Tumor-educated platelet as liquid biopsy in lung cancer patients

Journal Pre-proof Tumor-educated platelet as liquid biopsy in lung cancer patients Lian Liu, Fang Lin, Xiaoting Ma, Zhaoxin Chen, Jing Yu

PII:

S1040-8428(20)30001-9

DOI:

https://doi.org/10.1016/j.critrevonc.2020.102863

Reference:

ONCH 102863

To appear in:

Critical Reviews in Oncology / Hematology

Received Date:

17 October 2019

Revised Date:

28 December 2019

Accepted Date:

1 January 2020

Please cite this article as: Liu L, Lin F, Ma X, Chen Z, Yu J, Tumor-educated platelet as liquid biopsy in lung cancer patients, Critical Reviews in Oncology / Hematology (2020), doi: https://doi.org/10.1016/j.critrevonc.2020.102863

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Tumor-educated platelet as liquid biopsy in lung cancer patients. Lian Liu1, Fang Lin2, Xiaoting Ma3, Zhaoxin Chen3, Jing Yu3*

1. Cancer Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China (current address: Comprehensive Cancer Center, University Hospital, LMU Munich, Munich, Germany) 2. Respiratory department, Beijing Friendship Hospital, Capital Medical University, Beijing, China 3. Cancer Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China

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* To whom correspondence should be addressed. Cancer Center, Beijing Friendship Hospital, Capital Medical University, No. 95 Yong An Road, Xicheng District, Beijing, 100050, China. Phone: +86-1063139326, Fax: +86-10-63139326. Email: [email protected]

Abstract

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Lung cancer is the most frequent cancer for males and third most frequent cancer for females. Targeted therapy drugs based on molecular alterations, such

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as angiogenesis inhibitors, epidermal growth factor receptor (EGFR) inhibitors, and anaplastic lymphoma kinase (ALK) inhibitors are important part of treatment of NSCLC. However, the quality of the available tumor biopsy and/or

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cytology material is sometimes not adequate to perform the necessary molecular testing, which has prompted the search for alternatives. This review examines

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the use of tumor-educated platelet (TEP) as a liquid biopsy in lung cancer patients. The development of sensitive and accurate techniques have made it possible to detect the specific genetic alterations for which targeted therapies are already available. Liquid biopsy offers opportunities to detect resistance mechanisms at an early stage. To conclude, tumor-educated platelet has the potential to be used as liquid biopsy for a variety of clinical and investigational applications. 1

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Keywords: tumor educated platelets; liquid biopsy; lung cancer; methodologies

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1. Introduction Lung cancer is the predominant cause of cancer incidence and cancer-related mortality worldwide, with 2.1 million new lung cancer cases and 1.8 million deaths predicted in 2018, representing 18.4% cancer deaths in the world (F. Bray et al., 2018). It´s the leading cause of death among males and second most leading cause of death among females (F. Bray et al., 2018). It is classified as small cell (13% of cases) or non-small cell (83%) for the purposes of treatment (Miller et al., 2016). Most patients with small cell lung cancer receive

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chemotherapy (Miller et al., 2016). In addition, some are also treated with radiotherapy. For stage I and II non-small cell lung cancers (NSCLC), the

majority of patients (69%) undergo surgery, and about 25% of surgical cases

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also receiving chemo- and/or radiotherapy. Most patients with III and IV

NSCLC receive chemotherapy with or without radiation (53%). Targeted

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therapy also play an important role for NSCLC, such as angiogenesis inhibitors, epidermal growth factor receptor (EGFR) inhibitors, and anaplastic lymphoma

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kinase (ALK) inhibitors. Targeted therapy has been found to have a much better clinical efficiency compared with standard therapy (Maemondo et al., 2010). This personalized treatment bases on molecular alterations of tumor DNA and

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consists of screening for genetic aberrations. Open surgical tissue biopsy is the gold standard for detecting tumor genotype. The overall success rate for EGFR

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mutation analysis was 94.2%, for KRAS mutation 91.6% and for ALK FISH 91.6% (Vanderlaan et al., 2014). However, there are several limitations doing

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mutational analysis using tumor tissues. One reason is the invasive procedure limited to certain locations, which makes inconvenience in the clinic and brings difficulties for early detection of cancer and further observation of tumor resistance mutations. Primary tumor tissue does not always provide enough information to stratify individual patients to the most promising therapy. The tissue obtained from image-guided percutaneous transthoracic core-needle biopsies provide high failure rates for mutation evaluation (31.8%, 27.3%, and 3

