Circulating biomarkers in patients with glioblastoma

Juliana Müller Bark; Arutha Kulasinghe; Benjamin Chua; Bryan W. Day; Chamindie Punyadeera

doi:10.1038/s41416-019-0603-6

Circulating biomarkers in patients with glioblastoma

Müller Bark, Juliana; Kulasinghe, Arutha; Chua, Benjamin; Day, Bryan W.; Punyadeera, Chamindie 2020-03-02 00:00:00 www.nature.com/bjc REVIEW ARTICLE 1,2 1,2 3,4 3,5,6 1,2 Juliana Müller Bark , Arutha Kulasinghe , Benjamin Chua , Bryan W. Day and Chamindie Punyadeera Gliomas are the most common tumours of the central nervous system and the most aggressive form is glioblastoma (GBM). Despite advances in treatment, patient survival remains low. GBM diagnosis typically relies on imaging techniques and postoperative pathological diagnosis; however, both procedures have their inherent limitations. Imaging modalities cannot differentiate tumour progression from treatment-related changes that mimic progression, known as pseudoprogression, which might lead to misinterpretation of therapy response and delay clinical interventions. In addition to imaging limitations, tissue biopsies are invasive and most of the time cannot be performed over the course of treatment to evaluate ‘real-time’ tumour dynamics. In an attempt to address these limitations, liquid biopsies have been proposed in the ﬁeld. Blood sampling is a minimally invasive procedure for a patient to endure and could provide tumoural information to guide therapy. Tumours shed tumoural content, such as circulating tumour cells, cell-free nucleic acids, proteins and extracellular vesicles, into the circulation, and these biomarkers are reported to cross the blood–brain barrier. The use of liquid biopsies is emerging in the ﬁeld of GBM. In this review, we aim to summarise the current literature on circulating biomarkers, namely circulating tumour cells, circulating tumour DNA and extracellular vesicles as potential non-invasively sampled biomarkers to manage the treatment of patients with GBM. British Journal of Cancer (2020) 122:295–305; https://doi.org/10.1038/s41416-019-0603-6 15–17 BACKGROUND are being trialled to improve treatment in GBM patients. Early Gliomas are the most common type of tumours originating from data have shown beneﬁt from the use of a PD-1 inhibitor, in other 18,19 20 the central nervous system (CNS) and can be classiﬁed according tumour types, as melanoma and non-small-cell lung cancer to the cells that give rise to them: oligodendrocytes give rise to in patients with a high tumour mutational burden. However, GBM oligodendrogliomas, ependymal cells generate ependymomas is thought to have a low mutational burden and to be 1 21 and astrocytes produce astrocytomas. Astrocytomas can be immunologically cold. Moreover, other studies have reported further classiﬁed according to the WHO deﬁnitions, based on that immunotherapies can alter the tumour microenvironment in the degree of malignancy, ranging from grade I to IV; grade IV GBM, which may inﬂuence patients’ response to treatment and 1 15,22 tumours are also called glioblastomas (GBM). Among astrocyto- culminate in beneﬁts from combined therapies. mas, GBM is the most frequent and fatal form; the incidence rate The diagnosis of GBM is currently based on imaging techniques in the United States is 3.20 per 100,000 population, and GBM and tissue biopsies. However, imaging techniques cannot reliably 2,3 accounts for 60–70% of malignant gliomas. On the basis of differentiate lesions caused by actual tumour progression from genome, transcriptome and proteome proﬁling, Phillips et al. pseudoprogression—treatment-related lesions that mimic tumour have categorised GBM into three molecular subtypes: proneural, progression and might resolve spontaneously over time. Similarly, 4–8 classic and mesenchymal. Each subtype shows important tissue biopsies entail a highly invasive procedure, yet might only genetic changes, reﬂecting altered signalling pathways, leading capture a static snapshot of an ever-changing tumour. By to differences in treatment responses, ultimately affecting a contrast, liquid biopsies that enable the detection of circulating patient’s prognosis. biomarkers confer the beneﬁt of being non-invasive, thereby Current therapeutic modalities for GBM consist of a combina- facilitating serial sampling and the ability to monitor potential 23–25 tion of surgery (which aims for maximal resection of the tumour), dynamic changes in the tumour over the course of therapy. radiotherapy and chemotherapy. The standard chemotherapeutic Tumours in general, including GBM, shed tumoural content into 9,10 26 27 drug used is temozolomide (TMZ). However, even with TMZ, the blood and cerebrospinal ﬂuid (CSF). The detection of these patients have a low median survival of ~15 months. In addition, biomarkers, such as proteins, cell-free nucleic acids (cfNAs), GBM patients show high rates of resistance to therapies and high extracellular vesicles (EVs) and circulating tumour cells (CTCs), in 10–14 rates of relapse, which result in poor overall survival. Some a liquid biopsy can be used to complement standard risk- immunotherapies, such as nivolumab, which targets the immune stratiﬁcation methods, monitoring of treatment response and checkpoint molecule programmed cell protein 1 (PD-1), and disease progression in GBM patients. This review aims to bevacizumab, which targets vascular endothelial growth factor, summarise the current literature on circulating biomarkers that Saliva and Liquid Biopsy Translational Research Team, The School of Biomedical Sciences, Institute of Health and Biomedical Innovation, Queensland University of Technology, 2 3 Kelvin Grove, QLD 4059, Australia; Translational Research Institute, Woolloongabba, QLD 4102, Australia; Faculty of Medicine, University of Queensland, 288 Herston Road, 4 5 Herston, QLD 4006, Australia; Cancer Care Services, Royal Brisbane and Women’s Hospital, Herston, QLD 4029, Australia; School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Gardens Point, QLD 4000, Australia and Cell and Molecular Biology Department, Sid Faithfull Brain Cancer Laboratory, QIMR Berghofer MRI, Brisbane, QLD 4006, Australia Correspondence: Chamindie Punyadeera ([email protected]) Received: 22 May 2019 Revised: 23 August 2019 Accepted: 23 September 2019 Published online: 31 October 2019 © The Author(s) 2019 Published by Springer Nature on behalf of Cancer Research UK Circulating biomarkers in patients with glioblastoma J Müller Bark et al. ab Treatment ↑ Methylation of the MGMT gene promoter (91%) p53 (overexpression) Pseudoprogression cd 10–30% Interferon regulatory factor 9 (IRF9) and x-ray repair cross-complementing 1 True progression (XRCC1) MRI scan within 12 weeks Fig. 1 Pseudoprogression. After treatment, a brain MRI scan is performed in GBM patients. When the MRI is performed within 12 weeks of treatment, 10–30% of patients may present enhanced lesions that may improve with time, which are known as pseudoprogression. A correlation between the methylation of the MGMT gene promoter, and overexpression of p53, IRF9 and XRCC1, and the occurrence of 35–37 pseudoprogression has been observed. Example of pseudoprogression in a male patient diagnosed with glioblastoma at 51 years of age. Initial gadolinium-enhanced T1-weighted MRI prior to any treatment (a) demonstrated a heterogeneously enhancing right temporal mass (red arrow), which was resected; histopathology was consistent with glioblastoma. Immediate postoperative imaging (b) demonstrated near- complete resection of tumour, but MRI after adjuvant radiation and chemotherapy (c) was concerned with progression due to interval development of new irregular enhancement adjacent to the cavity (green arrow). On this basis, he proceeded to further debulking surgery, the histopathology consistent with necrosis only. Five years after diagnosis, his MRI (d) remained free of evidence of recurrence, consistent with the diagnosis of pseudoprogression. Figure produced using Servier Medical Art are found in the blood of GBM patients, with a focus on CTCs, ﬁrst MRI scan, usually within the ﬁrst 12 weeks of treatment. The circulating tumour DNA (ctDNA) and EVs. ability to differentiate between pseudoprogression and true progression is important, as it would help clinicians to avoid performing unnecessary operations and prescribing ineffective 25,28,30,35,36 CURRENT APPROACHES TO THE MANAGEMENT OF GBM therapies. However, currently, there are no biomarkers Diagnosis or clinical features to distinguish glioma true progression from The initial diagnosis of GBM is achieved by neuroimaging, pseudoprogression. Brandes et al. showed, by using tissue followed by resection or biopsy of tumour tissue to deﬁnitively biopsy samples from 103 patients with GBM, that patients with diagnose, grade and characterise the tumour. Currently, tissue methylation of the MGMT gene promoter had higher rates of biopsies are the gold-standard technique for GBM diagnosis. pseudoprogression (91%) than patients with unmethylated MGMT However, resection or biopsy from a brain tumour can present (41%), as shown by MRI. In their study, pseudoprogression was 37 38 risks to the patients, such as possible brain swelling within and found in 31% of patients. In addition, Kang et al. discovered around the tumour mass, or might even affect neurological that p53 overexpression in tumour tissue sections correlated with functions. Moreover, some tumours might be inaccessible owing pseudoprogression in 35 tumour samples from glioma patients. to their location. Furthermore, tissue biopsies can sometimes fail p53 was considered to be overexpressed when >10% of the to predict the heterogeneity of the whole tumour mass and might tumour cells stained positive for p53, and of the 13 patients whose not be a true representation of the tumour activity in real time. samples showed p53 overexpression, seven had pseudoprogres- Further conﬁrmatory and descriptive tests are performed on sion, three presented with non-progression and three showed tumour samples by using immunohistochemistry and molecular early progression. Pseudoprogression rates were higher in glioma 16,30–32 analyses, including the combined loss of chromosome arms patients who presented with p53 overexpression than in patients 1p and 19q, the mutation and/or expression of p53, the presence without p53 overexpression. In a total of 22 patients whose of isocitrate dehydrogenase 1 (IDH1) mutation (within exon 4 to samples did not show p53 overexpression, 14 showed non- codon 132, the most common being c.395 G > A (R132H) progression, one presented with pseudoprogression and seven 33 6 38 substitutions ) and epigenetic alterations, such as O -methylgua- presented with early progression. Following their analysis of 9,32 nine-DNA methyltransferase (MGMT) hypermethylation. tumour tissue from 17 glioma patients to identify a potential biomarker for pseudoprogression, Qian et al. suggested that Treatment higher expressions of interferon regulatory factor 9 (IRF9) and X- As alluded to above, current therapeutic modalities for GBM entail ray repair cross-complementing 1 (XRCC1) were associated with a combination of surgery followed by radiotherapy and/or pseudoprogression. However, despite these emerging data, more chemotherapy. In surgery, it is challenging to safely remove all studies in this ﬁeld are warranted to identify a biomarker that can tumour cells due to the high invasive capacity of GBM cells into be implemented into a clinical setting to better differentiate true normal tissue; as a result, GBM tumours recur in the majority of the progression from pseudoprogression. cases. In patients with recurrent GBM, the median overall In order to improve outcomes for patients with GBM new and survival is 6.2 months. merging strategies are used. These include non-invasive methods of sampling to help in the diagnosis and monitoring of GBM. Prognosis and pseudoprogression Some of the efforts are in the ﬁelds of advanced magnetic To obtain prognostic information, a brain MRI scan is performed resonance imaging and spectroscopy (MRI/MRS), plasma immu- after treatment. Contrast-enhancing lesions that appear on the noproﬁling and liquid biopsies. Currently, conventional MRI is a images can be caused by tumour progression, but might also be gold-standard technique for workup and treatment response, due to post-radiotherapy changes, referred to as pseudoprogres- providing detailed structural information enabling guided surgery sion, which might resolve spontaneously (Fig. 1). Pseudopro- and mapping of tumour tissues. Nevertheless, in the initial gression occurs in 10–30% of GBM patients who have had their workup, this technique