Blood-based biomarkers for the diagnosis and monitoring of gliomas

Blood-based biomarkers for the diagnosis and monitoring of gliomas Abstract Monitoring patient response to treatment is challenging for most cancers, but it is particularly difficult in glioblastoma multiform, the most common and aggressive form of malignant brain tumor. These tumors exhibit a high degree of heterogeneity which may not be reflected in a biopsy. To determine if the current standard of care is effective, glioma patients are monitored using MRI or CT scans, an effective but sometimes misleading approach due to the phenomenon of pseudoprogression. As such, there is incredible need for a minimally invasive “liquid biopsy” to assist in molecularly characterizing the tumors while also aiding in the identification of true progression in glioblastoma. This review details the status and potential impact for circulating tumor cells, extracellular vesicles, ctDNA, and ctRNA, putative circulating biomarkers found in the blood in glioblastoma patients. As mutation-based therapy becomes more prevalent in gliomas, blood-based analyses may offer a non-invasive method of identifying mutations. The ability to obtain serial “liquid biopsies” will provide unique opportunities to study the evolution of tumors and mechanisms of treatment resistance and monitor for mutational changes in response to therapy. CTCs, ctDNA, ctRNA, extracellular vesicles, liquid biopsy Advances in the mutational classification of brain tumors have led to a more targeted management of gliomas tailored to individual patients’ mutations.1,2 Patients must undergo surgery for tissue diagnosis, which has risks and is limited to mutational analysis at a single time point. After therapy, therapeutic response and recurrence are monitored using magnetic resonance imaging (MRI), which does not reliably distinguish tumor progression from radiation necrosis. As such, there is a great need for tools to diagnose, molecularly characterize, and determine treatment response while minimizing the morbidity associated with surgery and improving sensitivity and specificity compared with current imaging-based technologies. Recent advances in blood-based analyses, including circulating tumor cells, circulating DNA, and circulating extracellular vesicles, have demonstrated significant promise to address this need.3 Brain tumors have been characterized classically by histopathologic evaluation and more recently by genetic information obtained via molecular analysis. Given the heterogeneity of gliomas, a biopsy may not reflect the entire tumor. Gerlinger et al demonstrated that 60%–70% of mutations identified were not present across all regions of a tumor,4 and therefore individual biopsies often missed mutations present elsewhere. In addition to regional heterogeneity, scarcity of tissue may limit histopathologic and genetic analyses. Brain tumors may be difficult to access, and surgery may have morbidity. Processing of tissue samples, including fixation and paraffin embedding, may limit subsequent molecular analysis. Finally, tissue biopsy limits tumor analysis to a single point in time, whereas tumors constantly evolve potentially in response to treatment.5 Liquid biopsy, referring to analysis of patient liquid specimens such as blood and cerebrospinal fluid (CSF) in lieu of surgical biopsy specimens, offers the promise of diagnosis and mutational analysis in a less invasive manner. The use of liquid biopsies as a complement to tumor biopsy analysis offers advantages in confirming diagnosis, identifying mutations present, and monitoring tumor evolution and response to therapy (Table 1).6 Table 1 Sensitivity and specificity of circulating biomarkers reported by recent studies focusing on gliomas Study Biomarker Sensitivity Specificity Sullivan et al10 CTCs 39.4% 100% Muller et al11 CTCs 20.6% 96.6% Macarthur et al12 CTCs 72% pre-radiotherapy; 8% post-radiotherapy 100% Figueroa et al41 EVs 61% 98% Boisselier et al57 ctDNA 60% 100% Lavon et al58 ctDNA 51% 100% Majchrzak-Celińska et al59 ctDNA 81% 97% Study Biomarker Sensitivity Specificity Sullivan et al10 CTCs 39.4% 100% Muller et al11 CTCs 20.6% 96.6% Macarthur et al12 CTCs 72% pre-radiotherapy; 8% post-radiotherapy 100% Figueroa et al41 EVs 61% 98% Boisselier et al57 ctDNA 60% 100% Lavon et al58 ctDNA 51% 100% Majchrzak-Celińska et al59 ctDNA 81% 97% View Large Table 1 Sensitivity and specificity of circulating biomarkers reported by recent studies focusing on gliomas Study Biomarker Sensitivity Specificity Sullivan et al10 CTCs 39.4% 100% Muller et al11 CTCs 20.6% 96.6% Macarthur et al12 CTCs 72% pre-radiotherapy; 8% post-radiotherapy 100% Figueroa et al41 EVs 61% 98% Boisselier et al57 ctDNA 60% 100% Lavon et al58 ctDNA 51% 100% Majchrzak-Celińska et al59 ctDNA 81% 97% Study Biomarker Sensitivity Specificity Sullivan et al10 CTCs 39.4% 100% Muller et al11 CTCs 20.6% 96.6% Macarthur et al12 CTCs 72% pre-radiotherapy; 8% post-radiotherapy 100% Figueroa et al41 EVs 61% 98% Boisselier et al57 ctDNA 60% 100% Lavon et al58 ctDNA 51% 100% Majchrzak-Celińska et al59 ctDNA 81% 97% View Large Circulating Tumor Cells Cells that have left the primary tumor site and entered the blood are termed circulating tumor cells (CTCs). While long considered to be present in the blood of metastatic patients,7,8 recent advances in technology have led to the capture, identification, and analysis of CTCs.9–12 CTCs are exceedingly rare compared with the background of normal circulating cells and are thought to contain the genetic material of the parental tumor.13 CTCs have been identified in melanoma, breast, lung, and prostate cancers, as well as more recently in gliomas.10–12 CTCs were identified in 39% of GBM patients and could be analyzed to identify epidermal growth factor receptor (EGFR) amplification in the primary tumor.10 CTCs are believed to represent a portrait of tumor subclones, and several studies showed that even at a single cell level, CTCs can retain specific characteristics from the primary tumor, such as mutations and gene expression signatures.10–12 In terms of CTC detection, several methods are available for CTC detection and enumeration, including epithelial cell adhesion molecule (EpCAM)–based capture, size-based filtration, microfluidic devices, magnetic cell sorters, acoustic-based separation, and even complex systems using combinations of these technologies.14–18 CTCs may be obtained via positive selection (eg, EpCAM-based selection) or negative selection (eg, depletion of white blood cells), with both approaches possessing advantages and disadvantages.19 CTCs may represent an important source of tumor information when primary tumor is no longer available or when there is a technical challenge sampling the primary lesion, as is often the case in brain tumors. In gliomas, several independent groups have reported the presence of CTCs in blood samples.11,12,19 Sullivan et al described the detection of glioblastoma (GBM) CTCs using CTC-iChip technology, which includes a combination of microfluidic flow manipulations, including hydrodynamic flow sorting, to remove small particles, inertial flow to align and organize nucleated cells, and finally