35.3% for EGFR, KRAS, and ALK tests, respectively) (Vanderlaan et al., 2014). A single biopsy provides a limited snapshot in time, which is subjected to selection bias and often fails to reflect the heterogeneity of the disease. These intra-patient heterogeneity of individual lesions motivated the research of safe, least invasive and least costly test available. Liquid biopsy, which has emerged as a useful complementary technique allows for the analysis of several bloodbased biomarkers. Obvious advantages of liquid biopsy compared to tissue biopsy is the easy accessibility of the material to be obtained and the fact that

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tumor derived material in blood may cover the cancer heterogeneity where a tissue biopsy is limited to the alterations in the tumor punctured. The analysis of these blood components might provide a comprehensive real-time picture of the tumor-associated changes in an individual cancer patient, which could be used

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for screening and early detection of cancer and monitoring dynamic changes in

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the molecular landscape of the disease and resistance mechanisms. In the last decades, many studies focused on circulating tumor cells (CTCs),

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circulating tumor DNA (ctDNA) and circulating free DNA (cfDNA) (AlixPanabieres & Pantel, 2014). Investigating CTCs and comprehensive studies at the deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and protein level

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provides important information on therapeutic targets and drug resistance mechanisms in cancer patients. CTCs have the potential to independently predict

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survival, in both NSCLC and small cell lung cancer (SCLC), after adjusting for clinically significant factors including disease stage (Syrigos, Fiste, Charpidou,

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& Grapsa, 2018). However, CTCs are extremely rare and adequate number of tumor cells and multiple blood samples are needed to detect CTCs (Mamdani, Ahmed, Armstrong, Mok, & Jalal, 2017). The advantages of ctDNA are potential to predict prognosis and detect treatment resistance, however, they are less stable than non-tumor DNA and have short half-life (Mamdani et al., 2017). Besides tumor cells and their products, normal cells present in the tumor microenvironment are also released into the blood stream, and these cells can 4

harbor important information. The potential diagnostic role of tumor-educated platelets (TEPs) is described (Best et al., 2015) and the interaction between blood platelets and tumor cells is known to affect tumor growth and dissemination (Kuznetsov et al., 2012). Between cancer patients and healthy individuals, different expression of mRNA within platelets exists due to interaction of tumor cells and platelets. It could be exploited to be biomarker for early cancer detection or cancer metastasis (Asghar, Parvaiz, & Manzoor, 2019). This review examines the use of TEPs in the provision of personalized therapies

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for lung cancer patients. 2. Potential mechanism of tumor educated platelets

Although platelets protect people during wound healing, they are also driver of

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some diseases, such as cancer. Interactions between tumor cells and platelets are important for homogenous spread of cancer. The exact effects causing

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alterations in the TEP RNA remain unknown. The potential mechanisms are

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listed below (Figure 1).

Figure 1. Potential mechanism of tumor educated platelets

2.1 Cancer cell rely on platelets for metastasis Platelets are small discoid shaped non-nucleated cells mainly originated from bone marrow megakaryocytes (MKs) cytoplasm. Recent study showed that megakaryocytes circulate through lungs and release platelets. Lung contributes 5

to approximately 50% of total platelet production (Lefrancais et al., 2017). In normal healthy individuals, platelets are the second most abundant cells next to red blood cells (RBCs) (Seyoum, Enawgaw, & Melku, 2018). Approximately 70% of the platelets circulate in the blood and 30% are stored in spleen (Seyoum et al., 2018). Platelets remains alive for approximately 5 to 9 days in the circulation, after which they are degraded by phagocytosis in the spleen and liver (Ghoshal & Bhattacharyya, 2014). More recently, platelets have emerged as central players in the systemic and local responses to tumor growth. Platelets

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assist immune evasion of tumor cells and finally enhance tumor evasion and angiogenesis (Heeke, Mograbi, Alix-Panabieres, & Hofman, 2019). Platelets contain a plethora of growth factors and cytokines, including high

concentrations of transforming growth factor-beta (TGFβ), which is known to

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promote metastasis by enhancing epithelial-mesenchymal transition (EMT) and invasiveness in primary carcinomas (Oft, Heider, & Beug, 1998). Platelet-

