Platelets as messengers of early-stage cancer.

Platelets have an important role in tumor angiogenesis, growth, and metastasis. The reciprocal interaction between cancer and platelets results in changes of several platelet characteristics. It is becoming clear that analysis of these platelet features could offer a new strategy in the search for biomarkers of cancer. Here, we review the human studies in which platelet characteristics (e.g., count, volume, protein, and mRNA content) are investigated in early-stage cancer. The main focus of this paper is to evaluate which platelet features are suitable for the development of a blood test that could detect cancer in its early stages.

Quantitative and qualitative changes in platelet traits of sunitinib-treated patients with renal cell carcinoma in relation to circulating sunitinib levels: a proof-of-concept study.

BACKGROUND: Tyrosine kinase inhibitors (TKIs), such as sunitinib, are used for cancer treatment, but may also affect platelet count and function with possible hemostatic consequences. Here, we investigated whether patient treatment with the TKI sunitinib affected quantitative and qualitative platelet traits as a function of the sunitinib level and the occurrence of bleeding. METHODS: Blood was collected from 20 metastatic renal cell carcinoma (mRCC) patients before treatment, and at 2 weeks, 4 weeks and 3 months after sunitinib administration. We measured blood cell counts, platelet aggregation, and concentrations of sunitinib as well as its N-desethyl metabolite in plasma, serum and isolated platelets. Progression of disease (PD) and bleeding were monitored after 3 months. RESULTS: In sunitinib-treated mRCC patients, concentrations of (N-desethyl-)sunitinib in plasma and serum were highly correlated. In the patients' platelets the active metabolite levels were relatively increased as compared to sunitinib. On average, a sustained reduction in platelet count was observed on-treatment, which was significantly related to the inhibitor levels in plasma/serum. Principal component and correlational analysis showed that the (N-desethyl-)sunitinib levels in plasma/serum were linked to a reduction in both platelet count and collagen-induced platelet aggregation. The reduced aggregation associated in part with reported bleeding, but did not correlate to PD. CONCLUSION: The sunitinib-induced reduction in quantitative and qualitative platelet traits may reflect the effective sunitinib levels in the patient. These novel results may serve as a proof-of-principle for other TKI-related drugs, where both platelet count and functions are affected, which could be used for therapeutic drug monitoring.

Effects of Cancer Presence and Therapy on the Platelet Proteome.

Platelets are involved in tumor angiogenesis and cancer progression. Previous studies indicated that cancer could affect platelet content. In the current study, we investigated whether cancer-associated proteins can be discerned in the platelets of cancer patients, and whether antitumor treatment may affect the platelet proteome. Platelets were isolated from nine patients with different cancer types and ten healthy volunteers. From three patients, platelets were isolated before and after the start of antitumor treatment. Mass spectrometry-based proteomics of gel-fractionated platelet proteins were used to compare patients versus controls and before and after treatment initiation. A total of 4059 proteins were detected, of which 50 were significantly more abundant in patients, and 36 more in healthy volunteers. Eight of these proteins overlapped with our previous cancer platelet proteomics study. From these data, we selected potential biomarkers of cancer including six upregulated proteins (RNF213, CTSG, PGLYRP1, RPL8, S100A8, S100A9) and two downregulated proteins (GPX1, TNS1). Antitumor treatment resulted in increased levels of 432 proteins and decreased levels of 189 proteins. In conclusion, the platelet proteome may be affected in cancer patients and platelets are a potential source of cancer biomarkers. In addition, we found in a small group of patients that anticancer treatment significantly changes the platelet proteome.

Platelets: the holy grail in cancer blood biomarker research?

We would like to promote the fact that platelets are increasingly emerging as a rich source of potential biomarkers for cancer. Blood platelets contain vast amounts of bioactive proteins, such as growth factors, chemokines, and cytokines. These proteins are either synthesized by the megakaryocytes that produce the platelets or are sequestered by the circulating platelets from the blood, in which case these proteins may originate from the tumor. Recent studies in patients have demonstrated that the presence of cancer influences multiple platelet characteristics (e.g., platelet count, volume, activation status, proteins, and RNA content). Interestingly, these changes happened already in early stages of the disease before metastasis had occurred. Additionally, exploiting these platelet alterations enabled discrimination of patients with early-stage cancer from healthy sex- and age-matched individuals. Therefore, we challenge clinicians and researchers to look beyond traditional fluid sources such as plasma or serum, and to take platelets and their content into account as they may become the holy grail in cancer blood biomarker research.

Role of the tumor stroma in resistance to anti-angiogenic therapy.