the removal of contaminating white blood cells using magnetophoresis.10 The authors detected CTCs in the peripheral blood of 39.3% (13/33) of patients with GBM. The majority of the CTCs showed a signature of aggressive mesenchymal GBM subtype, with high expression of SERPINE1, TGFB1, TGFBR2, and VIM. Müller and collaborators employed differential centrifugation with Ficoll-Paque gradients followed by fluorescence immunocytochemistry using glial fibrillary acidic protein as a putative marker for CTCs.11 CTCs were found in 20.6% of patients (29 of 141), and the presence of CTCs was correlated with EGFR amplification. Macarthur and colleagues described the detection of GBM CTCs using nestin and human telomerase as markers.12 CTCs were detected in 71% of patients and were predictive of disease progression. Challenges limiting widespread clinical utilization of circulating tumor cell assays for GBM include the complex combination of technologies required and to the sensitivity of assays. GBM CTC isolation technology has incredible promise in brain tumors, as with other cancers. However, additional studies are needed to demonstrate the clinical significance of CTCs during a patient’s disease course. Currently, CTC capture from blood can be adversely affected by time from blood draw; this may complicate efforts to scale CTC capture to a clinical level, and efforts are under way to mitigate these limitations. The reported sensitivities vary between 20.6% and 71%, and it would be important to establish higher sensitivity prior to clinical usage. Ongoing and future work may address these issues. Extracellular Vesicles Extracellular vesicles (EVs) are small lipid bilayer enclosed vesicles released by both cancer and noncancer cells into the extracellular space.20 The most commonly studied categories of EVs include exosomes, which range from 30 to 100 nm in size, and microvesicles, which range from 100 to 1000 nm in size.21,22 EVs are shed via budding of plasma membrane or via fusion of intracellular vesicles with the plasma membrane.23 EVs secreted by tumor cells may be taken up by neighboring stromal cells, leading to alteration of cell program. This has been demonstrated in pancreatic, lung, prostate, breast, skin, and glial cancers.24–26 EVs may also be taken up by cells of the immune system, causing immunosuppression.27 More recently, EVs have gained a role in cancer diagnosis and therapy,28,29 allowing the identification of BRAF, KRAS, or EGFR mutations in a variety of primary tumors with high sensitivity and specificity.30 EVs possess a very short half-life in vivo, making them suitable to track rapid changes in tumor biology.31 Gliomas have been demonstrated to release EVs which transfer tumorigenic potential to other tumor cells, interact with endothelial cells to promote angiogenesis, and act in an autocrine fashion to stimulate GBM tumor cell growth.32–34 Glioblastoma EVs contain many of the same transcripts as primary tumor cells and can be used to detect EGFR variant III (EGFRvIII) transcripts and miRNAs.32,35 EGFRvIII is a frequent activating mutation in EGFR in gliomas which generates a truncated version of the EGFR protein, with an in-frame deletion of exons 2–7.36 EGFRvIII is a constitutively active form of EGFR that is able to activate downstream targets of EGFR, without the need of an increase in EGF levels.37 Furthermore, EGFRvIII protein carries a unique peptide sequence generated by the fusion of exons 1 and 8 that has been shown to serve as a tumor-specific target for immunotherapy approaches, including chimeric antigen receptor T cells.38 The transfer of EVs from GBM cells to microglia has been visualized in vitro and in a mouse model of GBM using 2-photon microscopy.39 Glioblastoma cells expressing EGFRvIII may transfer this receptor variant to nearby tumor cells that do not endogenously express the variant, thereby promoting tumor progression.33 Radiation promotes the release of EVs from GBM cells that promote a migratory phenotype upon uptake by recipient GBM cells.34 Glioma exosomes carry a variety of cargo.22 EV-associated microRNAs (including miR-451, miR-21, miR-29a, miR-222, miR-30a, miR-92b, miR-221, and miR-23a) may promote proliferation and resistance to apoptosis.22,40,41EGFRvIII mRNA and protein may support constitutively active signaling via AKT and mitogen-activated protein kinase (MAPK) pathways and thereby drive tumor progression.32 O6-methylguanine-DNA methyltransferase (MGMT) and alkylpurine DNA n-glycosylase (APNG) mRNA contained within EVs may promote resistance to chemotherapy. Notably, mRNA levels within EVs correlated with mRNA levels in cells from which the EVs were derived.42 Chen et al described the use of sensitive polymerase chain reaction (PCR) assays to detect isocitrate dehydrogenase 1 (IDH1) wild-type and mutant mRNA in EVs obtained from the CSF of patients with gliomas.43 Figueroa et al reported a notable multicenter study including 71 GBM patients where extracellular vesicles derived from CSF were obtained and analyzed for EGFRvIII status and EGFR amplification and compared with tissue from the primary tumor.44 They demonstrated a sensitivity of 61% and a specificity of 98% when using EVs to assess EGFRvIII status of the primary tumor. The majority of the research performed thus far on extracellular vesicles for GBM focuses on tumor biology or proof of principle and is not yet ready for clinical application. However, 2 of the areas with the most future promise include evaluation of EGFRvIII and IDH1 in exosomes. For EGFRvIII, 2 groups have achieved a high specificity of detection within exosomes derived from CSF.43,44 Notably, one study was performed in multiple centers with a total of 71 patients.44 However, sensitivity (<70%) would need to be improved. Similarly, IDH1 was detected in exosomes derived from CSF with a high specificity (100%) and intermediate sensitivity (62.5%) in a total of 14 patients.43 While the clinical implications of determining IDH1 status via CSF sampling are exciting, further work prior to clinical use should focus on improvement of sensitivity and inclusion of a larger number of patients. Circulating DNA The first description of circulating free DNA (cfDNA) dates to the 1940s, when Mandel and Metais described the presence of circulating nucleic acids in healthy individuals.45 Today, cfDNA is appreciated as an important source of information in diverse physiological and pathological states, such as exercise, trauma, surgery, stroke, renal failure, and cancer.46–48 Given the abundance of cfDNA released by normal cells, it is important to distinguish circulating tumor DNA (ctDNA) from cfDNA. Strategies targeting somatic mutations that are characteristic of certain tumors and not present in healthy individuals are often used to differentiate ctDNA from cfDNA.49 Given the technical challenge involved in identifying ctDNA, current methodologies merit attention. Next-generation sequencing and digital PCR technologies may be used to detect ctDNA among the background of DNA sloughed from normal cells.50–53 Next-generation sequencing technologies involve cutting DNA into short segments, which are separated spatially and sequenced in parallel