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derived TGFβ promote activation of the TGFβ/Smad and NF-κB pathway in a

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direct platelet-tumor cell contact manner, resulting in a prometastatic invasive phenotype in cancer cells and enhanced metastasis in vivo (Labelle, Begum, & Hynes, 2011). A recent study further explained that tumor necrosis factor

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receptor-associated factor (TRAF) family member-associated NF-kB activator (TANK)-binding kinase 1 (TBK1) mediates platelet-induced NF- κB signaling

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and EMT. Potential therapeutic application targeting TBK1 is important for prevention of metastatic disease (Zhang et al., 2019). Platelets stimulate CRC

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invasiveness by enhancing matrix metalloproteinases-9 (MMP-9) activation and expression via the p38 mitogen-activated protein kinase (p38MAPK) pathway (Radziwon-Balicka et al., 2014). Its effect was abolished in part by inhibition of platelet-secreted proteins such as clusterin and thrombospondin 1 (TSP1) (Radziwon-Balicka et al., 2014). Thus, cancer cell may rely on platelet-derived signals for efficient metastases outside of the primary tumor. 2.2 Platelets absorbed cancer-secreted cytokines 6

Clinical studies have also showed that platelet activation is a feature of some inflammatory diseases such as asthma, allergic rhinitis, and eczema (Page & Pitchford, 2014). Certain luminal breast cancer (LBC) secrete cytokines that are absorbed by platelets, which are recruited to tumor sites where they aid vessel formation (Kuznetsov et al., 2012). LBC tumors loaded platelets with proinflammatory and pro-angiogenic factors (Kuznetsov et al., 2012). Systemically acting cytokines result in tumor outgrowth and are rendered pro-tumorigenic by instigating triple negative breast tumors (Kuznetsov et al., 2012). This processes

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possibly resulting in tumor-associated biomolecules ('education') that may lead to transformation of naive platelets into pro-tumorigenic platelets. Platelets are able to take microparticles during their lifespan. Moreover, the content of

platelets-derived microparticles can be delivered to other cells, such as cancer,

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epithelial, stromal and immune cell, and contribute to cancer promotion and its metastasization (Dovizio, Bruno, Contursi, Grande, & Patrignani, 2018).

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Platelets derived products include mesenchymal phenotype in cancer cells, such

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as the transcription factors Twist1, Snail, and Zeb, and the down-regulation of epithelial marker expression such as E-cadherin (Dovizio et al., 2018), which led to the enhanced migratory capacity of cancer cells.

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2.3 Cancer cells release RNA into platelets Platelets have evolved unique adaptive molecular signals for maintenance of

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genetic and protein diversities (Denis et al., 2005). Cancer cells can release

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RNA into the circulation by a variety of macrovesicles types (Arroyo et al., 2011). These vehicles are capable of transferring tumor-derived RNA into platelets, which are capable of taking up protein and nucleotides from their surroundings during their lifespan (Best, Vancura, & Wurdinger, 2017; Nilsson et al., 2011). Platelets isolated from glioma and prostate cancer patients contain the cancer-associated RNA biomarkers EGFRvIII and PCA3, respectively (Nilsson et al., 2011). Platelets can be educated by tumor cells by the transfer of tumor associated biomolecules, mostly RNA (Mader & Pantel, 2017). Platelets 7

contain many different RNA species (P. F. Bray et al., 2013; Rowley et al., 2016), including messenger RNAs (mRNA) (P. F. Bray et al., 2013), microRNAs (Boilard & Belleannee, 2016; Nagalla et al., 2011) and circular RNAs (Alhasan et al., 2016). After comparative analysis between megakaryocyte and platelet RNA molecules, Gecchetti et al. proved that megakaryocytes selectively dispense transcripts for MMP and tissue inhibitor of metalloprotease (TIMPs) into platelets (Cecchetti et al., 2011). The TEP mRNA content appears to be regulated at multiple levels and is a highly dynamic and

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transient process (Best, Vancura, et al., 2017). Shifts in platelets subpopulations during cancer progression and treatment may be of interest for TEP based liquid biopsy.

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2.4 Direct contact between platelets and tumor cells.

Platelets interacting with tumor cells during blood dissemination leads to platelet

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activation (Heeke et al., 2019). However, platelet receptors on tumor cells are only partially characterized. CD97 expressed on tumor cells has been recently

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reported to be involved in platelets activation (Ward et al., 2018). CD97 is an adhesion g-protein coupled receptor overexpressed in several cancer types.