cannot distinguish between different 1234567890();,: Circulating biomarkers in patients with glioblastoma J Müller Bark et al. high-grade gliomas, such as glioblastoma from oligodendro- different stages of gliomas, as the progression of the disease 40 63 glioma, and there may also be difﬁculties in distinguishing aggravates its disruption and permeability increases. This infective causes, lymphoma and metastases from primary brain disruption can be seen on MRI by using a contrast medium such 41 63,64 tumours. After treatment of GBM, there remain signiﬁcant as gadolinium, which does not normally cross the intact BBB. imaging challenges in response assessment, such as pseudopro- However, some regions of GBM can have an intact BBB. Despite gression and pseudoresponse. Advanced MR techniques are the association between BBB dysfunctions and GBM disease being investigated to provide more detailed information on progression, EVs derived from glioma cells were shown to cross 40,42 66 tumour properties, but many of these remain investigational. the intact BBB and were detected in the blood of GBM patients. MR may also not predict molecular characteristics of primary brain This highlights the importance of EVs in liquid biopsies since EVs tumours, such as MGMT methylation and IDH mutation status. Han can be detected in cases in which the BBB is not compromised. et al. correlated the MGMT promoter methylation with tumour Liquid biopsy could therefore be a helpful tool to complement location and necrosis by using advanced MRI. However, Moon current strategies for predicting GBM prognosis, by allowing a et al. did not ﬁnd signiﬁcant association between MGMT more dynamic view of tumour characteristics, and response to methylation status and tumour location. Furthermore, small chemotherapy, by providing a platform (through multiple 49,51,67 cohort sizes were used in these studies. Therefore, there is a sampling) to monitor treatment responses. A schematic need for more research to be conducted to better understand the illustration of circulating biomarkers that could be investigated in role of advanced MR imaging. GBM patients’ blood is shown in Fig. 2; these biomarkers are discussed in more detail below. LIQUID BIOPSIES Tumours shed their tumoural content into circulation and can be CIRCULATING TUMOUR CELLS sampled in a number of body ﬂuids. Examples of these are CTCs, CTCs and metastasis cfNAs, ctDNA and EVs (comprising both microvesicles [MVs] and Glioma metastasis outside of the CNS is a very rare event, with 26,68 exosomes). The majority of these biomarkers have a short half-life, ~0.4–0.5% of gliomas metastasising to sites such as the lungs though—up to 3 h—and are rapidly degraded when present and pleura, regional lymph nodes, bones and liver. The low rates 25,46 freely in the plasma. However, some of them are packaged in of distant metastasis might be due to low survival rates of GBM EVs, such as MVs and exosomes, which offers protection from patients, or a possible suppression of tumour cell growth outside degradation by circulating proteases and nucleases. the CNS by the immune system or the BBB, the presence of which The sampling and analysis of these molecules in non-solid makes it more difﬁcult for the cells to intravasate into the biological ﬂuids is deﬁned as a liquid biopsy, ﬂuid biopsy or circulation. Despite these low rates, some cases of extracranial ﬂuid-phase biopsy. Although liquid biopsies are often carried metastases have been reported when patients have signed up to out by using a blood draw, other bioﬂuids, such as saliva and donate organs, providing evidence that distant metastasis can 49 26,68,70 urine, can be also used. Cerebrospinal ﬂuid (CSF) has also been occur with glioma patients. used to investigate tumour-speciﬁc biomarkers in brain In several solid tumour types, metastasis is normally related to 27,50 tumours, as it circulates along with the brain and spinal cord, the presence of CTCs—cells that are shed by either primary or 71,72 and therefore has close contact with the CNS, but CSF collection secondary tumours into the circulatory system. CTCs are requires an invasive lumbar puncture procedure. Liquid biopsy metastatic precursor cells that undergo epithelial–mesenchymal presents a minimally invasive way to capture tumour activities in transition (EMT), a cellular process that is characterised by a more 49,51 real time to diagnose and predict disease progression. mesenchymal phenotype and increased migratory potential. CTCs The use of liquid biopsies has been studied in different tumour can then extravasate from the circulatory system and colonise types, and this approach has been used in the prognosis of breast other organs. CTCs can be released as single cells, or in homotypic 52 53 54 cancer, head and neck and lung cancer. For lung cancer, for or heterotypic clusters, which have been reported to have a higher 73–76 example, when tumour tissue is limited, blood plasma can be used metastatic propensity compared with single CTCs. Szczerba to detect the presence of mutations in the epidermal growth et al. reported that CTC–neutrophil clusters injected into 55,56 factor receptor. The ﬁrst FDA-approved broad companion tumour-free mice induced faster tumour formation than single pan-cancer diagnostic test utilises the presence of ctDNA within a CTCs, as well as increased metastatic potential and shorter overall 76 74 liquid biopsy sample to test for the presence of a number of solid survival. Gkountela et al. observed in breast cancer that the tumours, including non-small-cell lung cancer, colorectal cancer, methylation status of CTC clusters is linked with the prognosis of 57,58 breast cancer, ovarian cancer and melanoma. the disease. Transcription factor binding sites in genes related to For liquid biopsies to be successfully used in GBM, it is assumed cell stemness and proliferation were differentially methylated in that tumour-speciﬁc material would cross the blood–brain–barrier CTC clusters (hypomethylated) in comparison with single CTCs (BBB). The BBB regulates the access and exchange of nutrients, (hypermethylated), and this alteration is reﬂected by an increased vitamins and other molecules into the brain. The integrity of the stemness phenotype and metastatic ability, culminating in a poor tight junctions of the BBB, which is determined by proteins such prognosis. CTCs have been detected and characterised in as claudin-3, claudin-5, claudin-12 and other transmembrane different tumour types, and their presence has been found to 77,78 proteins with scaffolding functions, deﬁnes the quality of a healthy correlate with poor overall survival. However, CTC research in BBB, and accordingly, a deﬁciency or mutation in claudin-1 or GBM is limited: the ﬁrst study on CTCs was published in 59 26,68,79 agrin has been related to BBB dysfunction in GBM. GBM induces 2014, and the ﬁrst paper on CTC clusters in GBM was a proangiogenic and inﬂamed microenvironment, which published in 2018. decreases tight junctions, helping to establish a more permeable BBB, supporting the access of pro-inﬂammatory immune cells Isolation and characterisation of CTCs such as tumour-associated macrophages. In addition, hypoxia, a CTCs can be isolated by using different approaches. One such typical feature of solid tumours such as GBM, is also correlated approach is to use label-free selection with speciﬁc protein with the disruption of the BBB. markers to select or to deplete those cells that express a particular 62 47,81 Zhao et al. reported that exosomes derived from the hypoxic marker. Other approaches to CTC isolation rely on differences GBM cell line U87 promoted the proliferation of brain micro- in their physical property and use techniques, such as ﬁltration, vascular endothelial cells (BMVECs), inducing BBB permeability in chip technology, density gradient centrifugation, electric ﬁeld, 82 54,83–85 an in vitro model. Changes in BBB permeability are seen within sound waves and microﬂuidic technology. Circulating biomarkers in patients with glioblastoma J Müller Bark et al. GBM BBB permeability GBM patient Blood Blood collection analysis Biomarkers shed into circulation CTCs Plasma/serum ctDNA Exosomes Tumour Circulating tumour cell Tumour cell ctDNA Proteins mRNA miRNA Exosomes Fig. 2 A schematic representation of biomolecular transportation from a tumour through the BBB into the circulation. (a) In patients with GBM, a leaky BBB allows circulating biomarkers—for example, circulating tumour cells (CTCs), circulating tumour DNA (ctDNA) and microvesicles—to enter the circulatory system, from where they can be collected, via blood draw, and further analysed. (b) A breakdown of the tumoural components found in the circulatory system. Several classes of biomarkers can be accessed and measured in liquid biopsies, including CTCs, which can be shed from a primary tumour; extracellular vesicles, which can be released by tumour cells (and can carry nucleic acids and proteins inside); ctDNA, which can also be released by tumour cells. These molecules carry tumoural information (e.g., mutational status, tumoural cargo), which can be sampled non-invasively. Figure produced using Servier Medical Art Characterisation of CTCs can be performed by using immuno- identiﬁed in patients with a progressive disease rather than in cytochemistry, molecular technologies and/or functional assays. patients with stable disease. Positive CTCs were then characterised Currently, the only FDA-approved platform for the isolation of by using a ‘cocktail’ of antibodies against SOX2, tubulin β-3, EGFR, CTCs is the CellSearch® system (Menarini Silicon Biosystems, Italy), A2B5 and c-MET based on GBM biomarkers identiﬁed in the which relies on the positive selection of tumour cells over- literature, before the expression of 25 genes, representing all the 45,86 expressing an epithelial cell adhesion marker, EpCAM. Cells molecular subtypes of GBM (proneural, neural, classical and from GBM tumours tend to adopt a more mesenchymal mesenchymal), was assessed; the results of the analysis concluded phenotype compared with the epithelial nature of cells needed that CTCs from GBM show more of a mesenchymal phenotype. for detection by using the CellSearch. Therefore, alternative This phenotype is associated with a higher invasion capacity, approaches for the isolation of CTCs need to be explored in GBM allowing cells to intravasate into the circulation, which may patients. explain the rare cases of extracranial metastases in GBM. Gao et al. used a matrix for separation followed by a negative CTCs in glioblastoma depletion of white blood cells by immunomagnetic beads. A Muller et al. detected CTCs in the peripheral blood of 29 out of polyploidy chromosome-8-positive detection was used as a 141 GBM patients (20.6%). Following their isolation by using positive criterion for CTCs, along with GFAP-positive or -negative density-gradient centrifugation, CTCs were stained for glial cells and CD45-negative cell status to conﬁrm glioma origin. CTCs ﬁbrillary acidic protein (GFAP) as a GBM marker, as well as were detected in peripheral blood from 24 out of 31 (77%) verifying ampliﬁcation of the EGFR gene and demonstrating gains patients with seven different subtypes of glioma, including and losses in genomic regions of chromosomes 7 and 10. The astrocytomas, oligodendrogliomas and oligoastrocytomas. In authors also used single-cell genomic analysis to identify common GBM patients speciﬁcally, CTCs were detected in nine out of 11 mutations found in CTCs and in tumour tissue, to prove that the patients (82%). No correlation was found between the number of CTCs were most likely derived from GBM. Similarly, MacArthur CTC and the different grade of glioma, but interestingly, when the et al. isolated CTCs from glioma patients by density-gradient authors investigated patients with new enhancing mass lesions centrifugation by using the OncoQuick® system (Greiner Bio-One, (ﬁve patients), the results correlated with CTC counts. Of the ﬁve Frickenhausen, Germany). As telomerase activity is elevated in patients in total, three had GBM. Two of these GBM patients had tumour cells but not in normal cells, a telomerase-based test was CTC counts of two and three, respectively, and showed recurrence used to detect CTCs, as well as testing for nestin expression as a of the disease; the CTC count of the other patient that showed glioma cell marker. CTCs were detected in eight out of 11 (72%) possible recurrence by imaging was zero. As the patient presented patients prior to radiotherapy, whereas the detection rate for post- with no clinical symptoms, no treatment was given. The patient 