Several angiogenesis inhibitors are currently used in the clinic for treatment of cancer. While anti-angiogenesis treatment can improve treatment outcome, the overall benefit on patient survival is still rather limited. This is partially explained by intrinsic or acquired resistance of tumor cells to angiostatic drugs. In addition, it has become evident that extrinsic mechanisms are also involved in resistance to angiostatic therapy. Most of these extrinsic mechanisms reside in the tumor stroma, which is composed of different cell types, including endothelial (progenitor) cells, smooth muscle cells, pericytes, (myo)fibroblasts, immune cells and platelets. In the current review, we describe the role of these stromal cells in the resistance to anti-angiogenic drugs and discuss possible strategies to overcome resistance and enhance the efficacy of angiostatic therapy.

Tumor-educated platelet blood tests for Non-Small Cell Lung Cancer detection and management.

Liquid biopsy approaches offer a promising technology for early and minimally invasive cancer detection. Tumor-educated platelets (TEPs) have emerged as a promising liquid biopsy biosource for the detection of various cancer types. In this study, we processed and analyzed the TEPs collected from 466 Non-small Cell Lung Carcinoma (NSCLC) patients and 410 asymptomatic individuals (controls) using the previously established thromboSeq protocol. We developed a novel particle-swarm optimization machine learning algorithm which enabled the selection of an 881 RNA biomarker panel (AUC 0.88). Herein we propose and validate in an independent cohort of samples (n = 558) two approaches for blood samples testing: one with high sensitivity (95% NSCLC detected) and another with high specificity (94% controls detected). Our data explain how TEP-derived spliced RNAs may serve as a biomarker for minimally-invasive clinical blood tests, complement existing imaging tests, and assist the detection and management of lung cancer patients.

The role of blood platelets in tumor angiogenesis.

Coagulation abnormalities occur frequently in cancer patients. It is becoming evident that blood platelets have an important function in this process. However, understanding of the underlying mechanisms is still very modest. In this review, we discuss the role of platelets in tumor angiogenesis and growth and suggest their potential significance in malignancies. Platelets contain various pro-and antiangiogenic molecules, which seem to be endocytosed and sequestered in different populations of α-granules. Furthermore, tumor endothelial cells are phenotypically and functionally different from endothelial cells in healthy tissue, stimulating local platelet adhesion and subsequent activation. As a consequence, platelets are able to secrete their angiogenic and angiostatic content, most likely in a regulated manner. The overall effect of these platelet-endothelium interactions appears to be proangiogenic, stimulating tumor angiogenesis. We favor the view that local adhesion and activation of blood platelets and dysregulation of coagulation represent underestimated pathways in the progression of cancer.

The Analysis of Platelet-Derived circRNA Repertoire as Potential Diagnostic Biomarker for Non-Small Cell Lung 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.

Tyrosine Kinase Inhibitor Pazopanib Inhibits Platelet Procoagulant Activity in Renal Cell Carcinoma Patients.

Pazopanib is an angiostatic tyrosine kinase inhibitor (TKI) presently used for cancer treatment, particularly in patients with renal cell carcinoma (RCC). This treatment can be accompanied by mild bleeding as an adverse effect. Given the role of protein tyrosine kinases in platelet activation processes, we investigated whether and how pazopanib can affect platelet functions in purified systems and during treatment of advanced RCC patients. In isolated platelets from healthy volunteers, pazopanib dose-dependently reduced collagen-induced integrin activation and secretion, as well as platelet aggregation. Pazopanib addition diminished glycoprotein (GP) VI-dependent tyrosine phosphorylation of multiple platelet proteins, including the tyrosine kinase Syk. Furthermore, pazopanib inhibited GPVI-induced Ca2+ elevation, resulting in reduced exposure of the procoagulant phospholipid phosphatidylserine (PS). Upon perfusion of control blood over a collagen surface, pazopanib inhibited thrombus size as well as PS exposure. Blood samples from 10 RCC patients were also analyzed before and after 14 days of pazopanib treatment as monotherapy. This treatment caused an overall lowering in platelet count, with 3 out of 10 patients experiencing mild bleeding. Platelets isolated from pazopanib-treated patients showed a significant lowering of PS exposure upon activation. In addition, platelet procoagulant activity was inhibited in thrombi formed under flow conditions. Control experiments indicated that higher pazopanib concentrations were required to inhibit GPVI-mediated PS exposure in the presence of plasma. Together, these results indicated that pazopanib suppresses GPVI-induced platelet activation responses in a way partly antagonized by the presence of plasma. In treated cancer patients, pazopanib effects were confined to a reduction in GPVI-dependent PS exposure. Together with the reduced platelet count, this may explain the mild bleeding tendency observed in pazopanib-treated patients.

Exploration of the platelet proteome in patients with early-stage cancer.