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CD97 is able to activate platelets, leading to several mediators of the endothelial barrier secretion, including the release of ATP, which disrupts the endothelial barrier and consequently promotes metastasis (Ward et al., 2018). However, the

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interaction of CD97 and platelets are proved to be necessary for metastasis only

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in pre-clinical models, further studies are needed to validate this finding in clinical setting.

2.5 Platelets-coated CTC Other prometastatic mechanisms of platelets have been described, such as protection of tumor cells from immune elimination within the circulatory system, protection tumor cell arrest within the vasculature and contribution of tumor cell survival, thus supporting the metastasis (Gay & Felding-Habermann, 8

2011). Studies show the existence of platelets-coated CTC in patients with metastatic cancer. Platelets can protect CTC from various attacks, such as immune response, apoptosis and shear stress and regulate CTCs intravasation and extravasation. Placke et al showed that coating might cause transfer of MHC class 1 onto the tumor cell surface, which resulted in tumor cells expression of high level platelet-derived MHC class 1 (Placke et al., 2012). The majority of CTCs are killed by NK/T cells. The false presents of MHC class 1 disrupts recognition of tumor cell, thereby escaping T-cell mediated immunity and

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impairing cytotoxicity (Jiang et al., 2017; Placke et al., 2012). Xiao-Liang Lou showed that platelets secret several cytokines, such as lysophosphatidic acid

(LPA), a lipid with growth factor-like signaling properties (Lou et al., 2015).

These cytokines facilitate the detachment of tumor cells from the primary tumor

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site and extravasation into the blood. Activated platelets secret α-granules which release high levels of TGFβ, a powerful activator of EMT state (Heeke et al.,

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2019).

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3. Tumor educated platelets as liquid biopsy in lung cancer 3.1 Distinguishing cancer from normal tissue

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The capacity of platelets to directly ingest circulating mRNA can provide TEPs with a highly dynamic mRNA repertoire, with potential applicability to cancer

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diagnostics. Best and colleagues showed by performing mRNA sequencing on TEPs that cancer patients of diverse entities could be differentiated from healthy

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individuals with 96% accuracy (Best et al., 2015). The location of six primary tumor including NSCLC, colorectal cancer, glioblastoma, pancreatic cancer, hepatobiliary cancer and breast cancer was correctly identified with an accuracy of 71%. Furthermore, biomarkers including MET or HER2, KRAS, EGFR, or PIK3CA were identified in surrogate TEP mRNA profiles, which might guide clinical diagnostics for non-invasive detection of cancer (Best et al., 2015). A hyperactive state of TEPs was revealed in a functional analysis from certain 9

cancer (Sol & Wurdinger, 2017), which seems to be discriminated in cancer patients compared to patients with non-cancerous inflammatory diseases. In further study, they investigated the potential and origin of spliced RNA profiles from TEPs for detection of early and last stage NSCLC and found that TEP RNA biomarker discriminates patients with NSCLC from healthy individuals and non-cancerous inflammatory conditions (Best, Sol, et al., 2017; Lood et al., 2010). These studies compared TEPs from patients with non-cancerous inflammatory disease and healthy individuals and gave us more insight into the

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function of platelets in different diseases including cancer. Different primary tumors showed different RNA profiles, making it possible to determine malignant lesions as a primary tumor or as a metastasis (Sol &

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Wurdinger, 2017). Several clinical studies confirmed the function of TEPs on detection of mutations as liquid biopsy (F. Lessi, 03 May 2019; D. Li et al.,

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2018; Liefaard et al., 2019; Sheng, Dong, & Xie, 2018; Xing et al., 2019; Xue, Xie, Song, & Song, 2018a). RNA-sequencing data of TEPs in 402 NSCLC

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patients and 231 healthy individuals were analyzed and a total of 48 biomarker genes were selected with advanced minimal-redundancy, maximal-relevance, and incremental feature-selection (IFS) methods (Sheng et al., 2018). Specificity

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and accuracy of the 48-gene classifier were 0.827 and 0.889, respectively (Sheng et al., 2018). The 48-gene TEP liquid-biopsy biomarkers may facilitate

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early screening of NSCLC. The expression of apoptotic chromatin condensation inducer 1 (ACIN1) mRNA in TEPs of lung cancer was higher than that in