79 68 radiotherapy patients was one out of 8 (8%). Sullivan et al. was asked to return after 1 month, whereupon the new imaging showed evidence of CTCs in 39% of peripheral blood samples results showed a decrease in enhancing lesion, which is indicative from patients with GBM. In this study, CTC–iCHIP technology was of pseudoprogression and not true progression. used to enrich for CTCs by depleting haematopoietic cells from In 2018, Krol et al. observed the ﬁrst evidence of CTC clusters blood specimens. Interestingly, higher counts of CTCs were in GBM and could detect CTCs in seven out of 13 patients with Circulating biomarkers in patients with glioblastoma J Müller Bark et al. progressive GBM (53.8%). Blood collections were made at seven ctDNA in glioblastoma different time points during disease progression in an open-label Schwaederle et al. conducted a study in which ctDNA in plasma Phase 1/2a study testing the compound BAL101553 (a micro- samples collected from 171 patients with different tumour types tubule inhibitor), and CTCs were isolated by Parsortix microﬂuidic was analysed on a targeted panel (54-gene panel) by using next- technology. The authors identiﬁed positive CTCs as cells that generation sequencing (NGS). Of these 171 patients, 33 had GBM met at least one of the following criteria: they were at least 9 µm in diagnosis (representing 19% of samples). Unaltered ctDNA was size, and were negative for CD45 staining; they were positive for detected in 73% of the patients with GBM; 24% had one alteration, EGFR, Ki67 or the microtubule-associated protein EB1, as well as and 3% had two or more alterations. In a separate study, 89 46 being CD45 negative. Liu et al. , also in 2018, isolated CTCs from Bettegowda et al. detected ctDNA in <10% of 27 patients with GBM patients as previously described and characterised their glioma by using PCR (ten out of the 27 were diagnosed with GBM). stemness by immunohistochemistry by using Olig2 and CD133. Glioma was the tumour type with the lowest frequency of cases A mouse model was used to show the capacity of CTCs to reseed with detectable ctDNA. These studies demonstrate a low yield of the primary tumour site when injected intravenously. Also, by ctDNA in glioma patients’ blood, mostly justiﬁed by the presence using cell viability and apoptosis assays, the authors analysed the of the BBB. By contrast, by using a NGS panel, Piccioni et al. resistance of CTCs to radiotherapy and chemotherapy with TMZ analysed 419 patients with primary brain tumours, including 222 and concluded that CTCs are more resistant to treatments and to patients with GBM, and detected ctDNA mutations in blood stress induced in the circulation than other tumour cells. Malara samples collected from 50% of all brain-tumour patients—55% et al. reported a case of a 67-year-old patient presenting with an among the GBM patients. The authors concluded that for patients intracranial lesion that was subsequently conﬁrmed to be GBM. with detectable ctDNA levels, the results of plasma ctDNA analysis The patient’s blood was collected before surgery and 2 months could provide a viable option to pursue treatment alternatives. If post-operation, and CTCs were captured by using density-gradient ctDNA is detected, speciﬁc mutations can be tracked during centrifugation. The ﬁrst blood sample showed 4.5 CTCs/ml of treatment, which might reveal an increase in tumour-speciﬁc DNA blood, but this ﬁgure increased to 7 CTCs/ml in the second or a change in DNA methylation status. The DNA methylation sample. After 9 months, the patient presented with tumour status can indicate acquired resistance to a certain treatment, and recurrence, and 5 months later had succumbed to the disease. therefore tracking those alterations could guide the modiﬁcation 95 96 Along with imaging results, CTC analysis proved to be relevant in of treatment. When Wang et al. analysed the serum and CSF of monitoring the patient’s intracranial lesion. patients with different grades of glioma, they detected the The number of studies showing the detection of CTCs in GBM presence of methylation in the MGMT promoter, by using patients is still limited, and the use of different approaches to methylation-speciﬁc PCR, in 38 patients with GBM out of 89 isolate and characterise these cells makes it difﬁcult to compare glioma patients (42.6%). This analysis showed a higher sensitivity their results. Also, the number of patient samples remains a when using CSF instead of blood samples (serum), with MGMT limitation. However, these studies demonstrate an increased promoter methylation detected in 19 out of 89 patients (21.3%) in application of CTCs in GBM studies with the potential for serum samples, and in 26 out of 78 patients (33.3%) by using investigation in clinical trials, but clearly, larger trials are CSF. This higher sensitivity found in CSF suggests that the use of warranted. CSF may present an advantage in comparison with serum and might be due to the BBB presence that prevents ctDNA clearing. In a pilot study conducted by Salkeni et al., plasma samples CIRCULATING TUMOUR NUCLEIC ACIDS from three out of 13 patients (23%) newly diagnosed with GBM Cells may release DNA and RNA (including mRNA and non-coding contained the EGFRvIII deletion variant. This variant, which is RNA) content into the circulation. Cell-free DNA (cfDNA) comprises frequently detected in GBM patients with EGFR ampliﬁcation, small fragments of DNA (180–200 base pairs) released by cells contains a deletion in exons 2–7, which generates a constitutively under physiological and pathological conditions. It is suggested active form of EGFR to confer activation of EGFR downstream that the main source of cfDNA derives from apoptotic cells. targets. The authors suggested that the levels of EGFRvIII DNA in When released by normal cells, these fragments are generally patients’ blood might correlate with the tumour resection status, cleared by phagocytosis, and consequently, cfDNA levels are as the levels of EGFRvIII DNA were higher in a patient who showed 23 97 98 typically low in healthy individuals. In cancer patients, a incomplete tumour removal. Faria et al. observed that the proportion of cfDNA will comprise ctDNA. The amount of ctDNA cfDNA levels were signiﬁcantly increased (by ~30-fold) in patients varies and is thought to reﬂect the burden of disease, with greater with GBM or brain metastases who were receiving a treatment of amounts of ctDNA present in more advanced/late-stage intranasal administration of perillyl alcohol compared with the patients. ctDNA might carry tumour-speciﬁc mutations, reﬂect- healthy control group. After treatment, patients who survived ing the mutational landscape of the primary tumour, and more than 6 months had 2.7-fold lower cfDNA levels than patients therefore represents an important means by which to sample who survived <6 months. This result indicates that the relevance tumour tissue non-invasively. However, there are challenges of cfDNA levels might present to prognostic assessment in associated with ctDNA analysis, such as the sensitive detection GBM. By using a patient-derived orthotopic xenograft model, Mair technologies needed to distinguish mutant from wild-type alleles et al. showed that tumour size and cell proliferation inﬂuence and the development of thresholds for mutations (e.g., variant the release of ctDNA in mice prior to treatment, while BBB allele frequency (VAF)). Furthermore, ctDNA fragments present a integrity does not. However, they also found that cell death post short half-life of <1.5 h and require to be processed rapidly. therapy is an additional factor that can augment ctDNA release. Tumour cells can also shed different classes of RNA into the These results suggest that BBB may not play the main role in circulation, such as protein-coding mRNA, and non-coding ctDNA release as it has been suggested in previous studies. More microRNAs (miRNA) that are small and contain around 21–24 studies need to be conducted in order to deﬁne the exact nucleotides, and long non-coding RNAs (lncRNA) that consist of contribution of BBB to ctDNA release. In addition, the authors 200 nucleotides or greater. These noncoding RNAs play a reported that whereas ctDNA was detected in 24% of mice (15/ signiﬁcant role in gene regulation and can be found as circulating 64), plasma tumour mitochondrial DNA (tmtDNA) was detected in cell-free nucleic acids or inside of EVs, which provide more 82% of the animals (52/64). tmtDNA was also detected in CSF and stability. Circulating RNAs have been found in blood and CSF of urine, while ctDNA was not detectable in urine. This study glioma patients, and may act as biomarkers for prognosis, demonstrates the potential use of tmtDNA analysis in GBM, and 24,91,92 diagnosis and treatment monitoring. moreover, helps to elucidate different factors that inﬂuence ctDNA Circulating biomarkers in patients with glioblastoma J Müller Bark et al. Exosomes Microvesicles 30–150 nm 50–1300 nm Fig. 3 A schematic representation of the two main classes of EVs. Exosomes and microvesicles differ mainly in size and origin. The diameter of exosomes is smaller (30–150 nm), and are derived from the endosomal membrane. The diameter of microvesicles ranges from (50 to 1300 nm), and are released from cell membrane budding. Figure produced using Servier Medical Art concentration in the circulation. In addition, this study highlights and low levels of GAS5 in serum associated with a reduced some advantages of the use of tmtDNA in comparison with probability of 2-year survival, suggesting its potential as prog- ctDNA, such as high sensitivity of detection, high copy number nostic biomarkers. present in glioma and low cost. Zhao et al. studied the response of 66 patients with GBM recurrent to the PD-1 immune checkpoint inhibitors nivolumab or EXTRACELLULAR VESICLES pembrolizumab. The authors analysed DNA and RNA from plasma, EVs are membrane-bound vesicles that are released by cells under in the form of cfNAs, tumour tissue and clinical data. Genomic and physiological and pathological conditions. EVs can carry cargo, transcriptomic analysis correlated with the patients’ response to such as mRNA, miRNA, DNA and cellular proteins, and they can be 28,110,111 immunotherapy: those who did not respond showed mutations in detected by using cell-surface markers. Previously, EVs 112,113 PTEN that are associated with immunosuppressive expression were considered as artefacts or fragments of dead cells, but signatures, whereas responders carried mutations in components they are now known to play a major role in cell–cell communica- 100 114 of the mitogen-activated protein kinase (MAPK) pathway. These tion, as their released cargo—such as mRNA, miRNA and results demonstrate that the response to immunotherapy by using angiogenic proteins—can be taken up by other cells, even from PD-1 inhibitor varies according to speciﬁc molecular alterations, distant sites, thereby enabling genetic information, as well as and the therapy may beneﬁt a subgroup of GBM patients, proteins to be delivered to, and inﬂuencing the phenotype of suggesting a molecular and personalised selection of patients for recipient cells, such as endothelial cells. One of the advantages of immunotherapies. these circulating biomarkers in liquid biopsy studies would be the protection of biomolecules within the EVs. Circulating RNA in glioblastoma There are two broad types of EVs, exosomes and MVs, which miR-21 is an important miRNA studied in cancer, and its differ mainly in their size and origin. Exosomes are smaller 101 102 upregulation has been reported in the plasma and tissue (30–150-nm diameter) and are derived from the endosomal of GBM patients, and associates with lower overall survival and membrane, while MVs range from 50 to ~1300 nm and are 103 104 115 tumour grading. Wang et al. analysed the plasma of ten GBM released directly from budding of the cell membrane (Fig. 3). patients before and after therapy, and described two miRNAs, Exosomes can be detected by transmission electron microscopy miR-128 and miR-342-3p, which are downregulated in patients (TEM), nanoparticle-tracking analysis (NTA) and the presence of a when compared with healthy controls. miR-128 and miR-342-3p number of membrane-associated proteins, such as CD63, CD81, levels correlated with glioma grades and increased after surgery CD9, CD37, CD53, CD82, ICAM-1 and integrins, all of which can be and chemoradiation, suggesting their use as biomarkers to identiﬁed by ﬂow cytometry or Western blot. Currently, there assess tumour grading and to monitor treatment response. are no standard protocols in consensus to speciﬁcally