Platelets play an important role in tumor growth and, at the same time, platelet characteristics are affected by cancer presence. Therefore, we investigated whether the platelet proteome harbors differentially expressed proteins associated with early-stage cancer. For this proof-of-concept study, patients with early-stage lung (n = 8) or head of pancreas cancer (n = 4) were included, as were healthy sex- and age-matched controls for both subgroups. Blood samples were collected from controls and from patients before surgery. Furthermore, from six of the patients, a second sample was collected two months after surgery. NanoLC-MS/MS-based proteomics of gel-fractionated platelet proteins was used for comparative spectral count analyses of patients to controls and before to after surgery samples. The total platelet proteome dataset included 4384 unique proteins of which 85 were significantly (criteria Fc > 1.5 and p < 0.05) changed in early-stage cancer compared to controls. In addition, the levels of 81 platelet proteins normalized after tumor resection. When filtering for the most discriminatory proteins, we identified seven promising platelet proteins associated with early-stage cancer. In conclusion, this pioneering study on the platelet proteome in cancer patients clearly identifies platelets as a new source of candidate protein biomarkers of early-stage cancer. BIOLOGICAL SIGNIFICANCE: Currently, most blood-based diagnostics/biomarker research is performed in serum or plasma, while the content of blood cells is usually neglected. It is known that especially blood platelets, which are the main circulating pool of many bioactive proteins, such as growth factors, chemokines, and cytokines, are a potentially rich source of biomarkers. The current study is the first to measure the effect of early-stage cancer on the platelet proteome of patients. Our study demonstrates that the platelet proteome of patients with early-stage lung or head of pancreas cancer differs considerably compared to that of healthy individuals of matched sex and age. In addition, the platelet proteome of cancer patients normalized after surgical resection of the tumor. Exploiting platelet proteome differences linked to both tumor presence and disease status, we were able to demonstrate that the platelet proteome can be mined for potential biomarkers of cancer.

A combination of platelet features allows detection of early-stage cancer.

BACKGROUND: Detection of early-stage cancer significantly improves patient survival. As platelets play an important role in cancer progression, we aimed to investigate whether platelets can be used for the discovery of early-stage cancer. METHODS: Patients with lung (n = 86) or head of pancreas (n = 42) cancer were included, as were healthy sex- and age-matched controls (n = 92). Blood was collected before initiation of treatment. Platelet count, volume and activation status were quantified in whole blood. Next, concentrations of vascular endothelial growth factor, platelet-derived growth factor, platelet factor 4, thrombospondin-1 and connective tissue-activating peptide III were measured in both platelets and plasma. Using the results, two multivariable diagnostic models were developed and internally validated. FINDINGS: Multiple platelet features, including platelet count, volume and protein content, were significantly changed in lung and head of pancreas cancer patients. However, the pattern of changes differed between both groups. The diagnostic model developed for lung cancer discriminated very well between patients and controls (AUC = 88.7%). Addition of smoking as a variable significantly increased the AUC of the model to 94.5%. The diagnostic model for head of pancreas cancer also performed well (AUC = 82.7%). Both models were internally validated, resulting in optimism-corrected AUC's of 86.8% and 80.8%, respectively. INTERPRETATION: In patients with lung or head of pancreas cancer, several platelet characteristics are changed compared to healthy sex- and age-matched controls. A cancer type-specific combination of these platelet features can be used to discriminate between patients with early-stage cancer and healthy individuals.

Sunitinib uptake inhibits platelet function in cancer patients.

BACKGROUND: Sunitinib is an oral tyrosine kinase inhibitor used for cancer treatment. Patients treated with sunitinib are at higher bleeding risk. As tyrosine kinases are essential for platelet signalling, the effects of sunitinib on platelet function in vitro and in cancer patients on treatment were investigated. PATIENTS AND METHODS: Blood samples were collected from eight healthy volunteers and eight patients diagnosed with metastatic renal cell cancer (RCC) before and 2 weeks on treatment with sunitinib. Platelets from 15 additional healthy individuals were preincubated with sunitinib or vehicle to perform in vitro experiments. Immunofluorescence imaging, western blotting, light transmission aggregometry, whole blood perfusion over collagen, flow cytometry and ELISA were performed. RESULTS: Confocal microscopy indicated that platelets sequester sunitinib in vitro and in patients. In platelets from healthy controls, tyrosine phosphorylation was inhibited by sunitinib. Also, sunitinib dose dependently reduced collagen- and ADP-induced aggregation, collagen-dependent thrombus formation and collagen-induced secretion of platelet-derived growth factor and ß-thromboglobulin. In blood from RCC patients before treatment, thrombus formation and procoagulant activity under flow were 47% and 80% higher than in healthy controls. After 14 d of sunitinib treatment, platelet count was moderately, but significantly decreased (from 243 to 144 × 10(9)/l). At the same time, collagen-induced platelet aggregation as well as thrombus formation and phosphatidylserine exposure under flow were significantly reduced (by 45%, 16% and 61%, respectively). CONCLUSIONS: Sunitinib uptake by platelets inhibits collagen receptor-induced aggregation and thrombus formation via reduction of protein tyrosine phosphorylation and α-granule secretion. This dysfunction may contribute to the higher bleeding tendency observed in sunitinib-treated patients.