factors are taken into account such as age and comorbidity. We show that particle-swarm optimization (PSO)-enhanced algorithms enable efficient selection of RNA biomarker panels. In a validation cohort we apply these algorithms to non-small-cell lung cancer and reach an accuracy of 88% in late stage (n=518) and early-stage 81% accuracy. Finally, in chapter 7 we describe our wet- and dry-lab protocols in detail. This includes platelet RNA isolation, mRNA amplification, and preparation for next-generation sequencing. The dry-lab protocol describes the automated FASTQ file pre-processing to quantified gene counts, quality controls, data normalization and correction, and swarm intelligence-enhanced support vector machine (SVM) algorithm development. Part 4 focuses on central nervous system (CNS) malignancies especially on glioblastoma. Chapter 8 gives an overview of the different liquid biomarkers for diffuse glioma, the most common primary CNS malignancy. In chapter 9 we assess the specificity of the platelet education due to glioblastoma by comparing the RNA profile of TEPs from glioblastoma patients with a neuroinflammatory disease and brain metastasis patients. This results in a detection accuracy of 80%. Secondly, analysis of patients with glioblastoma versus healthy controls in an independent validation series provide a detection accuracy of 95%. Furthermore, we describe the potential value of platelets as a monitoring biomarker for patients with glioma, distinguishing pseudoprogression from real tumor progression. In part 5 thromboSeq is applied to breast cancer diagnostics both as a screening tool in the general population and in a high risk population, BRCA mutated women. In chapter 11 we first apply our technique to an inflammatory condition, multiple sclerosis (MS). Platelet RNA is used as input for the development of a diagnostic MS classifier capable of detecting MS with 80% accuracy in the independent validation series. In the final part we conclude this thesis with a general discussion of the main findings and suggestions for future research

Liquid biopsies in patients with diffuse glioma

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

Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): a double-blind, randomised, placebo-controlled trial

Background: Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes. Methods: We did this double-blind, randomised, placebo-controlled trial at 689 sites in 41 countries. We enrolled adults aged 18–85 years with type 2 diabetes, estimated glomerular filtration rate (eGFR)25–75 mL/min per 1·73 m 2 of body surface area, and a urine albumin-to-creatinine ratio (UACR)of 300–5000 mg/g who had received maximum labelled or tolerated renin–angiotensin system inhibition for at least 4 weeks. Participants were given atrasentan 0·75 mg orally daily during an enrichment period before random group assignment. Those with a UACR decrease of at least 30% with no substantial fluid retention during the enrichment period (responders)were included in the double-blind treatment period. Responders were randomly assigned to receive either atrasentan 0·75 mg orally daily or placebo. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of doubling of serum creatinine (sustained for ≥30 days)or end-stage kidney disease (eGFR <15 mL/min per 1·73 m 2 sustained for ≥90 days, chronic dialysis for ≥90 days, kidney transplantation, or death from kidney failure)in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials.gov, number NCT01858532. Findings: Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325)or placebo group (n=1323). Median follow-up was 2·2 years (IQR 1·4–2·9). 79 (6·0%)of 1325 patients in the atrasentan group and 105 (7·9%)of 1323 in the placebo group had a primary composite renal endpoint event (hazard ratio [HR]0·65 [95% CI 0·49–0·88]; p=0·0047). Fluid retention and anaemia adverse events, which have been previously attributed to endothelin receptor antagonists, were more frequent in the atrasentan group than in the placebo group. Hospital admission for heart failure occurred in 47 (3·5%)of 1325 patients in the atrasentan group and 34 (2·6%)of 1323 patients in the placebo group (HR 1·33 [95% CI 0·85–2·07]; p=0·208). 58 (4·4%)patients in the atrasentan group and 52 (3·9%)in the placebo group died (HR 1·09 [95% CI 0·75–1·59]; p=0·65). Interpretation: Atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 diabetes at high risk of developing end-stage kidney disease. Funding: AbbVie

Detection and localization of early- and late-stage cancers using platelet RNA

Cancer patients benefit from early tumor detection since treatment outcomes are more favorable for less advanced cancers. Platelets are involved in cancer progression and are considered a promising biosource for cancer detection, as they alter their RNA content upon local and systemic cues. We show that tumor-educated platelet (TEP) RNA-based blood tests enable the detection of 18 cancer types. With 99% specificity in asymptomatic controls, thromboSeq correctly detected the presence of cancer in two-thirds of 1,096 blood samples from stage I–IV cancer patients and in half of 352 stage I–III tumors. Symptomatic controls, including inflammatory and cardiovascular diseases, and benign tumors had increased false-positive test results with an average specificity of 78%. Moreover, thromboSeq determined the tumor site of origin in five different tumor types correctly in over 80% of the cancer patients. These results highlight the potential properties of TEP-derived RNA panels to supplement current approaches for blood-based cancer screening