isolate EV Zhi et al. analysed the serum of patients and established that subtypes and separate exosomes and MVs. Therefore, the the upregulation of miR-20a-5p, miR-106a-5p and miR-181b-5p International Society for Extracellular Vesicles (ISEV) recommends correlated with tumour grading, and miR-19a-3p, miR-106a-5p to consider the physical/biochemical characteristics of EVs in order and miR-181b-5p were linked with poor prognosis. In addition, to name them, for example, ‘small EVs' or ‘medium/large EVs' or 106 116 Zhao et al. isolated miRNA from the serum of patients and CD63+/CD81+ EVs. In this review, we separated the EV classes, described that miR-222-3p, miR-182, miR-20a-5p, miR-106a-5p and MVs and exosomes, based on the terminology used in the original miR-145-5p correlated with poor patient outcome. Along with research papers. miRNA class, another noncoding RNA class, circulating lncRNAs, is 107 108 emerging as potential cancer biomarkers. Tan et al. studied Microvesicles in glioblastoma the prognostic value of a long non-coding RNA, HOX transcript Koch et al. investigated whether blood-derived MVs could aid antisense intergenic RNA (HOTAIR) in patients’ serum. HOTAIR is in differentiating GBM recurrence from tumour pseudoprogres- known to be overexpressed in GBM and to induce cell sion on the basis of a difference in MV number. In blood collected proliferation. Higher HOTAIR levels were detected in patients’ from seven healthy controls or 11 patients with GBM at different total serum and in exosomes when compared with healthy treatment times, the quantity of MVs from patients with stable 108 109 controls. Shen et al. also reported that high levels of HOTAIR disease or pseudoprogression was signiﬁcantly lower than Circulating biomarkers in patients with glioblastoma J Müller Bark et al. in patients who underwent true tumour progression. high-grade gliomas from other tumours’ brain metastases. Never- Evans et al. also correlated an increase in MV number with theless, when in combination, the panel differentiated high-grade poor overall survival and with earlier disease recurrence. Skog gliomas from other tumours’ brain metastases, suggesting that it et al., having isolated MVs from tumour samples and serum of can represent an alternative for inconclusive biopsy results or in 25 GBM patients by centrifugation, identiﬁed the EGFRvIII deletion cases in which the tumour is located in critical brain areas. variant in MVs from seven out of the 25 patients, whereas no Manterola et al. also analysed exosomal small non-coding RNA EGFRvIII was detected in the control healthy group. The authors signature from the serum of 75 patients with GBM. The authors also concluded that GBM cells shed MVs, and that their content— concluded that miR-320 and miR-574-3p, as well as a small no- that included angiogenic proteins in addition to EGFRvIII—can ncoding RNA, RNU6-1, are upregulated and are able to enhance the angiogenic phenotype of normal brain endothelial discriminate GBM patients from healthy controls. Most of the cells and proliferation in other glioma cells. EV studies in GBM present a limitation of the small size of cohorts. Therefore, there is still the need for validation of these ﬁndings in Exosomes in glioblastoma larger cohorts. Osti et al. demonstrated that the concentration of EVs was increased in GBM patients in comparison with healthy controls and patients with other CNS diseases. When comparing EV CONCLUSIONS AND FUTURE DIRECTIONS concentrations over different time points, an increase in EV There remains a need for non-invasive sampling to capture brain- concentrations correlated with tumour recurrence, suggesting tumour activity in real time to better inform prognosis of the that exosomes could help to predict GBM recurrence. Similarly, disease and to monitor treatment responses. Current diagnosis of Andre-Gregoire et al. observed a higher concentration of EVs in GBM relies on imaging and tumour tissue data; however, there are patients with GBM, as well as showing that EVs from patient- some challenges and limitations. Conventional MRI can guide derived glioblastoma stem cells, which are thought to be involved surgery; however, it cannot distinguish between high-grade in tumour initiation, expansion, resistance to treatments and gliomas and may provide imaging ﬁndings that are challenging relapse, had increased cargo relating to cell adhesion after TMZ to interpret. Tumour tissue biopsies are invasive and cannot be treatment, indicating that TMZ had the potential to promote the repeated easily. Liquid biopsies present advantages when increased release of factors favouring tumour progression. compared with the current approaches, and these include the Manda et al. investigated the expression of EGFR and EGFRvIII ability to repeat sampling over the course of treatment in a non- in serum exosomes and tumour tissue in 96 patients with high- invasive manner, and the fact that the BBB may be more grade glioma. They detected EGFRvIII in 39.5% of tumour tissue permeable in the presence of a high-grade tumour, allowing samples and in 44.7% of their paired serum exosome samples, molecular transportation. A liquid biopsy may be able to reveal 133,134 whereas 28.1% of tumour biopsy samples had EGFR and EGFRvIII tumour information prior to clinical progression. However, co-expression. Although the co-expression of EGFR and EGFRvIII is the tumour morphological features and the microenvironment are rare in GBM cells, this co-expression is suggested to cooperate more readily available in the tissue biopsy. Therefore, a liquid with tumour growth and induce macrophage inﬁltration. biopsy aims to provide additional and complementary data to Also, the presence of EGFRvIII in exosomes correlated with improve upon the diagnosis and follow-up of GBM patients. a lower overall survivor pattern—21.1 months—compared with CTCs, ctDNA and MVs have been demonstrated to be able to 28.6 months for patients with no EGFRvIII expression in be sampled from different bioﬂuids for a number of tumour exosomes. types, and studies have demonstrated that these biomarkers can Chandran et al. reported that syndecan-1 found in plasma be found in GBM patients, and that their mutational proﬁles EVs can be used to distinguish low-grade glioma from high-grade represent those of the GBM in origin. There is a pressing need to GBM with a sensitivity of 71% and a speciﬁcity of 80%, and improve the technologies involved in regularly and reliably provided strong support for plasma–EV-derived syndecan-1 being isolating and characterising these biomarkers, and larger studies derived from GBM tumours. Yang et al. isolated exosomes from in GBM investigating these biomarkers are warranted, with tumours generated in mice by using four GBM patient-derived clinical correlatives measured over time to determine the effects samples collected during surgery, and reported an increase in the on clinical outcome. Unlike for many other tumour types, the expression of the genes encoding dynamin-3, p65 and CD117, use of CTCs in GBM as a diagnostic screening tool is not ideal, alongside a decrease in the expression of the genes encoding because by the time a patient with GBM experiences clinical PTEN and p53, in the tumour tissue and blood of mice. In another symptoms and receives a positive diagnosis from a treating study of 60 glioma patients, including 27 diagnosed with GBM, physician, their disease is already at an advanced stage. miRNA was detected in exosomes isolated from the serum by However, the liquid biopsy approach shows great potential in centrifugation and quantitated by using real-time PCR. The managing GBM patients. authors found that in comparison with low-grade gliomas, miR- Currently, no clinically validated circulating biomarkers for 301a levels were higher in high-grade gliomas. They also observed managing GBM patients exist. One reason for the relative lack of that the serum exosomal miR-301a levels were lower after surgical circulating biomarkers in this ﬁeld is because of the BBB, resection of the tumour, but were increased during GBM restricting the transportation of molecules from blood to the recurrence, indicating that serum exosomal miR-301a could be a brain and vice versa. Along with biological difﬁculties, there are potential biomarker for diagnosis/prognosis for GBM technical limitations for the establishment of a role for CTCs in 127,128 129 patients. Ebrahimkhani et al. used a panel of seven GBM. Only a few studies have been carried out by using brain- exosomal miRNAs—miR-182-5p, miR-328-3p, miR-339-5p, miR- tumour-derived CTCs, and they show that the detection rates vary 340-5p, miR-485-3p, miR-486-5p and miR-543—to differentiate from 20 to 77% in GBM patients, depending on the CTC isolation GBM patients from healthy controls with an accuracy rate of techniques used. However, other studies do warrant further 91.7%. In addition, Santangelo et al. analysed a miRNA signature investigation of CTCs in GBM. For example, the ﬁrst report in exosomes from the serum of glioma patients in an attempt to detecting CTC clusters in GBM, published in 2018, also indicates differentiate tumour grading and gliomas from brain metastases. the capacity of GBM clusters to cross the BBB. This is an The authors demonstrated the upregulation of three miRNAs, miR- important clinical ﬁnding that requires large studies to test the 21, miR-222 and miR-124-3p in glioma. miR-21 is known to play a reproducibility of these data. role in GBM pathogenesis. In their study, miR-21 differentiated The detection rates for ctDNA in GBM patients’ blood also vary healthy controls from glioma patients, but could not distinguish (10–55%), highlighting the need for more studies with larger Circulating biomarkers in patients with glioblastoma J Müller Bark et al. Table 1. Summary of advantages and disadvantages of using CTCs, ctDNA and exosomes as biomarkers in cancer Advantages Disadvantages Reference 9 25,28,47,75 CTCs � Information can be provided at the protein, DNA and � CTCs are rare (1 cell in 10 blood cells) RNA levels � Possibility of carrying out functional assays � May represent only part of the tumour mass heterogeneity � There are new technologies in the development � Process to isolate them is challenging phase to isolate CTCs 23,25,46,75 ctDNA � Higher ctDNA levels compared with CTC � Short half-life, <1.5 h � Levels correlate with disease stage � Released mainly by cells undergoing necrosis or apoptosis � Easy detection 25,115,135 Exosomes � Can be released by all cells, including tumour cells � The release is not exclusive from tumour cells � Can carry proteins, DNA, RNA and miRNA � Possible presence of contaminants by current isolation methods � Present protection for their content cohorts to better understand ctDNA in GBM. GBM patients often AUTHOR CONTRIBUTIONS J.M.B., A.K., B.C., B.W.D. and C.P. all contributed to the writing of the paper. develop resistance to treatment. Monitoring patients over the course of treatment, by serial sampling, and detecting speciﬁc tumour mutations and changes in DNA methylation pattern, ADDITIONAL INFORMATION might prove valuable for understanding tumour behaviour. These Competing interests: The authors declare no competing interests. parameters might complement the current conventional meth- odologies used in managing GBM patients. Moreover, multiple Ethical approval and consent to participate: Not applicable collections would enable tumour progression to be monitored, or pseudoprogression to be detected in a minimally invasive manner. Funding: J.M.B. is funded by ATM LATAM QUT Postgraduate Research Scholarship. When different bioﬂuid sources are compared—for example, ctDNA detected in blood or in CSF—CSF appears to be more representative, possibly owing to the proximity of CSF to the Consent to publish: Not applicable brain. Nevertheless, CSF collection is much more invasive and risky compared with blood collection. Data availability: Not applicable The exosome ﬁeld in GBM is also emerging and has been producing promising data, such as detection of the EGFRvIII Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. deletion variant in tumour tissue (39.5%) matching with EGFRvIII expression in exosomes (44.7%), and in both cases correlating with poor survival. However, some technical limitations also need to be addressed in the future for this ﬁeld. In addition, the cohort REFERENCES size of the majority of the studies is small, and currently there are 1. Louis, D. 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Publisher: Springer Journals
Copyright: Copyright © The Author(s) 2019