using fluorescence or other readouts for each base.50 Next-generation sequencing is rapid and less expensive than Sanger sequencing.54 In analysis of circulating nucleic acids, next-generation sequencing offers the benefit of being able to detect mutations without knowing exactly what they are and being able to detect a large number of possible mutations in a particular gene. Digital droplet PCR (ddPCR) technologies involve partition of a sample into many subsamples, each of which undergoes an individual PCR reaction that reads out as positive or negative.52,55 This readout undergoes a transformation based on the Poisson distribution, giving the absolute number of copies of nucleic acid present in the original sample. Digital droplet PCR is extremely sensitive and specific, able to work with smaller amounts of nucleic acid than next-generation sequencing, even in samples with degradation or high wild-type background.52 Digital PCR is faster and more cost effective than next-generation sequencing.54 However, digital PCR may not be able to detect all possible mutations, due to specific location of some single nucleotide variants that can interfere with the primers and probe to bind to the mutation site. With multiplexing, up to 4 assays may be run at once within a particular sample (usually allowing the detection of up to 4 possible distinct mutations per gene), whereas next-generation sequencing may be run in a massively parallel fashion.50 In upcoming clinical assays, digital PCR and next-generation sequencing will likely both be employed depending on the context.56 Digital PCR will be the preferred method in the setting of known specific mutations (eg, IDH1 p.R132H) and will be used to confirm mutations identified via next-generation sequencing. Next-generation sequencing will be used when there are a vast number of possible mutations in a particular gene or when there is suspicion of mutations that are not detected by known digital PCR primers. Next-generation sequencing and digital PCR have been employed successfully to detect ctDNA in a variety of cancer types, allowing the identification of clinically actionable mutations and the understanding of mechanisms underlying resistance to therapy.57 Lebofsky et al performed de novo detection of somatic mutations using plasma DNA compared with metastasis biopsies in 34 patients with a variety of nonglial cancers.58 Of the 34 patients examined, 27 underwent biopsies that produced enough material to evaluate mutations. Among the 27 patients with successful biopsies, 97% of mutations identified by biopsy were also identified by evaluation of ctDNA. There was one mutation identified by ctDNA that was not seen on biopsy. Interestingly, ctDNA demonstrated mutations in the 7 patients with a failed biopsy. These results suggest that in the evaluated subset of patients, ctDNA may be nearly as effective at identifying tumor mutations as biopsy. In another study, comprising 18 patients with colorectal cancer, Diehl et al compared ctDNA with carcinoembryonic antigen (CEA), the gold standard biomarker for disease progression. Interestingly, levels of ctDNA correlated with complete versus incomplete resection after surgery. Levels of ctDNA decreased by 99% on average in complete resections, whereas incomplete resection led to a slight decrease or increase in ctDNA levels. Notably, ctDNA level predicted recurrent disease more accurately than did CEA level.58 These studies suggest that ctDNA holds great promise for eventual clinical application in some nonglial cancers. The use of ctDNA for detection of mutations in gliomas has been more challenging. Bettegowda et al employed digital PCR to identify ctDNA in 640 patients with various cancer types.59 Circulating tumor DNA was detectable in >75% of patients with advanced pancreatic, ovarian, colorectal bladder, melanoma, and head and neck cancer but was detectable in only <10% of patients with gliomas (n = 27). Finally, the authors examined a subset of 206 patients with colorectal cancer. KRAS mutations could be detected by ctDNA with a sensitivity of 87.2% and a specificity of 99.2%. Interestingly, ctDNA identified mutations that could mechanistically explain development of resistance to EGFR blockade in 23 of 24 patients who objectively responded to therapy but subsequently relapsed. Several groups have focused on ctDNA specifically in glioma patients.60–62 Boisselier et al employed a variant of digital PCR to evaluate IDH mutations based on ctDNA in glioma patients, detecting IDH1 p.R132H mutations in 15 out of 25 patients with mutated tumors (60%) and in none of the 14 patients with wild-type tumors.60 The sensitivity increased with enhancing tumor volume (6/6 for enhancing volume ≥10 cm3). Specificity was 100% (zero of 14 potential false positives). Lavon et al examined a cohort of 70 patients with high-grade gliomas or oligodendrogliomas.61 Examining MGMT promoter methylation or loss of heterozygosity in chromosome 1p, 19q, and 10q, they identified tumor-specific cfDNA alteration in 51% of gliomas and 55% of oligodendrogliomas. When including only patients with measurable tumor by MRI near the time of serum collection, the rate of detection was 76% for both glial and oligoglial tumors. Of note, no tumor-specific cfDNA alterations were identified in healthy controls. Similarly, the Majchrzak-Celińska group analyzed methylation status in MGMT, RASSF1A, p15INK4B, and p14ARF in serum ctDNA of 33 patients with newly diagnosed CNS tumors, including 17 gliomas, 6 meningiomas, and 10 metastatic malignancies.62 Of the 17 ctDNA samples from glioma patients, 12 (71%) demonstrated methylation in at least 1 of the 4 tumor suppressor genes: MGMT (18%), RASSF1A (47%), p15INK4B (12%), and p14ARF (41%). Compared with the tissue gold standard, ctDNA identified hypermethylation with a sensitivity of 81% and specificity of 97% overall. In conclusion, recent work has demonstrated progress in identifying promoter hypermethylation and mutations via ctDNA analysis of glioma patients. Tumor-derived DNA may be identified from CSF as well as from the blood. Wang et al described the detection of tumor DNA from CSF samples in 35 patients with gliomas and other primary tumors of the CNS and coined the term “CSF-tDNA” (CSF-derived tumor DNA).63 The authors employed next-generation sequencing of primary tumor samples to identify mutations present. Guided by this analysis, the authors searched for these mutations in the CSF of the same patients. CSF-tDNA was identified in 74% of cases. Interestingly, CSF-tDNA was detectable in all patients with medulloblastomas, ependymomas, and high-grade gliomas that abutted a CSF space. Of note, the authors’ method involved initial identification of mutations from primary tumor samples, and therefore the method would be useful for tracking recurrence after surgery but not for initial diagnosis and molecular characterization. Prior to clinical use, further work would be necessary to establish specificity and to evaluate this method’s ability to distinguish between recurrent tumor and radiation necrosis. Two additional papers reported identification of mutations associated with specific brain tumors in CSF. Bing–Neel syndrome