The use of blood-based liquid biopsies to detect, diagnose, and monitor cancer may enable earlier diagnosis of cancer, lower costs by tailoring molecular targeted treatments, improve convenience for cancer patients, and ultimately supplements clinical oncological decision-making. Current blood-based biosources under evaluation demonstrate suboptimal sensitivity for cancer diagnostics, in particular in patients with localized disease. So far, none of the current blood-based biosources, including plasma DNA, exosomes, and CTCs, have been employed for multiclass cancer diagnostics (Alix-Panabières and Pantel, 2014, Bettegowda et al., 2014, Skog et al., 2008), hampering its implementation for early cancer detection. Here, we report that molecular interrogation of blood platelet mRNA can offer valuable diagnostics information for all cancer patients analyzed—spanning six different tumor types. Our results suggest that platelets may be employable as an all-in-one biosource to broadly scan for molecular traces of cancer in general and provide a strong indication on tumor type and molecular subclass. This includes patients with localized disease possibly allowing for targeted diagnostic confirmation using routine clinical diagnostics for each particular tumor type.
Since the discovery of circulating tumor material in blood of patients with cancer (Leon et al., 1977) and the recognition of the clinical utility of blood-based liquid biopsies, a wealth of studies has assessed the use of blood for cancer diagnostics, prognostication and treatment monitoring (Alix-Panabières et al., 2012, Bidard et al., 2014, Crowley et al., 2013, Haber and Velculescu, 2014). By development of highly sensitive targeted detection methods, such as targeted deep sequencing (Newman et al., 2014), droplet digital PCR (Bettegowda et al., 2014), and allele-specific PCR (Maheswaran et al., 2008, Thierry et al., 2014), the utility and applicability of liquid biopsies for clinical implementation has accelerated. These advances previously allowed for a pan-cancer comparison of various biosources and revealed that in >75% of cancers, including advanced stage pancreas, colorectal, breast, and ovarian cancer, cell-free DNA is detectable although detection rates are dependent on the grade of the tumor and depth of analysis (Bettegowda et al., 2014). Here, we show that the platelet RNA profiles are affected in nearly all cancer patients, regardless of the type of tumor, although the abundance of tumor-associated RNAs seems variable among cancer patients. In addition, surrogate RNA onco-signatures of tissue biomarkers, also in 88% of localized KRAS mutant cancer patients as measured by the tumor-specific and pan-cancer SVM/LOOCV procedures, are readily available from a minute amount (100–500 pg) of platelet RNA. As whole blood can be stored up to 48 hr on room temperature prior to isolation of the platelet pellet, while maintaining high-quality RNA and the dominant cancer RNA signatures, TEPs can be more readily implemented in daily clinical laboratory practice and could potentially be shipped prior to further blood sample processing.
Blood platelets are widely involved in tumor growth and cancer progression (Gay and Felding-Habermann, 2011). Platelets sequester solubilized tumor-associated proteins (Klement et al., 2009) and spliced and unspliced mRNAs (Calverley et al., 2010, Nilsson et al., 2011), whereas platelets do also directly interact with tumor cells (Labelle et al., 2011), neutrophils (Sreeramkumar et al., 2014), circulating NK-cells (Palumbo et al., 2005, Placke et al., 2012), and circulating tumor cells (Ting et al., 2014, Yu et al., 2013). Interestingly, in vivo experiments have revealed breast cancer-mediated systemic instigation by supplying circulating platelets with pro-inflammatory and pro-angiogenic proteins, supporting outgrowth of dormant metastatic foci (Kuznetsov et al., 2012). Using a gene ontology methodology, CAGE, we correlated TEP-cancer signatures with publicly available curated datasets. Indeed, we identified widespread correlations with cancer tissues, hypoxia, platelet-signatures, and cytoskeleton, possibly reflecting the “alert” and pro-tumorigenic state of TEPs. We observed strong negative correlations with RNAs implicated in RNA translation, T cell immunity, and interleukin-signaling, implying diminished needs of TEPs for RNAs involved in these biological processes or orchestrated translation of these RNAs to proteins (Denis et al., 2005). We observed that the tumor-specific educational programs in TEPs are predominantly influenced by tumor type and, to a lesser extent, by tumor progression and metastases. Although we were not able to measure significant differences between non-metastasized and metastasized tumors, we do not exclude that the use of larger sample sets could allow for the generation of SVM algorithms that do have the power to discriminate between certain stages of cancer, including those with in situ carcinomas and even pre-malignant lesions. In addition, different molecular tumor subtypes (e.g., HER2-amplified versus wild-type BrCa) result in different effects on the platelet profiles, possibly caused by different “educational” stimuli generated by the different molecular tumor subtypes (Koboldt et al., 2012). Altogether, the RNA content of platelets in patients with cancer is dependent on the transcriptional state of the bone-marrow megakaryocyte (Calverley et al., 2010, McAllister and Weinberg, 2014), complemented by sequestration of spliced RNA (Nilsson et al., 2011), release of RNA (Clancy and Freedman, 2014, Kirschbaum et al., 2015, Rak and Guha, 2012, Risitano et al., 2012), and possibly queue-specific pre-mRNA splicing during platelet circulation. Partial or complete normalization of the platelet profiles following successful treatment of the tumor would enable TEP-based disease recurrence monitoring, requiring the analysis of follow-up platelet samples. Future studies will be required to address the tumor-specific “educated” profiles on both an (small non-coding) RNA (Laffont et al., 2013, Landry et al., 2009, Leidinger et al., 2014, Lu et al., 2005) and protein (Burkhart et al., 2014,Geiger et al., 2013, Klement et al., 2009) level and determine the ability of gene ontology, blood-based cancer classification.
In conclusion, we provide robust evidence for the clinical relevance of blood platelets for liquid biopsy-based molecular diagnostics in patients with several types of cancer. Further validation is warranted to determine the potential of surrogate TEP profiles for blood-based companion diagnostics, therapy selection, longitudinal monitoring, and disease recurrence monitoring. In addition, we expect the self-learning algorithms to further improve by including significantly more samples. For this approach, isolation of the platelet fraction from whole blood should be performed within 48 hr after blood withdrawal, the platelet fraction can subsequently be frozen for cancer diagnosis. Also, future studies should address causes and anticipated risks of outlier samples identified in this study, such as healthy donors classified as cancer patients. Systemic factors such as chronic or transient inflammatory diseases, or cardiovascular events and other non-cancerous diseases may also influence the platelet mRNA profile and require evaluation in follow-up studies, possibly also including individuals predisposed for cancer.
2012pharmaceutical
sjwilliamspa