Subject: Biomedicine; Biomedicine, general; Cancer Research; Epidemiology; Molecular Medicine; Oncology; Drug Resistance
ISSN: 0007-0920
eISSN: 1532-1827
DOI: 10.1038/s41416-019-0603-6
Publisher site: See Article on Publisher Site

Abstract

www.nature.com/bjc REVIEW ARTICLE 1,2 1,2 3,4 3,5,6 1,2 Juliana Müller Bark , Arutha Kulasinghe , Benjamin Chua , Bryan W. Day and Chamindie Punyadeera Gliomas are the most common tumours of the central nervous system and the most aggressive form is glioblastoma (GBM). Despite advances in treatment, patient survival remains low. GBM diagnosis typically relies on imaging techniques and postoperative pathological diagnosis; however, both procedures have their inherent limitations. Imaging modalities cannot differentiate tumour progression from treatment-related changes that mimic progression, known as pseudoprogression, which might lead to misinterpretation of therapy response and delay clinical interventions. In addition to imaging limitations, tissue biopsies are invasive and most of the time cannot be performed over the course of treatment to evaluate ‘real-time’ tumour dynamics. In an attempt to address these limitations, liquid biopsies have been proposed in the ﬁeld. Blood sampling is a minimally invasive procedure for a patient to endure and could provide tumoural information to guide therapy. Tumours shed tumoural content, such as circulating tumour cells, cell-free nucleic acids, proteins and extracellular vesicles, into the circulation, and these biomarkers are reported to cross the blood–brain barrier. The use of liquid biopsies is emerging in the ﬁeld of GBM. In this review, we aim to summarise the current literature on circulating biomarkers, namely circulating tumour cells, circulating tumour DNA and extracellular vesicles as potential non-invasively sampled biomarkers to manage the treatment of patients with GBM. British Journal of Cancer (2020) 122:295–305; https://doi.org/10.1038/s41416-019-0603-6 15–17 BACKGROUND are being trialled to improve treatment in GBM patients. Early Gliomas are the most common type of tumours originating from data have shown beneﬁt from the use of a PD-1 inhibitor, in other 18,19 20 the central nervous system (CNS) and can be classiﬁed according tumour types, as melanoma and non-small-cell lung cancer to the cells that give rise to them: oligodendrocytes give rise to in patients with a high tumour mutational burden. However, GBM oligodendrogliomas, ependymal cells generate ependymomas is thought to have a low mutational burden and to be 1 21 and astrocytes produce astrocytomas. Astrocytomas can be immunologically cold. Moreover, other studies have reported further classiﬁed according to the WHO deﬁnitions, based on that immunotherapies can alter the tumour microenvironment in the degree of malignancy, ranging from grade I to IV; grade IV GBM, which may inﬂuence patients’ response to treatment and 1 15,22 tumours are also called glioblastomas (GBM). Among astrocyto- culminate in beneﬁts from combined therapies. mas, GBM is the most frequent and fatal form; the incidence rate The diagnosis of GBM is currently based on imaging techniques in the United States is 3.20 per 100,000 population, and GBM and tissue biopsies. However, imaging techniques cannot reliably 2,3 accounts for 60–70% of malignant gliomas. On the basis of differentiate lesions caused by actual tumour progression from genome, transcriptome and proteome proﬁling, Phillips et al. pseudoprogression—treatment-related lesions that mimic tumour have categorised GBM into three molecular subtypes: proneural, progression and might resolve spontaneously over time. Similarly, 4–8 classic and mesenchymal. Each subtype shows important tissue biopsies entail a highly invasive procedure, yet might only genetic changes, reﬂecting altered signalling pathways, leading capture a static snapshot of an ever-changing tumour. By to differences in treatment responses, ultimately affecting a contrast, liquid biopsies that enable the detection of circulating patient’s prognosis. biomarkers confer the beneﬁt of being non-invasive, thereby Current therapeutic modalities for GBM consist of a combina- facilitating serial sampling and the ability to monitor potential 23–25 tion of surgery (which aims for maximal resection of the tumour), dynamic changes in the tumour over the course of therapy. radiotherapy and chemotherapy. The standard chemotherapeutic Tumours in general, including GBM, shed tumoural content into 9,10 26 27 drug used is temozolomide (TMZ). However, even with TMZ, the blood and cerebrospinal ﬂuid (CSF). The detection of these patients have a low median survival of ~15 months. In addition, biomarkers, such as proteins, cell-free nucleic acids (cfNAs), GBM patients show high rates of resistance to therapies and high extracellular vesicles (EVs) and circulating tumour cells (CTCs), in 10–14 rates of relapse, which result in poor overall survival. Some a liquid biopsy can be used to complement standard risk- immunotherapies, such as nivolumab, which targets the immune stratiﬁcation methods, monitoring of treatment response and checkpoint molecule programmed cell protein 1 (PD-1), and disease progression in GBM patients. This review aims to bevacizumab, which targets vascular endothelial growth factor, summarise the current literature on circulating biomarkers that Saliva and Liquid Biopsy Translational Research Team, The School of Biomedical Sciences, Institute of Health and Biomedical Innovation, Queensland University of Technology, 2 3 Kelvin Grove, QLD 4059, Australia; Translational Research Institute, Woolloongabba, QLD 4102, Australia; Faculty of Medicine, University of Queensland, 288 Herston Road, 4 5 Herston, QLD 4006, Australia; Cancer Care Services, Royal Brisbane and Women’s Hospital, Herston, QLD 4029, Australia; School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Gardens Point, QLD 4000, Australia and Cell and Molecular Biology Department, Sid Faithfull Brain Cancer Laboratory, QIMR Berghofer MRI, Brisbane, QLD 4006, Australia Correspondence: Chamindie Punyadeera ([email protected]) Received: 22 May 2019 Revised: 23 August 2019 Accepted: 23 September 2019 Published online: 31 October 2019 © The Author(s) 2019 Published by Springer Nature on behalf of Cancer Research UK Circulating biomarkers in patients with glioblastoma J Müller Bark et al. ab Treatment ↑ Methylation of the MGMT gene promoter (91%) p53 (overexpression) Pseudoprogression cd 10–30% Interferon regulatory factor 9 (IRF9) and x-ray repair cross-complementing 1 True progression (XRCC1) MRI scan within 12 weeks Fig. 1 Pseudoprogression. After treatment, a brain MRI scan is performed in GBM patients. When the MRI is performed within 12 weeks of treatment, 10–30% of patients may present enhanced lesions that may improve with time, which are known as pseudoprogression. A correlation between the methylation of the MGMT gene promoter, and overexpression of p53, IRF9 and XRCC1, and the occurrence of 35–37 pseudoprogression has been observed. Example of pseudoprogression in a male patient diagnosed with glioblastoma at 51 years of age. Initial gadolinium-enhanced T1-weighted MRI prior to any treatment (a) demonstrated a heterogeneously enhancing right temporal mass (red arrow), which was resected; histopathology was consistent with glioblastoma. Immediate postoperative imaging (b) demonstrated near- complete resection of tumour, but MRI after adjuvant radiation and chemotherapy (c) was concerned with progression due to interval development of new irregular enhancement adjacent to the cavity (green arrow). On this basis, he proceeded to further debulking surgery, the histopathology consistent with necrosis only. Five years after diagnosis, his MRI (d) remained free of evidence of recurrence, consistent with the diagnosis of pseudoprogression. Figure produced using Servier Medical Art are found in the blood of GBM patients, with a focus on CTCs, ﬁrst MRI scan, usually within the ﬁrst 12 weeks of treatment. The circulating tumour DNA (ctDNA) and EVs. ability to differentiate between pseudoprogression and true progression is important, as it would help clinicians to avoid performing unnecessary operations and prescribing ineffective 25,28,30,35,36 CURRENT APPROACHES TO THE MANAGEMENT OF GBM therapies. However, currently, there are no biomarkers Diagnosis or clinical features to distinguish glioma true progression from The initial diagnosis of GBM is achieved by neuroimaging, pseudoprogression. Brandes et al. showed, by using tissue followed by resection or biopsy of tumour tissue to deﬁnitively biopsy samples from 103 patients with GBM, that patients with diagnose, grade and characterise the tumour. Currently, tissue methylation of the MGMT gene promoter had higher rates of biopsies are the gold-standard technique for GBM diagnosis. pseudoprogression (91%) than patients with unmethylated MGMT However, resection or biopsy from a brain tumour can present (41%), as shown by MRI. In their study, pseudoprogression was 37 38 risks to the patients, such as possible brain swelling within and found in 31% of patients. In addition, Kang et al. discovered around the tumour mass, or might even affect neurological that p53 overexpression in tumour tissue sections correlated with functions. Moreover, some tumours might be inaccessible owing pseudoprogression in 35 tumour samples from glioma patients. to their location. Furthermore, tissue biopsies can sometimes fail p53 was considered to be overexpressed when >10% of the to predict the heterogeneity of the whole tumour mass and might tumour cells stained positive for p53, and of the 13 patients whose not be a true representation of the tumour activity in real time. samples showed p53 overexpression, seven had pseudoprogres- Further conﬁrmatory and descriptive tests are performed on sion, three presented with non-progression and three showed tumour samples by using immunohistochemistry and molecular early progression. Pseudoprogression rates were higher in glioma 16,30–32 analyses, including the combined loss of chromosome arms patients who presented with p53 overexpression than in patients 1p and 19q, the mutation and/or expression of p53, the presence without p53 overexpression. In a total of 22 patients whose of isocitrate dehydrogenase 1 (IDH1) mutation (within exon 4 to samples did not show p53 overexpression, 14 showed non- codon 132, the most common being c.395 G > A (R132H) progression, one presented with pseudoprogression and seven 33 6 38 substitutions ) and epigenetic alterations, such as O -methylgua- presented with early progression. Following their analysis of 9,32 nine-DNA methyltransferase (MGMT) hypermethylation. tumour tissue from 17 glioma patients to identify a potential biomarker for pseudoprogression, Qian et al. suggested that Treatment higher expressions of interferon regulatory factor 9 (IRF9) and X- As alluded to above, current therapeutic modalities for GBM entail ray repair cross-complementing 1 (XRCC1) were associated with a combination of surgery followed by radiotherapy and/or pseudoprogression. However, despite these emerging data, more chemotherapy. In surgery, it is challenging to safely remove all studies in this ﬁeld are warranted to identify a biomarker that can tumour cells due to the high invasive capacity of GBM cells into be implemented into a clinical setting to better differentiate true normal tissue; as a result, GBM tumours recur in the majority of the progression from pseudoprogression. cases. In patients with recurrent GBM, the median overall In order to improve outcomes for patients with GBM new and survival is 6.2 months. merging strategies are used. These include non-invasive methods of sampling to help in the diagnosis and monitoring of GBM. Prognosis and pseudoprogression Some of the efforts are in the ﬁelds of advanced magnetic To obtain prognostic information, a brain MRI scan is performed resonance imaging and spectroscopy (MRI/MRS), plasma immu- after treatment. Contrast-enhancing lesions that appear on the noproﬁling and liquid biopsies. Currently, conventional MRI is a images can be caused by tumour progression, but might also be gold-standard technique for workup and treatment response, due to post-radiotherapy changes, referred to as pseudoprogres- providing detailed structural information enabling guided surgery sion, which might resolve spontaneously (Fig. 1). Pseudopro- and mapping of tumour tissues. Nevertheless, in the initial gression occurs in 10–30% of GBM patients who have had their workup, this technique cannot distinguish between different 1234567890();,: Circulating biomarkers in patients with glioblastoma J Müller Bark et al. high-grade gliomas, such