and primary central nervous system lymphoma (PCNSL) are associated with the MYD88 p.L265P mutation. Hiemcke-Jiwa et al validated ddPCR as a method of identifying MYD88 p.L265P mutation in formalin-fixed, paraffin-embedded brain tumor specimens.64 Next, they isolated DNA and performed ddPCR on CSF samples from 9 patients with clinical suspicion of Bing–Neel syndrome and 3 patients with PCNSL. MYD88 p.L265P mutations were identified in 8 of 9 patients with clinical suspicion of Bing–Neel syndrome and in 1 of 3 patients with PCNSL. Prior to widestream use, specificity would need to be evaluated with CSF from nontumor control patients and sensitivity would need to be improved for PCNSL. Histone H3K27M mutations are associated with diffuse midline glioma. Huang et al were able to isolate DNA from CSF samples taken from 5 children with diffuse midline glioma, and histone H3K27M mutations were identified in CSF from 4 of the 5 children.65 The authors report a sensitivity of 87.5% and specificity of 100%. While these results are quite promising, larger clinical studies would be required prior to entry into the clinic. In summary, ctDNA is most commonly detected using next-generation sequencing and digital PCR technologies.50–53 Blood-based detection of ctDNA has been demonstrated to be sensitive and specific in a variety of nonglial cancers.48,57,58 In gliomas, blood-based detection of ctDNA is specific but not nearly as sensitive (10%–60%) as in other cancers.59–62 Interestingly, higher sensitivity is seen with large enhancing tumor volume.60 Identification of glioma ctDNA has been performed with higher sensitivity using CSF (33%–89%) compared with blood.63–65 Areas that appear most promising for clinical use include detection from CSF (rather than blood) and the subset of patients with large enhancing tumors. Larger trials should be performed, and sensitivity will need to be improved. Circulating MicroRNA Circulating microRNA (miRNA) is another circulating biomarker that has attracted attention. Ranging in length from 21 to 24 nucleotides, miRNAs represent small noncoding RNA molecules that possess an important regulatory role in transcription in both normal and cancer cells.66 While miRNAs regulate transcription within the cell of origin, recent evidence points to an important role for miRNAs in intercellular communication as pro- and anti-oncogenic regulators and immunosuppressors.67,68 Analysis of miRNA accurately identified cancer tissue origin in a variety of cancers.69 Several recent studies have examined miRNA from CSF70 and blood71–75 as a diagnostic tool in gliomas. Dong et al examined miRNA in the serum of 3 patients with GBM and 3 age- and sex-matched healthy controls.72 They identified 115 miRNAs that were significantly upregulated and 24 miRNAs that were significantly downregulated in the serum of GBM patients but not healthy controls. Similarly, Tang et al demonstrated that the level of circulating miR-185 was significantly higher in 66 glioma patients compared with normal controls.73 Interestingly, levels of circulating miR-185 returned to normal levels after surgery and chemoradiation. Additionally, Lai et al demonstrated a 7-fold increase in circulating miR-210 levels in 210 patients with GBM compared with healthy controls,74 whereas Yue et al showed decreased levels of circulating miR-205 in 64 patients with glioma.75 Notably both studies demonstrated a correlation between level of the miRNA studied and outcome. Examining pediatric gliomas, López-Aguilar et al found that circulating miR-130a and miR-145 were significantly elevated, whereas miR-335 and miR-1303 were significantly decreased compared with healthy controls.71 These results suggest that circulating miRNAs hold early promise as tools for the diagnosis and monitoring of gliomas. Current State of Circulating Biomarkers While a great deal of optimism surrounds circulating biomarkers as a means of liquid biopsy for gliomas, technical limitations need to be addressed prior to clinical use. Current techniques allow isolation of CTCs with a sensitivity of 20%–72% of GBM patients.10–12 EVs allowed for the determination of IDH1 status when isolated from CSF but not when isolated from blood.43EGFR amplification was identified using circulating EVs with a sensitivity of 61% and a specificity of 98%.44 Analysis of ctDNA has a sensitivity of 51%–81%60–62 and specificity as high as 97% but has been shown to have a low detection frequency in patients with gliomas. Evaluation of circulating miRNAs identified several candidate biomarkers for glioma diagnosis and monitoring71–75; however, further evaluation of sensitivity and specificity must be done. Sensitivity in particular needs to be addressed for all types of liquid biopsy prior to general clinical use. Future Promise Blood-based analyses will be critical in the diagnosis of inoperable tumors, where safe biopsy may be difficult or impossible. As mutation-based therapy becomes more prevalent in gliomas, blood-based analyses may offer a non-invasive method of identifying mutations. The ability to obtain serial “liquid biopsies” will provide unique opportunities to study the evolution of tumors and mechanisms of treatment resistance and monitor for mutational changes in response to therapy. Combination of techniques (eg, ctDNA in combination with EVs) may allow for improved sensitivity and specificity.30,76,77 Importantly, patients who undergo chemotherapy and radiation frequently develop increase in enhancement on serial MRI examinations, which represents progression versus radiation necrosis (also known as pseudoprogression). Using current imaging technologies, tumor progression and radiation necrosis cannot be reliably differentiated, often requiring operation for diagnosis. A blood-based method of differentiating the 2 diagnoses would be very valuable in this subset of patients. Finally, a combination of blood-based techniques may improve the sensitivity and specificity for liquid biopsy and lead to a clinically useful tool that has the potential to diagnose, monitor, and identify mutations with the goal to identify patients who would benefit from new targeted therapies.30,76,77 Funding This work was supported by a US National Institutes of Health (NIH) Grant (EB002503), NIH National Institute of Biomedical Imaging and Bioengineering Grant (EB012493) and the Wang Pediatric Brain Tumor Collaborative (to B.V.N. and S.L.S.). Conflict of interest statement. None declared. References 1. Louis DN , Perry A , Reifenberger G , et al. The 2016 World Health Organization classification of tumors of the central nervous system: a summary . Acta Neuropathol . 2016 ; 131 ( 6 ): 803 – 820 . Google Scholar CrossRef Search ADS PubMed 2. 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Improved EGFR mutation detection using combined exosomal RNA and circulating tumor DNA in NSCLC patient plasma . Ann Oncol . 2018 ; 29 ( 3 ): 700 – 706 . Google Scholar CrossRef Search ADS PubMed 77. Akers JC , Hua W , Li H , et al. A cerebrospinal fluid microRNA signature as biomarker for glioblastoma . Oncotarget . 2017 ; 8 ( 40 ): 68769 – 68779 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Neuro-Oncology Oxford University Press