Platelet RNA signatures for the detection of cancer

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

Re: A Word of Caution on New and Revolutionary Diagnostic Tests

In his letter “A word of caution on new and revolutionary diagnostic tests,” Dr. Diamandis argues that discoveries and progress in the field of biomarker discovery for the early detection of cancer are hampered by multiple factors, including pre- and post-analytical and bioinformatics artifacts. He further indicates that careful patient selection is crucial

Re: A Word of Caution on New and Revolutionary Diagnostic Tests

In his letter “A word of caution on new and revolutionary diagnostic tests,” Dr. Diamandis argues that discoveries and progress in the field of biomarker discovery for the early detection of cancer are hampered by multiple factors, including pre- and post-analytical and bioinformatics artifacts. He further indicates that careful patient selection is crucial

Platelet RNA in Cancer Diagnostics

Platelets are involved in several steps of cancer metastasis. During this process, platelets are exposed to the tumor and its environment, thereby exchanging biomolecules with the tumor cells and resulting in tumor-mediated education of the platelets and a change in their RNA profile. Analysis of platelet RNA profiles or direct measurement of tumor-derived biomarkers within platelets can provide information on ongoing cancer-related processes in the individual (e.g., whether the patient has cancer, the tumor type, and possibly identify oncogenic alterations driving the disease for treatment selection). The close interaction with the disease process and the ability to respond to systemic alterations make platelets an interesting biosource for implementation in precision medicine

RNA sequencing and swarm intelligence–enhanced classification algorithm development for blood-based disease diagnostics using spliced blood platelet RNA

Blood-based diagnostics tests, using individual or panels of biomarkers, may revolutionize disease diagnostics and enable minimally invasive therapy monitoring. However, selection of the most relevant biomarkers from liquid biosources remains an immense challenge. We recently presented the thromboSeq pipeline, which enables RNA sequencing and cancer classification via self-learning and swarm intelligence–enhanced bioinformatics algorithms using blood platelet RNA. Here, we provide the wet-lab protocol for the generation of platelet RNA-sequencing libraries and the dry-lab protocol for the development of swarm intelligence–enhanced machine-learning-based classification algorithms. The wet-lab protocol includes platelet RNA isolation, mRNA amplification, and preparation for next-generation sequencing. The dry-lab protocol describes the automated FASTQ file pre-processing to quantified gene counts, quality controls, data normalization and correction, and swarm intelligence–enhanced support vector machine (SVM) algorithm development. This protocol enables platelet RNA profiling from 500 pg of platelet RNA and allows automated and optimized biomarker panel selection. The wet-lab protocol can be performed in 5 d before sequencing, and the algorithm development can be completed in 2 d, depending on computational resources. The protocol requires basic molecular biology skills and a basic understanding of Linux and R. In all, with this protocol, we aim to enable the scientific community to test platelet RNA for diagnostic algorithm development

Analysis of AKT and ERK1/2 protein kinases in extracellular vesicles isolated from blood of patients with cancer

Background: Extracellular vesicles (EVs) are small nanometre-sized vesicles that are circulating in blood. They are released by multiple cells, including tumour cells. We hypothesized that circulating EVs contain protein kinases that may be assessed as biomarkers during treatment with tyrosine kinase inhibitors. Methods: EVs released by U87 glioma cells, H3255 and H1650 non-small-cell lung cancer (NSCLC) cells were profiled by tandem mass spectrometry. Total AKT/protein kinase B and extracellular signal regulated kinase 1/2 (ERK1/2) levels as well as their relative phosphorylation were measured by western blot in isogenic U87 cells with or without mutant epidermal growth factor receptor (EGFRvIII) and their corresponding EVs. To assess biomarker potential, plasma samples from 24 healthy volunteers and 42 patients with cancer were used. Results: In total, 130 different protein kinases were found to be released in EVs including multiple drug targets, such as mammalian target of rapamycin (mTOR), AKT, ERK1/2, AXL and EGFR. Overexpression of EGFRvIII in U87 cells results in increased phosphorylation of EGFR, AKT and ERK1/2 in cells and EVs, whereas a decreased phosphorylation was noted upon treatment with the EGFR inhibitor erlotinib. EV samples derived from patients with cancer contained significantly more protein (p=0.0067) compared to healthy donors. Phosphorylation of AKT and ERK1/2 in plasma EVs from both healthy donors and patients with cancer was relatively low compared to levels in cancer cells. Preliminary analysis of total AKT and ERK1/2 levels in plasma EVs from patients with NSCLC before and after sorafenib/metformin treatment (n=12) shows a significant decrease in AKT levels among patients with a favourable treatment response (p<0.005). Conclusion: Phosphorylation of protein kinases in EVs reflects their phosphorylation in tumour cells. Total AKT protein levels may allow monitoring of kinase inhibitor responses in patients with cancer