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healthy individuals, with a sensitivity, specificity and accuracy to detect lung cancer of 0.827, 0.448, and 0.724, respectively (Xue et al., 2018a). Platelet RNAs were isolated from blood of 9 early stage NSCLC patients and 8 healthy controls and analyzed using RNA-seq. ITGA2B was selected as candidate marker with higher levels in NSCLC patients than in control groups. The diagnostic accuracy of ITGA2B was area under the curve (AUC) of 0.922, sensitivity of 92.8% and specificity of 78.6% in the test group of 152 NSCLC 10

patients, 109 benign pulmonary nodules (BPN) and 97 healthy controls by quantitative real time PCR (q-PCR) (Xing et al., 2019). These results were validated in another cohort comprising 91 NSCLC patients, 32 BPN patients and 53 HC through more precise method droplet digital PCR (ddPCR) and similar results were observed (Xing et al., 2019). RNA-seq data from normal, adenocarcinoma in situ (AIS) and invasive lung cancer tissues were used to identify a gene module that represents the distinguishing characteristics of AIS as AIS-specific genes (D. Li et al., 2018). 107 AIS-specific genes were

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identified and 20 of the AIS-specific genes were selected as early stage lung cancer signatures using the dataset obtained from The Cancer Genome Atlas

(TCGA) lung adenocarcinoma samples, which consistently yielded about 98%

accuracy for distinguishing early stage lung cancer from normal cases (D. Li et

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al., 2018). However, the prediction accuracy for the blood platelet samples was only 64.35%, with sensitivity, specificity and AUC of 78.1%, 50.59% and

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0.747, respectively (D. Li et al., 2018). TEP mRNAs profiles from breast cancer

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patients can be discriminated from those with other tumor types (Liefaard et al., 2019). RNA signatures from TEPs enable detection of early breast cancer with AUC of 0.78, and warrant validation in a confirmatory and screening setting

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(Liefaard et al., 2019). This TEPs may allow for blood-based highly sensitive early-stage cancer screening but need more high quality, large scale clinical

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trials for validation. Several ongoing studies regarding early detection of cancer have currently implemented TEPs RNA analysis in their protocols, e.g., the

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PLATO-VTE study (ClinicalTrials.gov identifier: NCT02739867). Among patients with a first episode of unprovoked venous thromboembolism (VTE), the contemporary one-year risk of detecting occult cancer is approximately 4% to 7%. Of these cases, 30% to 60% are missed by routine limited screening for cancer. The objective of their study is to evaluate the diagnostic accuracy of TEPs RNA profiling in detecting occult cancer in patients with VTE. Except for RNA signature in TEPs for liquid biopsy, proteomics analysis in TEPs also 11

shown to be an effective biomarker for detecting cancer. Partial Least Squares Discriminant Analysis (PLS-DA) of platelets protein expression suggested differences of stages III-IV of ovarian cancer with a sensitivity of 96% and a specificity of 88% compared to benign adnexal lesions (Lomnytska et al., 2018). Platelet proteins of patients with early stage lung and head of pancreas cancer are different from healthy individuals of matched sex and age (Sabrkhany et al., 2018). Thus, blood platelets offer a promising new source of potential biomarkers distinguishing cancer cells from normal tissue and further studies are

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needed in future blood-based biomarker research. 3.2 Detecting mutations during treatment

Tumor platelets serve not only as potential biomarker for NSCLC diagnosis, but

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also as candidates for general molecular traces for cancer surveillance (Xue, Xie, Song, & Song, 2018b). Detecting for EGFR mutations and ALK

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rearrangements should be considered for all adenocarcinomas regardless of histological grade or pattern and mixed carcinomas with an adenocarcinoma

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component (Dacic, Shuai, Yousem, Ohori, & Nikiforova, 2010; Ohtsuka et al., 2007).