Blood-based biomarkers for the diagnosis and monitoring of gliomas

Neuro-Oncology , Volume 20 (9) – Sep 1, 2018

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
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1522-8517
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1523-5866
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10.1093/neuonc/noy074
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Abstract

Abstract Monitoring patient response to treatment is challenging for most cancers, but it is particularly difficult in glioblastoma multiform, the most common and aggressive form of malignant brain tumor. These tumors exhibit a high degree of heterogeneity which may not be reflected in a biopsy. To determine if the current standard of care is effective, glioma patients are monitored using MRI or CT scans, an effective but sometimes misleading approach due to the phenomenon of pseudoprogression. As such, there is incredible need for a minimally invasive “liquid biopsy” to assist in molecularly characterizing the tumors while also aiding in the identification of true progression in glioblastoma. This review details the status and potential impact for circulating tumor cells, extracellular vesicles, ctDNA, and ctRNA, putative circulating biomarkers found in the blood in glioblastoma patients. As mutation-based therapy becomes more prevalent in gliomas, blood-based analyses may offer a non-invasive method of identifying mutations. The ability to obtain serial “liquid biopsies” will provide unique opportunities to study the evolution of tumors and mechanisms of treatment resistance and monitor for mutational changes in response to therapy. CTCs, ctDNA, ctRNA, extracellular vesicles, liquid biopsy Advances in the mutational classification of brain tumors have led to a more targeted management of gliomas tailored to individual patients’ mutations.1,2 Patients must undergo surgery for tissue diagnosis, which has risks and is limited to mutational analysis at a single time point. After therapy, therapeutic response and recurrence are monitored using magnetic resonance imaging (MRI), which does not reliably distinguish tumor progression from radiation necrosis. As such, there is a great need for tools to diagnose, molecularly characterize, and determine treatment response while minimizing the morbidity associated with surgery and improving sensitivity and specificity compared with current imaging-based technologies. Recent advances in blood-based analyses, including circulating tumor cells, circulating DNA, and circulating extracellular vesicles, have demonstrated significant promise to address this need.3 Brain tumors have been characterized classically by histopathologic evaluation and more recently by genetic information obtained via molecular analysis. Given the heterogeneity of gliomas, a biopsy may not reflect the entire tumor. Gerlinger et al demonstrated that 60%–70% of mutations identified were not present across all regions of a tumor,4 and therefore individual biopsies often missed mutations present elsewhere. In addition to regional heterogeneity, scarcity of tissue may limit histopathologic and genetic analyses. Brain tumors may be difficult to access, and surgery may have morbidity. Processing of tissue samples, including fixation and paraffin embedding, may limit subsequent molecular analysis. Finally, tissue biopsy limits tumor analysis to a single point in time, whereas tumors constantly evolve potentially in response to treatment.5 Liquid biopsy, referring to analysis of patient liquid specimens such as blood and cerebrospinal fluid (CSF) in lieu of surgical biopsy specimens, offers the promise of diagnosis and mutational analysis in a less invasive manner. The use of liquid biopsies as a complement to tumor biopsy analysis offers advantages in confirming diagnosis, identifying mutations present, and monitoring tumor evolution and response to therapy (Table 1).6 Table 1 Sensitivity and specificity of circulating biomarkers reported by recent studies focusing on gliomas Study Biomarker Sensitivity Specificity Sullivan et al10 CTCs 39.4% 100% Muller et al11 CTCs 20.6% 96.6% Macarthur et al12 CTCs 72% pre-radiotherapy; 8% post-radiotherapy 100% Figueroa et al41 EVs 61% 98% Boisselier et al57 ctDNA 60% 100% Lavon et al58 ctDNA 51% 100% Majchrzak-Celińska et al59 ctDNA 81% 97% Study Biomarker Sensitivity Specificity Sullivan et al10 CTCs 39.4% 100% Muller et al11 CTCs 20.6% 96.6% Macarthur et al12 CTCs 72% pre-radiotherapy; 8% post-radiotherapy 100% Figueroa et al41 EVs 61% 98% Boisselier et al57 ctDNA 60% 100% Lavon et al58 ctDNA 51% 100% Majchrzak-Celińska et al59 ctDNA 81% 97% View Large Table 1 Sensitivity and specificity of circulating biomarkers reported by recent studies focusing on gliomas Study Biomarker Sensitivity Specificity Sullivan et al10 CTCs 39.4% 100% Muller et al11 CTCs 20.6% 96.6% Macarthur et al12 CTCs 72% pre-radiotherapy; 8% post-radiotherapy 100% Figueroa et al41 EVs 61% 98% Boisselier et al57 ctDNA 60% 100% Lavon et al58 ctDNA 51% 100% Majchrzak-Celińska et al59 ctDNA 81% 97% Study Biomarker Sensitivity Specificity Sullivan et al10 CTCs 39.4% 100% Muller et al11 CTCs 20.6% 96.6% Macarthur et al12 CTCs 72% pre-radiotherapy; 8% post-radiotherapy 100% Figueroa et al41 EVs 61% 98% Boisselier et al57 ctDNA 60% 100% Lavon et al58 ctDNA 51% 100% Majchrzak-Celińska et al59 ctDNA 81% 97% View Large Circulating Tumor Cells Cells that have left the primary tumor site and entered the blood are termed circulating tumor cells (CTCs). While long considered to be present in the blood of metastatic patients,7,8 recent advances in technology have led to the capture, identification, and analysis of CTCs.9–12 CTCs are exceedingly rare compared with the background of normal circulating cells and are thought to contain the genetic material of the parental tumor.13 CTCs have been identified in melanoma, breast, lung, and prostate cancers, as well as more recently in gliomas.10–12 CTCs were identified in 39% of GBM patients and could be analyzed to identify epidermal growth factor receptor (EGFR) amplification in the primary tumor.10 CTCs are believed to represent a portrait of tumor subclones, and several studies showed that even at a single cell level, CTCs can retain specific characteristics from the primary tumor, such as mutations and gene expression signatures.10–12 In terms of CTC detection, several methods are available for CTC detection and enumeration, including epithelial cell adhesion molecule (EpCAM)–based capture, size-based filtration, microfluidic devices, magnetic cell sorters, acoustic-based separation, and even complex systems using combinations of these technologies.14–18 CTCs may be obtained via positive selection (eg, EpCAM-based selection) or negative selection (eg, depletion of white blood cells), with both approaches possessing advantages and disadvantages.19 CTCs may represent an important source of tumor information when primary tumor is no longer available or when there is a technical challenge sampling the primary lesion, as is often the case in brain tumors. In gliomas, several independent groups have reported the presence of CTCs in