as glioblastoma from oligodendro- different stages of gliomas, as the progression of the disease 40 63 glioma, and there may also be difﬁculties in distinguishing aggravates its disruption and permeability increases. This infective causes, lymphoma and metastases from primary brain disruption can be seen on MRI by using a contrast medium such 41 63,64 tumours. After treatment of GBM, there remain signiﬁcant as gadolinium, which does not normally cross the intact BBB. imaging challenges in response assessment, such as pseudopro- However, some regions of GBM can have an intact BBB. Despite gression and pseudoresponse. Advanced MR techniques are the association between BBB dysfunctions and GBM disease being investigated to provide more detailed information on progression, EVs derived from glioma cells were shown to cross 40,42 66 tumour properties, but many of these remain investigational. the intact BBB and were detected in the blood of GBM patients. MR may also not predict molecular characteristics of primary brain This highlights the importance of EVs in liquid biopsies since EVs tumours, such as MGMT methylation and IDH mutation status. Han can be detected in cases in which the BBB is not compromised. et al. correlated the MGMT promoter methylation with tumour Liquid biopsy could therefore be a helpful tool to complement location and necrosis by using advanced MRI. However, Moon current strategies for predicting GBM prognosis, by allowing a et al. did not ﬁnd signiﬁcant association between MGMT more dynamic view of tumour characteristics, and response to methylation status and tumour location. Furthermore, small chemotherapy, by providing a platform (through multiple 49,51,67 cohort sizes were used in these studies. Therefore, there is a sampling) to monitor treatment responses. A schematic need for more research to be conducted to better understand the illustration of circulating biomarkers that could be investigated in role of advanced MR imaging. GBM patients’ blood is shown in Fig. 2; these biomarkers are discussed in more detail below. LIQUID BIOPSIES Tumours shed their tumoural content into circulation and can be CIRCULATING TUMOUR CELLS sampled in a number of body ﬂuids. Examples of these are CTCs, CTCs and metastasis cfNAs, ctDNA and EVs (comprising both microvesicles [MVs] and Glioma metastasis outside of the CNS is a very rare event, with 26,68 exosomes). The majority of these biomarkers have a short half-life, ~0.4–0.5% of gliomas metastasising to sites such as the lungs though—up to 3 h—and are rapidly degraded when present and pleura, regional lymph nodes, bones and liver. The low rates 25,46 freely in the plasma. However, some of them are packaged in of distant metastasis might be due to low survival rates of GBM EVs, such as MVs and exosomes, which offers protection from patients, or a possible suppression of tumour cell growth outside degradation by circulating proteases and nucleases. the CNS by the immune system or the BBB, the presence of which The sampling and analysis of these molecules in non-solid makes it more difﬁcult for the cells to intravasate into the biological ﬂuids is deﬁned as a liquid biopsy, ﬂuid biopsy or circulation. Despite these low rates, some cases of extracranial ﬂuid-phase biopsy. Although liquid biopsies are often carried metastases have been reported when patients have signed up to out by using a blood draw, other bioﬂuids, such as saliva and donate organs, providing evidence that distant metastasis can 49 26,68,70 urine, can be also used. Cerebrospinal ﬂuid (CSF) has also been occur with glioma patients. used to investigate tumour-speciﬁc biomarkers in brain In several solid tumour types, metastasis is normally related to 27,50 tumours, as it circulates along with the brain and spinal cord, the presence of CTCs—cells that are shed by either primary or 71,72 and therefore has close contact with the CNS, but CSF collection secondary tumours into the circulatory system. CTCs are requires an invasive lumbar puncture procedure. Liquid biopsy metastatic precursor cells that undergo epithelial–mesenchymal presents a minimally invasive way to capture tumour activities in transition (EMT), a cellular process that is characterised by a more 49,51 real time to diagnose and predict disease progression. mesenchymal phenotype and increased migratory potential. CTCs The use of liquid biopsies has been studied in different tumour can then extravasate from the circulatory system and colonise types, and this approach has been used in the prognosis of breast other organs. CTCs can be released as single cells, or in homotypic 52 53 54 cancer, head and neck and lung cancer. For lung cancer, for or heterotypic clusters, which have been reported to have a higher 73–76 example, when tumour tissue is limited, blood plasma can be used metastatic propensity compared with single CTCs. Szczerba to detect the presence of mutations in the epidermal growth et al. reported that CTC–neutrophil clusters injected into 55,56 factor receptor. The ﬁrst FDA-approved broad companion tumour-free mice induced faster tumour formation than single pan-cancer diagnostic test utilises the presence of ctDNA within a CTCs, as well as increased metastatic potential and shorter overall 76 74 liquid biopsy sample to test for the presence of a number of solid survival. Gkountela et al. observed in breast cancer that the tumours, including non-small-cell lung cancer, colorectal cancer, methylation status of CTC clusters is linked with the prognosis of 57,58 breast cancer, ovarian cancer and melanoma. the disease. Transcription factor binding sites in genes related to For liquid biopsies to be successfully used in GBM, it is assumed cell stemness and proliferation were differentially methylated in that tumour-speciﬁc material would cross the blood–brain–barrier CTC clusters (hypomethylated) in comparison with single CTCs (BBB). The BBB regulates the access and exchange of nutrients, (hypermethylated), and this alteration is reﬂected by an increased vitamins and other molecules into the brain. The integrity of the stemness phenotype and metastatic ability, culminating in a poor tight junctions of the BBB, which is determined by proteins such prognosis. CTCs have been detected and characterised in as claudin-3, claudin-5, claudin-12 and other transmembrane different tumour types, and their presence has been found to 77,78 proteins with scaffolding functions, deﬁnes the quality of a healthy correlate with poor overall survival. However, CTC research in BBB, and accordingly, a deﬁciency or mutation in claudin-1 or GBM is limited: the ﬁrst study on CTCs was published in 59 26,68,79 agrin has been related to BBB dysfunction in GBM. GBM induces 2014, and the ﬁrst paper on CTC clusters in GBM was a proangiogenic and inﬂamed microenvironment, which published in 2018. decreases tight junctions, helping to establish a more permeable BBB, supporting the access of pro-inﬂammatory immune cells Isolation and characterisation of CTCs such as tumour-associated macrophages. In addition, hypoxia, a CTCs can be isolated by using different approaches. One such typical feature of solid tumours such as GBM, is also correlated approach is to use label-free selection with speciﬁc protein with the disruption of the BBB. markers to select or to deplete those cells that express a particular 62 47,81 Zhao et al. reported that exosomes derived from the hypoxic marker. Other approaches to CTC isolation rely on differences GBM cell line U87 promoted the proliferation of brain micro- in their physical property and use techniques, such as ﬁltration, vascular endothelial cells (BMVECs), inducing BBB permeability in chip technology, density gradient centrifugation, electric ﬁeld, 82 54,83–85 an in vitro model. Changes in BBB permeability are seen within sound waves and microﬂuidic technology. Circulating biomarkers in patients with glioblastoma J Müller Bark et al. GBM BBB permeability GBM patient Blood Blood collection analysis Biomarkers shed into circulation CTCs Plasma/serum ctDNA Exosomes Tumour Circulating tumour cell Tumour cell ctDNA Proteins mRNA miRNA Exosomes Fig. 2 A schematic representation of biomolecular transportation from a tumour through the BBB into the circulation. (a) In patients with GBM, a leaky BBB allows circulating biomarkers—for example, circulating tumour cells (CTCs), circulating tumour DNA (ctDNA) and microvesicles—to enter the circulatory system, from where they can be collected, via blood draw, and further analysed. (b) A breakdown of the tumoural components found in the circulatory system. Several classes of biomarkers can be accessed and measured in liquid biopsies, including CTCs, which can be shed from a primary tumour; extracellular vesicles, which can be released by tumour cells (and can carry nucleic acids and proteins inside); ctDNA, which can also be released by tumour cells. These molecules carry tumoural information (e.g., mutational status, tumoural cargo), which can be sampled non-invasively. Figure produced using Servier Medical Art Characterisation of CTCs can be performed by using immuno- identiﬁed in patients with a progressive disease rather than in cytochemistry, molecular technologies and/or functional assays. patients with stable disease. Positive CTCs were then characterised Currently, the only FDA-approved platform for the isolation of by using a ‘cocktail’ of antibodies against SOX2, tubulin β-3, EGFR, CTCs is the CellSearch® system (Menarini Silicon Biosystems, Italy), A2B5 and c-MET based on GBM biomarkers identiﬁed in the which relies on the positive selection of tumour cells over- literature, before the expression of 25 genes, representing all the 45,86 expressing an epithelial cell adhesion marker, EpCAM. Cells molecular subtypes of GBM (proneural, neural, classical and from GBM tumours tend to adopt a more mesenchymal mesenchymal), was assessed; the results of the analysis concluded phenotype compared with the epithelial nature of cells needed that CTCs from GBM show more of a mesenchymal phenotype. for detection by using the CellSearch. Therefore, alternative This phenotype is associated with a higher invasion capacity, approaches for the isolation of CTCs need to be explored in GBM allowing cells to intravasate into the circulation, which may patients. explain the rare cases of extracranial metastases in GBM. Gao et al. used a matrix for separation followed by a negative CTCs in glioblastoma depletion of white blood cells by immunomagnetic beads. A Muller et al. detected CTCs in the peripheral blood of 29 out of polyploidy chromosome-8-positive detection was used as a 141 GBM patients (20.6%). Following their isolation by using positive criterion for CTCs, along with GFAP-positive or -negative density-gradient centrifugation, CTCs were stained for glial cells and CD45-negative cell status to conﬁrm glioma origin. CTCs ﬁbrillary acidic protein (GFAP) as a GBM marker, as well as were detected in peripheral blood from 24 out of 31 (77%) verifying ampliﬁcation of the EGFR gene and demonstrating gains patients with seven different subtypes of glioma, including and losses in genomic regions of chromosomes 7 and 10. The astrocytomas, oligodendrogliomas and oligoastrocytomas. In authors also used single-cell genomic analysis to identify common GBM patients speciﬁcally, CTCs were detected in nine out of 11 mutations found in CTCs and in tumour tissue, to prove that the patients (82%). No correlation was found between the number of CTCs were most likely derived from GBM. Similarly, MacArthur CTC and the different grade of glioma, but interestingly, when the et al. isolated CTCs from glioma patients by density-gradient authors investigated patients with new enhancing mass lesions centrifugation by using the OncoQuick® system (Greiner Bio-One, (ﬁve patients), the results correlated with CTC counts. Of the ﬁve Frickenhausen, Germany). As telomerase activity is elevated in patients in total, three had GBM. Two of these GBM patients had tumour cells but not in normal cells, a telomerase-based test was CTC counts of two and three, respectively, and showed recurrence used to detect CTCs, as well as testing for nestin expression as a of the disease; the CTC count of the other patient that showed glioma cell marker. CTCs were detected in eight out of 11 (72%) possible recurrence by imaging was zero. As the patient presented patients prior to radiotherapy, whereas the detection rate for post- with no clinical symptoms, no treatment was given. The patient 79 68 radiotherapy patients was one out of 8 (8%). Sullivan et al. was asked to return after 1 month, whereupon the new imaging showed evidence of CTCs