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ALK rearrangements occur in about 4% of patients with lung adenocarcinoma (Rodig et al., 2009; Shaw et al., 2009) and are associated with high sensitivity to

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ALK TKIs crizotinib (Solomon et al., 2014). The majority of the ALK rearrangements in lung adenocarcinoma result in the EML4–ALK fusion gene

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product (Dacic, 2013). Nilsson and colleges were the first to detect EML4-ALK rearrangements in the platelets of NSCLC patients (Nilsson et al., 2016). EML4ALK rearrangements were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) in platelets isolated from blood obtained from 77 patients with NSCLC, 38 of whom had EML4-ALK-rearranged tumors. In total, RTPCR demonstrated 65% sensitivity and 100% specificity for the detection of EML4-ALK rearrangements in platelets (Nilsson et al., 2016), which proved the 12

function of platelets for the non-invasive detection of EML4-ALK rearrangements. Although most patients with ALK-positive NSCLC derive substantial clinical benefit from crizotinib, the benefit is relatively short-lived because of the development of acquired resistance (Katayama et al., 2012). Typically, patients with ALK rearrangement NSCLC relapse in a year after treatment due to acquired resistance (Katayama et al., 2012). This suggests the importance for identification of resistance mutations for crizotinib in NSCLC using TEPs.

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At present, the most widely known tyrosine kinase inhibitor (TKI) targets in

NSCLC are EGFR mutations, 90% of which consist of deletions in exon 19 and the L858R point mutation in exon 21 (Dacic, 2013). First line treatment of

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advanced NSCLC patients harboring EGFR mutations with EGFR TKIs

gefitinib, erlotinib or afatinib has resulted in superior progression free survival

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(PFS), overall response rate (ORR) and quality of life compared with chemotherapy (Keedy et al., 2011; Rosell et al., 2012; Wu et al., 2014).

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However, the majority of patients with lung cancer treated with EGFR TKI will develop acquired resistance within a year manifesting as recurrence or progression of disease (Ohashi, Maruvka, Michor, & Pao, 2013; Sequist et al.,

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2011). The acquisition of the T790M mutation is the most frequent resistance mechanism, responsible for nearly 60% of cases (Arcila et al., 2011; Taniguchi

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et al., 2011). Liquid biopsy offers opportunities to detect resistance mechanisms, such as the EGFR T790M mutation in the case of EGFR TKI use at an early

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stage and allows clinicians to adopt more effective, alternative treatment strategies, such as AZD9291 as irreversible EGFR TKIs that target the T790M resistance mutation (Finlay et al., 2014; Watanabe et al., 2015). Watanabe et al. were the first to use ultra-sensitive ddPCR to detect the pretreatment T790M mutation in 373 surgically resected cancer tissues from EGFR mutated NSCLC patients (Watanabe et al., 2015). Wild-type or T790M mutation-containing DNA fragments were cloned into plasmids as establishment of ddPCR. Approximately 13

80% of the patients with NSCLC harboring EGFR-activating mutations had the pretreatment T790M mutation. Mutation was more frequently detected in patients with larger tumors (Watanabe et al., 2015). However, accuracy of evaluation of T790M mutation using TEPs method remains unclear, which is of great clinical significance and could be future perspective. 3.3 Predicting response to treatment In 77 patients with EML4-ALK-rearrangment NSCLC treated with crizotinib,

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EML4-ALK rearrangements in platelets were correlated with PFS and OS (Nilsson et al., 2016). PFS was 3.7 months for patients with EML4-ALK+

platelets and 16 months for these with EML4-ALK- platelets (P = 0.02), median OS was not reached for EML4-ALK- patients and 6.7 months for the EML4-

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ALK+ patients, indicating function of platelets for predicting and monitoring

outcome for crizotinib, thereby improving clinical decisions. The presence of a

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preexisting EGFR T790M mutation prior to any treatment was significantly associated with a longer PFS using ddPCR method (Vendrell et al., 2019).

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However, no studies are published about the effect of platelet detected EGFR mutations, as predictors for treatment response. More convincing large scale

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clinical trials are needed to validate this.

3.4 Methodologies for detecting platelet RNA

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Several methodologies have been used for platelet isolation and RNA profiling (P. F. Bray et al., 2013; Rolf, Knoefler, Suttorp, Kluter, & Bugert, 2005; Rowley

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et al., 2011). Procedure include platelet isolation from whole blood, platelet RNA isolation and RNA transcripts. Actually, RNA transcripts could be quantified using various methods, such as platelet-RNA-sequencing (P. F. Bray et al., 2013; Rowley et al., 2011; Schubert, Weyrich, & Rowley, 2014), microarray hybridization techniques (Edelstein et al., 2013; Nilsson et al., 2011; Simon et al., 2014) and reverse transcriptase-polymerase chain reaction (qRTPCR) (Table 1) (P. F. Bray et al., 2013; Clancy, Beaulieu, Tanriverdi, & 14