blood samples.11,12,19 Sullivan et al described the detection of glioblastoma (GBM) CTCs using CTC-iChip technology, which includes a combination of microfluidic flow manipulations, including hydrodynamic flow sorting, to remove small particles, inertial flow to align and organize nucleated cells, and finally the removal of contaminating white blood cells using magnetophoresis.10 The authors detected CTCs in the peripheral blood of 39.3% (13/33) of patients with GBM. The majority of the CTCs showed a signature of aggressive mesenchymal GBM subtype, with high expression of SERPINE1, TGFB1, TGFBR2, and VIM. Müller and collaborators employed differential centrifugation with Ficoll-Paque gradients followed by fluorescence immunocytochemistry using glial fibrillary acidic protein as a putative marker for CTCs.11 CTCs were found in 20.6% of patients (29 of 141), and the presence of CTCs was correlated with EGFR amplification. Macarthur and colleagues described the detection of GBM CTCs using nestin and human telomerase as markers.12 CTCs were detected in 71% of patients and were predictive of disease progression. Challenges limiting widespread clinical utilization of circulating tumor cell assays for GBM include the complex combination of technologies required and to the sensitivity of assays. GBM CTC isolation technology has incredible promise in brain tumors, as with other cancers. However, additional studies are needed to demonstrate the clinical significance of CTCs during a patient’s disease course. Currently, CTC capture from blood can be adversely affected by time from blood draw; this may complicate efforts to scale CTC capture to a clinical level, and efforts are under way to mitigate these limitations. The reported sensitivities vary between 20.6% and 71%, and it would be important to establish higher sensitivity prior to clinical usage. Ongoing and future work may address these issues. Extracellular Vesicles Extracellular vesicles (EVs) are small lipid bilayer enclosed vesicles released by both cancer and noncancer cells into the extracellular space.20 The most commonly studied categories of EVs include exosomes, which range from 30 to 100 nm in size, and microvesicles, which range from 100 to 1000 nm in size.21,22 EVs are shed via budding of plasma membrane or via fusion of intracellular vesicles with the plasma membrane.23 EVs secreted by tumor cells may be taken up by neighboring stromal cells, leading to alteration of cell program. This has been demonstrated in pancreatic, lung, prostate, breast, skin, and glial cancers.24–26 EVs may also be taken up by cells of the immune system, causing immunosuppression.27 More recently, EVs have gained a role in cancer diagnosis and therapy,28,29 allowing the identification of BRAF, KRAS, or EGFR mutations in a variety of primary tumors with high sensitivity and specificity.30 EVs possess a very short half-life in vivo, making them suitable to track rapid changes in tumor biology.31 Gliomas have been demonstrated to release EVs which transfer tumorigenic potential to other tumor cells, interact with endothelial cells to promote angiogenesis, and act in an autocrine fashion to stimulate GBM tumor cell growth.32–34 Glioblastoma EVs contain many of the same transcripts as primary tumor cells and can be used to detect EGFR variant III (EGFRvIII) transcripts and miRNAs.32,35 EGFRvIII is a frequent activating mutation in EGFR in gliomas which generates a truncated version of the EGFR protein, with an in-frame deletion of exons 2–7.36 EGFRvIII is a constitutively active form of EGFR that is able to activate downstream targets of EGFR, without the need of an increase in EGF levels.37 Furthermore, EGFRvIII protein carries a unique peptide sequence generated by the fusion of exons 1 and 8 that has been shown to serve as a tumor-specific target for immunotherapy approaches, including chimeric antigen receptor T cells.38 The transfer of EVs from GBM cells to microglia has been visualized in vitro and in a mouse model of GBM using 2-photon microscopy.39 Glioblastoma cells expressing EGFRvIII may transfer this receptor variant to nearby tumor cells that do not endogenously express the variant, thereby promoting tumor progression.33 Radiation promotes the release of EVs from GBM cells that promote a migratory phenotype upon uptake by recipient GBM cells.34 Glioma exosomes carry a variety of cargo.22 EV-associated microRNAs (including miR-451, miR-21, miR-29a, miR-222, miR-30a, miR-92b, miR-221, and miR-23a) may promote proliferation and resistance to apoptosis.22,40,41EGFRvIII mRNA and protein may support constitutively active signaling via AKT and mitogen-activated protein kinase (MAPK) pathways and thereby drive tumor progression.32 O6-methylguanine-DNA methyltransferase (MGMT) and alkylpurine DNA n-glycosylase (APNG) mRNA contained within EVs may promote resistance to chemotherapy. Notably, mRNA levels within EVs correlated with mRNA levels in cells from which the EVs were derived.42 Chen et al described the use of sensitive polymerase chain reaction (PCR) assays to detect isocitrate dehydrogenase 1 (IDH1) wild-type and mutant mRNA in EVs obtained from the CSF of patients with gliomas.43 Figueroa et al reported a notable multicenter study including 71 GBM patients where extracellular vesicles derived from CSF were obtained and analyzed for EGFRvIII status and EGFR amplification and compared with tissue from the primary tumor.44 They demonstrated a sensitivity of 61% and a specificity of 98% when using EVs to assess EGFRvIII status of the primary tumor. The majority of the research performed thus far on extracellular vesicles for GBM focuses on tumor biology or proof of principle and is not yet ready for clinical application. However, 2 of the areas with the most future promise include evaluation of EGFRvIII and IDH1 in exosomes. For EGFRvIII, 2 groups have achieved a high specificity of detection within exosomes derived from CSF.43,44 Notably, one study was performed in multiple centers with a total of 71 patients.44 However, sensitivity (<70%) would need to be improved. Similarly, IDH1 was detected in exosomes derived from CSF with a high specificity (100%) and intermediate sensitivity (62.5%) in a total of 14 patients.43 While the clinical implications of determining IDH1 status via CSF sampling are exciting, further work prior to clinical use should focus on improvement of sensitivity and inclusion of a larger number of patients. Circulating DNA The first description of circulating free DNA (cfDNA) dates to the 1940s, when Mandel and Metais described the presence of circulating nucleic acids in healthy individuals.45 Today, cfDNA is appreciated as an important source of information in diverse physiological and pathological states, such as exercise, trauma, surgery, stroke, renal failure, and cancer.46–48 Given the abundance of cfDNA released by normal cells, it is important to distinguish circulating tumor DNA (ctDNA) from cfDNA. Strategies targeting somatic mutations that are characteristic of certain tumors and not present in healthy individuals are often used to differentiate ctDNA from cfDNA.49 Given the technical challenge involved in identifying ctDNA, current