in 39% of peripheral blood samples results showed a decrease in enhancing lesion, which is indicative from patients with GBM. In this study, CTC–iCHIP technology was of pseudoprogression and not true progression. used to enrich for CTCs by depleting haematopoietic cells from In 2018, Krol et al. observed the ﬁrst evidence of CTC clusters blood specimens. Interestingly, higher counts of CTCs were in GBM and could detect CTCs in seven out of 13 patients with Circulating biomarkers in patients with glioblastoma J Müller Bark et al. progressive GBM (53.8%). Blood collections were made at seven ctDNA in glioblastoma different time points during disease progression in an open-label Schwaederle et al. conducted a study in which ctDNA in plasma Phase 1/2a study testing the compound BAL101553 (a micro- samples collected from 171 patients with different tumour types tubule inhibitor), and CTCs were isolated by Parsortix microﬂuidic was analysed on a targeted panel (54-gene panel) by using next- technology. The authors identiﬁed positive CTCs as cells that generation sequencing (NGS). Of these 171 patients, 33 had GBM met at least one of the following criteria: they were at least 9 µm in diagnosis (representing 19% of samples). Unaltered ctDNA was size, and were negative for CD45 staining; they were positive for detected in 73% of the patients with GBM; 24% had one alteration, EGFR, Ki67 or the microtubule-associated protein EB1, as well as and 3% had two or more alterations. In a separate study, 89 46 being CD45 negative. Liu et al. , also in 2018, isolated CTCs from Bettegowda et al. detected ctDNA in <10% of 27 patients with GBM patients as previously described and characterised their glioma by using PCR (ten out of the 27 were diagnosed with GBM). stemness by immunohistochemistry by using Olig2 and CD133. Glioma was the tumour type with the lowest frequency of cases A mouse model was used to show the capacity of CTCs to reseed with detectable ctDNA. These studies demonstrate a low yield of the primary tumour site when injected intravenously. Also, by ctDNA in glioma patients’ blood, mostly justiﬁed by the presence using cell viability and apoptosis assays, the authors analysed the of the BBB. By contrast, by using a NGS panel, Piccioni et al. resistance of CTCs to radiotherapy and chemotherapy with TMZ analysed 419 patients with primary brain tumours, including 222 and concluded that CTCs are more resistant to treatments and to patients with GBM, and detected ctDNA mutations in blood stress induced in the circulation than other tumour cells. Malara samples collected from 50% of all brain-tumour patients—55% et al. reported a case of a 67-year-old patient presenting with an among the GBM patients. The authors concluded that for patients intracranial lesion that was subsequently conﬁrmed to be GBM. with detectable ctDNA levels, the results of plasma ctDNA analysis The patient’s blood was collected before surgery and 2 months could provide a viable option to pursue treatment alternatives. If post-operation, and CTCs were captured by using density-gradient ctDNA is detected, speciﬁc mutations can be tracked during centrifugation. The ﬁrst blood sample showed 4.5 CTCs/ml of treatment, which might reveal an increase in tumour-speciﬁc DNA blood, but this ﬁgure increased to 7 CTCs/ml in the second or a change in DNA methylation status. The DNA methylation sample. After 9 months, the patient presented with tumour status can indicate acquired resistance to a certain treatment, and recurrence, and 5 months later had succumbed to the disease. therefore tracking those alterations could guide the modiﬁcation 95 96 Along with imaging results, CTC analysis proved to be relevant in of treatment. When Wang et al. analysed the serum and CSF of monitoring the patient’s intracranial lesion. patients with different grades of glioma, they detected the The number of studies showing the detection of CTCs in GBM presence of methylation in the MGMT promoter, by using patients is still limited, and the use of different approaches to methylation-speciﬁc PCR, in 38 patients with GBM out of 89 isolate and characterise these cells makes it difﬁcult to compare glioma patients (42.6%). This analysis showed a higher sensitivity their results. Also, the number of patient samples remains a when using CSF instead of blood samples (serum), with MGMT limitation. However, these studies demonstrate an increased promoter methylation detected in 19 out of 89 patients (21.3%) in application of CTCs in GBM studies with the potential for serum samples, and in 26 out of 78 patients (33.3%) by using investigation in clinical trials, but clearly, larger trials are CSF. This higher sensitivity found in CSF suggests that the use of warranted. CSF may present an advantage in comparison with serum and might be due to the BBB presence that prevents ctDNA clearing. In a pilot study conducted by Salkeni et al., plasma samples CIRCULATING TUMOUR NUCLEIC ACIDS from three out of 13 patients (23%) newly diagnosed with GBM Cells may release DNA and RNA (including mRNA and non-coding contained the EGFRvIII deletion variant. This variant, which is RNA) content into the circulation. Cell-free DNA (cfDNA) comprises frequently detected in GBM patients with EGFR ampliﬁcation, small fragments of DNA (180–200 base pairs) released by cells contains a deletion in exons 2–7, which generates a constitutively under physiological and pathological conditions. It is suggested active form of EGFR to confer activation of EGFR downstream that the main source of cfDNA derives from apoptotic cells. targets. The authors suggested that the levels of EGFRvIII DNA in When released by normal cells, these fragments are generally patients’ blood might correlate with the tumour resection status, cleared by phagocytosis, and consequently, cfDNA levels are as the levels of EGFRvIII DNA were higher in a patient who showed 23 97 98 typically low in healthy individuals. In cancer patients, a incomplete tumour removal. Faria et al. observed that the proportion of cfDNA will comprise ctDNA. The amount of ctDNA cfDNA levels were signiﬁcantly increased (by ~30-fold) in patients varies and is thought to reﬂect the burden of disease, with greater with GBM or brain metastases who were receiving a treatment of amounts of ctDNA present in more advanced/late-stage intranasal administration of perillyl alcohol compared with the patients. ctDNA might carry tumour-speciﬁc mutations, reﬂect- healthy control group. After treatment, patients who survived ing the mutational landscape of the primary tumour, and more than 6 months had 2.7-fold lower cfDNA levels than patients therefore represents an important means by which to sample who survived <6 months. This result indicates that the relevance tumour tissue non-invasively. However, there are challenges of cfDNA levels might present to prognostic assessment in associated with ctDNA analysis, such as the sensitive detection GBM. By using a patient-derived orthotopic xenograft model, Mair technologies needed to distinguish mutant from wild-type alleles et al. showed that tumour size and cell proliferation inﬂuence and the development of thresholds for mutations (e.g., variant the release of ctDNA in mice prior to treatment, while BBB allele frequency (VAF)). Furthermore, ctDNA fragments present a integrity does not. However, they also found that cell death post short half-life of <1.5 h and require to be processed rapidly. therapy is an additional factor that can augment ctDNA release. Tumour cells can also shed different classes of RNA into the These results suggest that BBB may not play the main role in circulation, such as protein-coding mRNA, and non-coding ctDNA release as it has been suggested in previous studies. More microRNAs (miRNA) that are small and contain around 21–24 studies need to be conducted in order to deﬁne the exact nucleotides, and long non-coding RNAs (lncRNA) that consist of contribution of BBB to ctDNA release. In addition, the authors 200 nucleotides or greater. These noncoding RNAs play a reported that whereas ctDNA was detected in 24% of mice (15/ signiﬁcant role in gene regulation and can be found as circulating 64), plasma tumour mitochondrial DNA (tmtDNA) was detected in cell-free nucleic acids or inside of EVs, which provide more 82% of the animals (52/64). tmtDNA was also detected in CSF and stability. Circulating RNAs have been found in blood and CSF of urine, while ctDNA was not detectable in urine. This study glioma patients, and may act as biomarkers for prognosis, demonstrates the potential use of tmtDNA analysis in GBM, and 24,91,92 diagnosis and treatment monitoring. moreover, helps to elucidate different factors that inﬂuence ctDNA Circulating biomarkers in patients with glioblastoma J Müller Bark et al. Exosomes Microvesicles 30–150 nm 50–1300 nm Fig. 3 A schematic representation of the two main classes of EVs. Exosomes and microvesicles differ mainly in size and origin. The diameter of exosomes is smaller (30–150 nm), and are derived from the endosomal membrane. The diameter of microvesicles ranges from (50 to 1300 nm), and are released from cell membrane budding. Figure produced using Servier Medical Art concentration in the circulation. In addition, this study highlights and low levels of GAS5 in serum associated with a reduced some advantages of the use of tmtDNA in comparison with probability of 2-year survival, suggesting its potential as prog- ctDNA, such as high sensitivity of detection, high copy number nostic biomarkers. present in glioma and low cost. Zhao et al. studied the response of 66 patients with GBM recurrent to the PD-1 immune checkpoint inhibitors nivolumab or EXTRACELLULAR VESICLES pembrolizumab. The authors analysed DNA and RNA from plasma, EVs are membrane-bound vesicles that are released by cells under in the form of cfNAs, tumour tissue and clinical data. Genomic and physiological and pathological conditions. EVs can carry cargo, transcriptomic analysis correlated with the patients’ response to such as mRNA, miRNA, DNA and cellular proteins, and they can be 28,110,111 immunotherapy: those who did not respond showed mutations in detected by using cell-surface markers. Previously, EVs 112,113 PTEN that are associated with immunosuppressive expression were considered as artefacts or fragments of dead cells, but signatures, whereas responders carried mutations in components they are now known to play a major role in cell–cell communica- 100 114 of the mitogen-activated protein kinase (MAPK) pathway. These tion, as their released cargo—such as mRNA, miRNA and results demonstrate that the response to immunotherapy by using angiogenic proteins—can be taken up by other cells, even from PD-1 inhibitor varies according to speciﬁc molecular alterations, distant sites, thereby enabling genetic information, as well as and the therapy may beneﬁt a subgroup of GBM patients, proteins to be delivered to, and inﬂuencing the phenotype of suggesting a molecular and personalised selection of patients for recipient cells, such as endothelial cells. One of the advantages of immunotherapies. these circulating biomarkers in liquid biopsy studies would be the protection of biomolecules within the EVs. Circulating RNA in glioblastoma There are two broad types of EVs, exosomes and MVs, which miR-21 is an important miRNA studied in cancer, and its differ mainly in their size and origin. Exosomes are smaller 101 102 upregulation has been reported in the plasma and tissue (30–150-nm diameter) and are derived from the endosomal of GBM patients, and associates with lower overall survival and membrane, while MVs range from 50 to ~1300 nm and are 103 104 115 tumour grading. Wang et al. analysed the plasma of ten GBM released directly from budding of the cell membrane (Fig. 3). patients before and after therapy, and described two miRNAs, Exosomes can be detected by transmission electron microscopy miR-128 and miR-342-3p, which are downregulated in patients (TEM), nanoparticle-tracking analysis (NTA) and the presence of a when compared with healthy controls. miR-128 and miR-342-3p number of membrane-associated proteins, such as CD63, CD81, levels correlated with glioma grades and increased after surgery CD9, CD37, CD53, CD82, ICAM-1 and integrins, all of which can be and chemoradiation, suggesting their use as biomarkers to identiﬁed by ﬂow cytometry or Western blot. Currently, there assess tumour grading and to monitor treatment response. are no standard protocols in consensus to speciﬁcally isolate EV Zhi et al. analysed the serum of patients and established that subtypes and separate exosomes and MVs. Therefore, the the upregulation of