Freedman, 2017). These techniques enabled potential role of platelet RNA for blood-based liquid biopsies. RNA sequencing as a relatively new application of next generation sequencing (NGS) is a favorite technique, since it allows analysis of several genes at the same time (Morganti et al., 2019). Complexities in platelet-isolation procedure bring difficulties in the implementation in routine clinical chemistry laboratories. Recently, a protocol named ThromboSeq pipeline was published using spliced blood platelet RNA to evaluate RNA sequencing and swarm intelligence–enhanced classification algorithm for blood-

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based disease diagnostics and cancer classification (Best, In 't Veld, Sol, & Wurdinger, 2019). Its advantage includes less restrictive time constraints, which allows the whole blood samples to be stored for up to 48 h before processing

and the ability to use low RNA yield for the platelet RNA profiling. However,

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the protocol cost is still expensive to be implemented in clinical setting and swarm-optimization algorithm is time consuming and requires extensive

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computational resources. The disadvantage of all these protocols is the difficulty

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to produce a pure biomarker during centrifugation. Most notable cofounders are the contamination of tumor-derived extracellular vesicles (tdEV) for TEPs (Rikkert, van der Pol, van Leeuwen, Nieuwland, & Coumans, 2018). Thus, care

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should be taken when the obtained results are interpreted, since results from TEPs may originate from other co-isolated biomarkers like tdEV which may

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enclose cfDNA. Optimized simplified procedure for platelets extraction and

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RNA sequencing with lower expenses is still needed in clinical implementation.

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Table 1. Methodologies for detecting platelet RNA protocols

Best et

thromboSeq pipeline

500 pg platelet RNA

Pros and Cons Pros:

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Presenter/Country/Year Amount of sample

1. wet-lab protocol for the generation 1. less restrictive time constraints

al/Netherland/2019

of

platelet

e-

(Best et al., 2019)

RNA-sequencing 2. the ability to use low RNA yield for the platelet RNA profiling

a) platelet RNA isolation

Cons:

b) mRNA amplification

1. expensive

c) preparation for NGS

2. time consuming

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Pr

libraries

1. dry-lab

protocol

development

for

of

the 3. extensive swarm

computational

resources

intelligence–enhanced machinelearning-based

classification

algorithms a) automated FASTQ file preprocessing b) quantified gene counts

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d) data normalization

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c) quality controls

e) correction swarm intelligence– support

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enhanced machine

(SVM)

vector

algorithm

Bray et al/USA/2013

2 ug of total RNA

1. RNA extraction and quality assessment

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(P. F. Bray et al., 2013)

e-

development

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2. RNA sequencing following

Pros: 1. the discovery of novel transcripts 2. the simultaneous highly sensitive

standard AB/Life Technologies

assessment of expression levels,

protocols

sequence variants and splice

3. Read sequencing using the Burrows-Wheeler Alignment

variants Cons:

(BWA) algorithm (H. Li &

1. high expenses

Durbin, 2009)

2. Sophisticated computational

4. qRT-PCR of Gene Expression

analyses not standardized or

5. Correlation between platelet

widely available

RNA-seq and microarray datasets

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PLT number about (1- 1. Platelets RNA isolation

al/USA/2011 (Rowley

3)*109

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Rowley et

Pros:

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2. mRNA Seq Sample Prep Kit 1. relatively low volume blood (Illumina) used to create libraries

sample

for the deep sequencing studies

Cons:

pr

et al., 2011)

3. Downstream analysis using a 1. many genes do not have a

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combination of programs found in

predicted ortholog

the University of Utah’s Useq 2. predicted

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analysis package and ad hoc perl programs

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are

occasionally incorrect 3. orthologs are selected based on

4. Sequencing on the Illumina GAIIx

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orthologs

sequencer

comparisons between the primary transcripts, regardless of multiple

5. RPKM assignment (reads per

transcripts or isoforms derived

kilobase of exon model per million

from the same primary transcript

mapped

reads),

ortholog

and 4. RPKM measurements based on

isoform choice, real-time PCR

transcript length may be falsely

performed as comparison

inflated or reduced

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blood sample;

leukocyte

Thrombocytopenic

automated

blood samples: 10ml

analyzer

of citrated whole-

counts

on

an

hematology

sample Cons: 1. A high yield of purified platelets

b) centrifugation of the blood sample

blood sample

Pros:

a) determination of PLT and 1. relatively low volume blood

pr

et al., 2005)

f

al/Germany/2005 (Rolf 3ml of citrated whole-

1. platelet-rich plasma (PRP)