methodologies merit attention. Next-generation sequencing and digital PCR technologies may be used to detect ctDNA among the background of DNA sloughed from normal cells.50–53 Next-generation sequencing technologies involve cutting DNA into short segments, which are separated spatially and sequenced in parallel using fluorescence or other readouts for each base.50 Next-generation sequencing is rapid and less expensive than Sanger sequencing.54 In analysis of circulating nucleic acids, next-generation sequencing offers the benefit of being able to detect mutations without knowing exactly what they are and being able to detect a large number of possible mutations in a particular gene. Digital droplet PCR (ddPCR) technologies involve partition of a sample into many subsamples, each of which undergoes an individual PCR reaction that reads out as positive or negative.52,55 This readout undergoes a transformation based on the Poisson distribution, giving the absolute number of copies of nucleic acid present in the original sample. Digital droplet PCR is extremely sensitive and specific, able to work with smaller amounts of nucleic acid than next-generation sequencing, even in samples with degradation or high wild-type background.52 Digital PCR is faster and more cost effective than next-generation sequencing.54 However, digital PCR may not be able to detect all possible mutations, due to specific location of some single nucleotide variants that can interfere with the primers and probe to bind to the mutation site. With multiplexing, up to 4 assays may be run at once within a particular sample (usually allowing the detection of up to 4 possible distinct mutations per gene), whereas next-generation sequencing may be run in a massively parallel fashion.50 In upcoming clinical assays, digital PCR and next-generation sequencing will likely both be employed depending on the context.56 Digital PCR will be the preferred method in the setting of known specific mutations (eg, IDH1 p.R132H) and will be used to confirm mutations identified via next-generation sequencing. Next-generation sequencing will be used when there are a vast number of possible mutations in a particular gene or when there is suspicion of mutations that are not detected by known digital PCR primers. Next-generation sequencing and digital PCR have been employed successfully to detect ctDNA in a variety of cancer types, allowing the identification of clinically actionable mutations and the understanding of mechanisms underlying resistance to therapy.57 Lebofsky et al performed de novo detection of somatic mutations using plasma DNA compared with metastasis biopsies in 34 patients with a variety of nonglial cancers.58 Of the 34 patients examined, 27 underwent biopsies that produced enough material to evaluate mutations. Among the 27 patients with successful biopsies, 97% of mutations identified by biopsy were also identified by evaluation of ctDNA. There was one mutation identified by ctDNA that was not seen on biopsy. Interestingly, ctDNA demonstrated mutations in the 7 patients with a failed biopsy. These results suggest that in the evaluated subset of patients, ctDNA may be nearly as effective at identifying tumor mutations as biopsy. In another study, comprising 18 patients with colorectal cancer, Diehl et al compared ctDNA with carcinoembryonic antigen (CEA), the gold standard biomarker for disease progression. Interestingly, levels of ctDNA correlated with complete versus incomplete resection after surgery. Levels of ctDNA decreased by 99% on average in complete resections, whereas incomplete resection led to a slight decrease or increase in ctDNA levels. Notably, ctDNA level predicted recurrent disease more accurately than did CEA level.58 These studies suggest that ctDNA holds great promise for eventual clinical application in some nonglial cancers. The use of ctDNA for detection of mutations in gliomas has been more challenging. Bettegowda et al employed digital PCR to identify ctDNA in 640 patients with various cancer types.59 Circulating tumor DNA was detectable in >75% of patients with advanced pancreatic, ovarian, colorectal bladder, melanoma, and head and neck cancer but was detectable in only <10% of patients with gliomas (n = 27). Finally, the authors examined a subset of 206 patients with colorectal cancer. KRAS mutations could be detected by ctDNA with a sensitivity of 87.2% and a specificity of 99.2%. Interestingly, ctDNA identified mutations that could mechanistically explain development of resistance to EGFR blockade in 23 of 24 patients who objectively responded to therapy but subsequently relapsed. Several groups have focused on ctDNA specifically in glioma patients.60–62 Boisselier et al employed a variant of digital PCR to evaluate IDH mutations based on ctDNA in glioma patients, detecting IDH1 p.R132H mutations in 15 out of 25 patients with mutated tumors (60%) and in none of the 14 patients with wild-type tumors.60 The sensitivity increased with enhancing tumor volume (6/6 for enhancing volume ≥10 cm3). Specificity was 100% (zero of 14 potential false positives). Lavon et al examined a cohort of 70 patients with high-grade gliomas or oligodendrogliomas.61 Examining MGMT promoter methylation or loss of heterozygosity in chromosome 1p, 19q, and 10q, they identified tumor-specific cfDNA alteration in 51% of gliomas and 55% of oligodendrogliomas. When including only patients with measurable tumor by MRI near the time of serum collection, the rate of detection was 76% for both glial and oligoglial tumors. Of note, no tumor-specific cfDNA alterations were identified in healthy controls. Similarly, the Majchrzak-Celińska group analyzed methylation status in MGMT, RASSF1A, p15INK4B, and p14ARF in serum ctDNA of 33 patients with newly diagnosed CNS tumors, including 17 gliomas, 6 meningiomas, and 10 metastatic malignancies.62 Of the 17 ctDNA samples from glioma patients, 12 (71%) demonstrated methylation in at least 1 of the 4 tumor suppressor genes: MGMT (18%), RASSF1A (47%), p15INK4B (12%), and p14ARF (41%). Compared with the tissue gold standard, ctDNA identified hypermethylation with a sensitivity of 81% and specificity of 97% overall. In conclusion, recent work has demonstrated progress in identifying promoter hypermethylation and mutations via ctDNA analysis of glioma patients. Tumor-derived DNA may be identified from CSF as well as from the blood. Wang et al described the detection of tumor DNA from CSF samples in 35 patients with gliomas and other primary tumors of the CNS and coined the term “CSF-tDNA” (CSF-derived tumor DNA).63 The authors employed next-generation sequencing of primary tumor samples to identify mutations present. Guided by this analysis, the authors searched for these mutations in the CSF of the same patients. CSF-tDNA was identified in 74% of cases. Interestingly, CSF-tDNA was detectable in all patients with medulloblastomas, ependymomas, and high-grade gliomas that abutted a CSF space. Of note, the authors’ method involved initial identification of mutations from primary tumor samples, and therefore the method would be useful for tracking recurrence after surgery but not for initial diagnosis and molecular