miR-20a-5p, miR-106a-5p and miR-181b-5p International Society for Extracellular Vesicles (ISEV) recommends correlated with tumour grading, and miR-19a-3p, miR-106a-5p to consider the physical/biochemical characteristics of EVs in order and miR-181b-5p were linked with poor prognosis. In addition, to name them, for example, ‘small EVs' or ‘medium/large EVs' or 106 116 Zhao et al. isolated miRNA from the serum of patients and CD63+/CD81+ EVs. In this review, we separated the EV classes, described that miR-222-3p, miR-182, miR-20a-5p, miR-106a-5p and MVs and exosomes, based on the terminology used in the original miR-145-5p correlated with poor patient outcome. Along with research papers. miRNA class, another noncoding RNA class, circulating lncRNAs, is 107 108 emerging as potential cancer biomarkers. Tan et al. studied Microvesicles in glioblastoma the prognostic value of a long non-coding RNA, HOX transcript Koch et al. investigated whether blood-derived MVs could aid antisense intergenic RNA (HOTAIR) in patients’ serum. HOTAIR is in differentiating GBM recurrence from tumour pseudoprogres- known to be overexpressed in GBM and to induce cell sion on the basis of a difference in MV number. In blood collected proliferation. Higher HOTAIR levels were detected in patients’ from seven healthy controls or 11 patients with GBM at different total serum and in exosomes when compared with healthy treatment times, the quantity of MVs from patients with stable 108 109 controls. Shen et al. also reported that high levels of HOTAIR disease or pseudoprogression was signiﬁcantly lower than Circulating biomarkers in patients with glioblastoma J Müller Bark et al. in patients who underwent true tumour progression. high-grade gliomas from other tumours’ brain metastases. Never- Evans et al. also correlated an increase in MV number with theless, when in combination, the panel differentiated high-grade poor overall survival and with earlier disease recurrence. Skog gliomas from other tumours’ brain metastases, suggesting that it et al., having isolated MVs from tumour samples and serum of can represent an alternative for inconclusive biopsy results or in 25 GBM patients by centrifugation, identiﬁed the EGFRvIII deletion cases in which the tumour is located in critical brain areas. variant in MVs from seven out of the 25 patients, whereas no Manterola et al. also analysed exosomal small non-coding RNA EGFRvIII was detected in the control healthy group. The authors signature from the serum of 75 patients with GBM. The authors also concluded that GBM cells shed MVs, and that their content— concluded that miR-320 and miR-574-3p, as well as a small no- that included angiogenic proteins in addition to EGFRvIII—can ncoding RNA, RNU6-1, are upregulated and are able to enhance the angiogenic phenotype of normal brain endothelial discriminate GBM patients from healthy controls. Most of the cells and proliferation in other glioma cells. EV studies in GBM present a limitation of the small size of cohorts. Therefore, there is still the need for validation of these ﬁndings in Exosomes in glioblastoma larger cohorts. Osti et al. demonstrated that the concentration of EVs was increased in GBM patients in comparison with healthy controls and patients with other CNS diseases. When comparing EV CONCLUSIONS AND FUTURE DIRECTIONS concentrations over different time points, an increase in EV There remains a need for non-invasive sampling to capture brain- concentrations correlated with tumour recurrence, suggesting tumour activity in real time to better inform prognosis of the that exosomes could help to predict GBM recurrence. Similarly, disease and to monitor treatment responses. Current diagnosis of Andre-Gregoire et al. observed a higher concentration of EVs in GBM relies on imaging and tumour tissue data; however, there are patients with GBM, as well as showing that EVs from patient- some challenges and limitations. Conventional MRI can guide derived glioblastoma stem cells, which are thought to be involved surgery; however, it cannot distinguish between high-grade in tumour initiation, expansion, resistance to treatments and gliomas and may provide imaging ﬁndings that are challenging relapse, had increased cargo relating to cell adhesion after TMZ to interpret. Tumour tissue biopsies are invasive and cannot be treatment, indicating that TMZ had the potential to promote the repeated easily. Liquid biopsies present advantages when increased release of factors favouring tumour progression. compared with the current approaches, and these include the Manda et al. investigated the expression of EGFR and EGFRvIII ability to repeat sampling over the course of treatment in a non- in serum exosomes and tumour tissue in 96 patients with high- invasive manner, and the fact that the BBB may be more grade glioma. They detected EGFRvIII in 39.5% of tumour tissue permeable in the presence of a high-grade tumour, allowing samples and in 44.7% of their paired serum exosome samples, molecular transportation. A liquid biopsy may be able to reveal 133,134 whereas 28.1% of tumour biopsy samples had EGFR and EGFRvIII tumour information prior to clinical progression. However, co-expression. Although the co-expression of EGFR and EGFRvIII is the tumour morphological features and the microenvironment are rare in GBM cells, this co-expression is suggested to cooperate more readily available in the tissue biopsy. Therefore, a liquid with tumour growth and induce macrophage inﬁltration. biopsy aims to provide additional and complementary data to Also, the presence of EGFRvIII in exosomes correlated with improve upon the diagnosis and follow-up of GBM patients. a lower overall survivor pattern—21.1 months—compared with CTCs, ctDNA and MVs have been demonstrated to be able to 28.6 months for patients with no EGFRvIII expression in be sampled from different bioﬂuids for a number of tumour exosomes. types, and studies have demonstrated that these biomarkers can Chandran et al. reported that syndecan-1 found in plasma be found in GBM patients, and that their mutational proﬁles EVs can be used to distinguish low-grade glioma from high-grade represent those of the GBM in origin. There is a pressing need to GBM with a sensitivity of 71% and a speciﬁcity of 80%, and improve the technologies involved in regularly and reliably provided strong support for plasma–EV-derived syndecan-1 being isolating and characterising these biomarkers, and larger studies derived from GBM tumours. Yang et al. isolated exosomes from in GBM investigating these biomarkers are warranted, with tumours generated in mice by using four GBM patient-derived clinical correlatives measured over time to determine the effects samples collected during surgery, and reported an increase in the on clinical outcome. Unlike for many other tumour types, the expression of the genes encoding dynamin-3, p65 and CD117, use of CTCs in GBM as a diagnostic screening tool is not ideal, alongside a decrease in the expression of the genes encoding because by the time a patient with GBM experiences clinical PTEN and p53, in the tumour tissue and blood of mice. In another symptoms and receives a positive diagnosis from a treating study of 60 glioma patients, including 27 diagnosed with GBM, physician, their disease is already at an advanced stage. miRNA was detected in exosomes isolated from the serum by However, the liquid biopsy approach shows great potential in centrifugation and quantitated by using real-time PCR. The managing GBM patients. authors found that in comparison with low-grade gliomas, miR- Currently, no clinically validated circulating biomarkers for 301a levels were higher in high-grade gliomas. They also observed managing GBM patients exist. One reason for the relative lack of that the serum exosomal miR-301a levels were lower after surgical circulating biomarkers in this ﬁeld is because of the BBB, resection of the tumour, but were increased during GBM restricting the transportation of molecules from blood to the recurrence, indicating that serum exosomal miR-301a could be a brain and vice versa. Along with biological difﬁculties, there are potential biomarker for diagnosis/prognosis for GBM technical limitations for the establishment of a role for CTCs in 127,128 129 patients. Ebrahimkhani et al. used a panel of seven GBM. Only a few studies have been carried out by using brain- exosomal miRNAs—miR-182-5p, miR-328-3p, miR-339-5p, miR- tumour-derived CTCs, and they show that the detection rates vary 340-5p, miR-485-3p, miR-486-5p and miR-543—to differentiate from 20 to 77% in GBM patients, depending on the CTC isolation GBM patients from healthy controls with an accuracy rate of techniques used. However, other studies do warrant further 91.7%. In addition, Santangelo et al. analysed a miRNA signature investigation of CTCs in GBM. For example, the ﬁrst report in exosomes from the serum of glioma patients in an attempt to detecting CTC clusters in GBM, published in 2018, also indicates differentiate tumour grading and gliomas from brain metastases. the capacity of GBM clusters to cross the BBB. This is an The authors demonstrated the upregulation of three miRNAs, miR- important clinical ﬁnding that requires large studies to test the 21, miR-222 and miR-124-3p in glioma. miR-21 is known to play a reproducibility of these data. role in GBM pathogenesis. In their study, miR-21 differentiated The detection rates for ctDNA in GBM patients’ blood also vary healthy controls from glioma patients, but could not distinguish (10–55%), highlighting the need for more studies with larger Circulating biomarkers in patients with glioblastoma J Müller Bark et al. Table 1. Summary of advantages and disadvantages of using CTCs, ctDNA and exosomes as biomarkers in cancer Advantages Disadvantages Reference 9 25,28,47,75 CTCs � Information can be provided at the protein, DNA and � CTCs are rare (1 cell in 10 blood cells) RNA levels � Possibility of carrying out functional assays � May represent only part of the tumour mass heterogeneity � There are new technologies in the development � Process to isolate them is challenging phase to isolate CTCs 23,25,46,75 ctDNA � Higher ctDNA levels compared with CTC � Short half-life, <1.5 h � Levels correlate with disease stage � Released mainly by cells undergoing necrosis or apoptosis � Easy detection 25,115,135 Exosomes � Can be released by all cells, including tumour cells � The release is not exclusive from tumour cells � Can carry proteins, DNA, RNA and miRNA � Possible presence of contaminants by current isolation methods � Present protection for their content cohorts to better understand ctDNA in GBM. GBM patients often AUTHOR CONTRIBUTIONS J.M.B., A.K., B.C., B.W.D. and C.P. all contributed to the writing of the paper. develop resistance to treatment. Monitoring patients over the course of treatment, by serial sampling, and detecting speciﬁc tumour mutations and changes in DNA methylation pattern, ADDITIONAL INFORMATION might prove valuable for understanding tumour behaviour. These Competing interests: The authors declare no competing interests. parameters might complement the current conventional meth- odologies used in managing GBM patients. Moreover, multiple Ethical approval and consent to participate: Not applicable collections would enable tumour progression to be monitored, or pseudoprogression to be detected in a minimally invasive manner. Funding: J.M.B. is funded by ATM LATAM QUT Postgraduate Research Scholarship. When different bioﬂuid sources are compared—for example, ctDNA detected in blood or in CSF—CSF appears to be more representative, possibly owing to the proximity of CSF to the Consent to publish: Not applicable brain. Nevertheless, CSF collection is much more invasive and risky compared with blood collection. Data availability: Not applicable The exosome ﬁeld in GBM is also emerging and has been producing promising data, such as detection of the EGFRvIII Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. deletion variant in tumour tissue (39.5%) matching with EGFRvIII expression in exosomes (44.7%), and in both cases correlating with poor survival. However, some technical limitations also need to be addressed in the future for this ﬁeld. In addition, the cohort REFERENCES size of the majority of the studies is small, and currently there are 1. Louis, D. 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Circulating biomarkers in patients with glioblastoma

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Circulating biomarkers in patients with glioblastoma

Circulating biomarkers in patients with glioblastoma

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