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Low volume samples:

e-

Rolf et

is also mandatory in thrombocytopenic patients

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c) leukocyte removal

2. determination of PLT counts on an

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automated hematology analyzer and of leukocyte counts by manual cell

counting

on

a

light

microscope. 3. RNA amplification

technology

SMART 4. RNA evaluation using Microarray hybridization analysis

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4. Advantages and Limits of the TEPs RNA as an assessment tool for lung cancer patients TEPs RNA as Liquid biopsies could provide a potential revolution in cancer detecting, monitoring and diagnostics as a minimally invasive method complementary to current tissue biopsy approaches. The idea that a simple blood test might allow to identify patients that are not responding to treatment and to discover targets for therapeutic intervention is intriguing. For lung cancer patients, TEPs RNA as liquid biopsy brings advantages since an analysis using

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blood samples can be first performed when a tissue biopsy cannot be performed (when the tumor site is not reachable by tumor biopsy or when the patients’ physical condition are poor to tolerate tumor biopsy). TEPs RNA as tumor

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biopsy hold several advantages compared to tissue biopsy within the context of follow-up of patients receiving targeted therapy for NSCLC patients, for

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example anti-EGFR TKIs. Although patients with EGFR mutation show good initial responses to anti-EGFR TKIs, most of them who respond to targeted

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therapy ultimately resistant after about 9 to 14 months of treatment. The most common resistance mechanism is substitution of threonine at the “gatekeeper” amino acid 790 to methionine (T790M) and it is detected in tumor cells from

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more than 50% of patients after progression (Cross et al., 2014). Osimertinib is an irreversible EGFR TKI for treatment of patients with metastatic, EGFR

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T790M mutation–positive NSCLC who have progressed during or after EGFR TKI therapy (Skoulidis & Papadimitrakopoulou, 2017). Importance would be

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detect the acquired resistance and adjust treatment according to patients´ gene mutations. During tumor progression, this non-invasive approach can investigate or detect resistance mutation onset in the EGFR gene and thereby treatment can be adapted with respect to the observed mutation for NSCLC patients (Cross et al., 2014). Importance will be to get the most out of the individual TEPs using the most sensitive techniques, including high-intensity sequencing. What is more, strong computational analysis is essential. Through self-learning 20

algorithms, biomarker files can be interrogated to calculate which combination of biomarkers yields the highest accuracy. These studies should focus on cancer detection, monitoring treatment response during treatment or detecting prognostics. The ideal liquid biopsy should be accurate, safe, cost-effective, easily reproducible, and not limited to large hospitals. However, we must admit that we have not yet sufficient evidence to use TEPs testing to guide treatment decision in our daily clinical practice. The implementation of TEPs as liquid

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biopsy in the clinic utility has its limitations. First, the detection of TEPs is not a standardized approach of daily practice and reproducibility requires good technical skills. Second, TEP detection techniques are not available in all

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hospitals and thus not easily accessible to all patients. Third, the value of liquid biopsy in immunotherapy remains to be determined. Fourth, the computational

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analysis necessary for the identification of the correct TEPs biomarkers is time consuming and requires extensive computational resources, especially large

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sized datasets. Nonetheless, TEPs can be potentially utilized as a blood-based biomarker of lung cancer in future.

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5. Conclusion

In conclusion, the TEPs as liquid biopsy is an expanding field in lung cancer and

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it is likely to contribute significantly to the management of lung cancer patients in different stages of disease. Nevertheless, the approach to TEPs testing

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requires more good-quality studies with adequate cohorts of patients and standardized methods of analysis. Experimentation in this field should follow the strict roles of clinical trials testing new drugs in order to produce results that can be transferred in the clinical practice. Conflict of interest None. 21

Conflict of interest None. Funding Supported by The Capital Health Research and Development of Special (No.2018-2-1113), National Natural Science Foundation of China (No.81774221), Research Foundation of Beijing Friendship Hospital, Capital Medical University (No.yyqdkt2016-4).

Acknowledgement

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The first author acknowledges the support of China Scholarship Council (No. 201908080031).

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Figure and table Figure 1. Potential mechanism of tumor educated platelets

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Table 1. methodologies for detecting platelet RNA

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