characterization. Prior to clinical use, further work would be necessary to establish specificity and to evaluate this method’s ability to distinguish between recurrent tumor and radiation necrosis. Two additional papers reported identification of mutations associated with specific brain tumors in CSF. Bing–Neel syndrome and primary central nervous system lymphoma (PCNSL) are associated with the MYD88 p.L265P mutation. Hiemcke-Jiwa et al validated ddPCR as a method of identifying MYD88 p.L265P mutation in formalin-fixed, paraffin-embedded brain tumor specimens.64 Next, they isolated DNA and performed ddPCR on CSF samples from 9 patients with clinical suspicion of Bing–Neel syndrome and 3 patients with PCNSL. MYD88 p.L265P mutations were identified in 8 of 9 patients with clinical suspicion of Bing–Neel syndrome and in 1 of 3 patients with PCNSL. Prior to widestream use, specificity would need to be evaluated with CSF from nontumor control patients and sensitivity would need to be improved for PCNSL. Histone H3K27M mutations are associated with diffuse midline glioma. Huang et al were able to isolate DNA from CSF samples taken from 5 children with diffuse midline glioma, and histone H3K27M mutations were identified in CSF from 4 of the 5 children.65 The authors report a sensitivity of 87.5% and specificity of 100%. While these results are quite promising, larger clinical studies would be required prior to entry into the clinic. In summary, ctDNA is most commonly detected using next-generation sequencing and digital PCR technologies.50–53 Blood-based detection of ctDNA has been demonstrated to be sensitive and specific in a variety of nonglial cancers.48,57,58 In gliomas, blood-based detection of ctDNA is specific but not nearly as sensitive (10%–60%) as in other cancers.59–62 Interestingly, higher sensitivity is seen with large enhancing tumor volume.60 Identification of glioma ctDNA has been performed with higher sensitivity using CSF (33%–89%) compared with blood.63–65 Areas that appear most promising for clinical use include detection from CSF (rather than blood) and the subset of patients with large enhancing tumors. Larger trials should be performed, and sensitivity will need to be improved. Circulating MicroRNA Circulating microRNA (miRNA) is another circulating biomarker that has attracted attention. Ranging in length from 21 to 24 nucleotides, miRNAs represent small noncoding RNA molecules that possess an important regulatory role in transcription in both normal and cancer cells.66 While miRNAs regulate transcription within the cell of origin, recent evidence points to an important role for miRNAs in intercellular communication as pro- and anti-oncogenic regulators and immunosuppressors.67,68 Analysis of miRNA accurately identified cancer tissue origin in a variety of cancers.69 Several recent studies have examined miRNA from CSF70 and blood71–75 as a diagnostic tool in gliomas. Dong et al examined miRNA in the serum of 3 patients with GBM and 3 age- and sex-matched healthy controls.72 They identified 115 miRNAs that were significantly upregulated and 24 miRNAs that were significantly downregulated in the serum of GBM patients but not healthy controls. Similarly, Tang et al demonstrated that the level of circulating miR-185 was significantly higher in 66 glioma patients compared with normal controls.73 Interestingly, levels of circulating miR-185 returned to normal levels after surgery and chemoradiation. Additionally, Lai et al demonstrated a 7-fold increase in circulating miR-210 levels in 210 patients with GBM compared with healthy controls,74 whereas Yue et al showed decreased levels of circulating miR-205 in 64 patients with glioma.75 Notably both studies demonstrated a correlation between level of the miRNA studied and outcome. Examining pediatric gliomas, López-Aguilar et al found that circulating miR-130a and miR-145 were significantly elevated, whereas miR-335 and miR-1303 were significantly decreased compared with healthy controls.71 These results suggest that circulating miRNAs hold early promise as tools for the diagnosis and monitoring of gliomas. Current State of Circulating Biomarkers While a great deal of optimism surrounds circulating biomarkers as a means of liquid biopsy for gliomas, technical limitations need to be addressed prior to clinical use. Current techniques allow isolation of CTCs with a sensitivity of 20%–72% of GBM patients.10–12 EVs allowed for the determination of IDH1 status when isolated from CSF but not when isolated from blood.43EGFR amplification was identified using circulating EVs with a sensitivity of 61% and a specificity of 98%.44 Analysis of ctDNA has a sensitivity of 51%–81%60–62 and specificity as high as 97% but has been shown to have a low detection frequency in patients with gliomas. Evaluation of circulating miRNAs identified several candidate biomarkers for glioma diagnosis and monitoring71–75; however, further evaluation of sensitivity and specificity must be done. Sensitivity in particular needs to be addressed for all types of liquid biopsy prior to general clinical use. Future Promise Blood-based analyses will be critical in the diagnosis of inoperable tumors, where safe biopsy may be difficult or impossible. As mutation-based therapy becomes more prevalent in gliomas, blood-based analyses may offer a non-invasive method of identifying mutations. The ability to obtain serial “liquid biopsies” will provide unique opportunities to study the evolution of tumors and mechanisms of treatment resistance and monitor for mutational changes in response to therapy. Combination of techniques (eg, ctDNA in combination with EVs) may allow for improved sensitivity and specificity.30,76,77 Importantly, patients who undergo chemotherapy and radiation frequently develop increase in enhancement on serial MRI examinations, which represents progression versus radiation necrosis (also known as pseudoprogression). Using current imaging technologies, tumor progression and radiation necrosis cannot be reliably differentiated, often requiring operation for diagnosis. A blood-based method of differentiating the 2 diagnoses would be very valuable in this subset of patients. Finally, a combination of blood-based techniques may improve the sensitivity and specificity for liquid biopsy and lead to a clinically useful tool that has the potential to diagnose, monitor, and identify mutations with the goal to identify patients who would benefit from new targeted therapies.30,76,77 Funding This work was supported by a US National Institutes of Health (NIH) Grant (EB002503), NIH National Institute of Biomedical Imaging and Bioengineering Grant (EB012493) and the Wang Pediatric Brain Tumor Collaborative (to B.V.N. and S.L.S.). Conflict of interest statement. None declared. References 1. Louis DN , Perry A , Reifenberger G , et al. The 2016 World Health Organization classification of tumors of the central nervous system: a summary . Acta Neuropathol . 2016 ; 131 ( 6 ): 803 – 820 . Google Scholar CrossRef Search ADS PubMed 2. 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Neuro-OncologyOxford University Press

Published: Sep 1, 2018

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