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Table of Contents
Year : 2021  |  Volume : 4  |  Issue : 3  |  Page : 179-187

Role of liquid biopsy in central nervous system tumors

Department of Pathology, All India Institute of Medical Sciences, New Delhi, India

Date of Web Publication02-Nov-2021

Correspondence Address:
Dr. Chitra Sarkar
Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/IJNO.IJNO_425_21

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Challenges in obtaining tissue specimens and tumor heterogeneity are major limitations for accurate diagnosis, molecular characterization, risk stratification, and development of biomarker-driven therapies in central nervous system (CNS) tumors. The potential of assessment of CNS tumors through analysis of corporeal fluids (liquid biopsy) is being explored to document tumor-related genetic/epigenetic alterations and protein expression to identify prognostic and therapeutic biomarkers. The quantity of circulating tumor DNA isolated also appears to be directly associated with tumor progression and response to treatment. In this review, we provide synopsis of the recent studies which have provided crucial insights into analyzing circulating tumor cells, cell-free nucleic acids, and extracellular vesicles for directing long-term disease control. We have also highlighted the stumbling blocks and gaps in technology that need to be overcome to translate research findings into a tool in the clinical setting.

Keywords: Liquid biopsy, cfDNA, ctDNA

How to cite this article:
Chakraborty R, Suri V, Dandapath I, Singh J, Sharma M C, Sarkar C. Role of liquid biopsy in central nervous system tumors. Int J Neurooncol 2021;4, Suppl S1:179-87

How to cite this URL:
Chakraborty R, Suri V, Dandapath I, Singh J, Sharma M C, Sarkar C. Role of liquid biopsy in central nervous system tumors. Int J Neurooncol [serial online] 2021 [cited 2022 May 22];4, Suppl S1:179-87. Available from: https://www.Internationaljneurooncology.com/text.asp?2021/4/3/179/329820

  Introduction Top

The evaluation of molecular genetic alterations in cancers is nowadays very important for diagnosis, prognostication, management, and treatment decisions, as well as assessment of treatment responses, treatment resistance, and relapse. Molecular profiling is generally done on resected or biopsy specimen of primary tumors. The main limitation of such analysis is that tissue biopsy/resection is a single fixed time point evaluation. Serial monitoring of tumor progression and evolution and dynamic follow-up of modifications in molecular alterations cannot be done on tissue biopsies/resections since these require surgical intervention, are invasive procedures and therefore not easily available. Further, formalin-fixed paraffin-embedded tissues present lot of variability in the quantity and quality of DNA and RNA depending on procedures of collection, storage, and preservation.[1],[2]

To circumvent such practical issues, liquid biopsies are becoming of increasing interest since they can be obtained by minimally invasive procedures. They allow real-time systematic and dynamic monitoring of molecular alterations in cancer patients as they can be done repeatedly. Hence, they are very useful for detecting, analyzing, and monitoring diagnostic, prognostic, and predictive cancer biomarkers at pretreatment, treatment, and posttreatment levels. Thus, they help in monitoring response to treatment as well as assessment of treatment resistance and disease relapse. [1],[2]

Liquid biopsies utilize liquid samples such as blood, urine, saliva, stool, and other biological material which can be obtained by minimally invasive techniques such as ascites fluid and cerebrospinal fluid (CSF).[3]

The biomarkers assessed in liquid biopsy include cell-free DNA (cfDNA), cell-free RNA, circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), tumor educated platelet, exosomes, proteins, and microRNA.[3]

CTCs are cancer cells that get detached from the primary tumor and/or metastatic lesion and travel by the bloodstream to other parts of the body. They comprise of DNA, RNA, and protein from tumors. Their number is very low (<10 cells per 1 ml of blood) and their half-life is also short (1–2.4 h.). Hence, CTC is tedious to isolate owing to their variable properties including size, clustering capability, and varied cell surface markers. However, they can be used to perform genomic and transcriptomic analysis in cancers. Certain innovative culturing approaches have also been developed to perform functional studies using these cells, including therapy testing, developing xenograft models, and three-dimensional tissues. As of 2018, CELL SEARCH CTC test based on the expression of epithelial cell adhesion molecule and the lack of CD45 on the cell surface is the only United States Food and Drug Administration approved CTC quantification platform. It is being used for metastatic prostate, breast, and colorectal cancers. [1],[2],[3]

cfDNA is released in liquid samples through secretion, apoptosis, or necrosis from cells in the body. The cfDNA concentration is approximately 1–10 ng/ml in the plasma. The tumor-derived DNA can be detected within this cfDNA fraction of blood and other liquid samples from patients with cancer. This is called ctDNA and its concentration can be <0.1% of the cfDNA. The difference between ctDNA and nontumor-derived cfDNA is that the former comprises of 134–144 bp DNA fragments versus 167 bp fragments in the latter. The main challenge of using ctDNA is to develop sufficiently sensitive assays for differentiating low ctDNA signals from high cfDNA background level.[3] The methods used for cfDNA analysis include quantitative polymerase chain reaction (PCR), digital PCR, and next-generation sequencing (NGS).[3]

Exosomes are used for analysis of nucleic acids in liquid biopsies. They are 42–200 mm in diameter and their content includes DNA, mRNA, microRNA, and protein. They are secreted from multivesicular bodies by the process of exocytosis in live cells. However, their method of isolation is laborious and it may be difficult to differentiate between tumor-derived exosomes and normal cells.[3]

Uses of liquid biopsy

  1. For early detection/diagnosis of cancer. This requires that specific DNA mutations are identified and validated as drivers for early-stage cancer
  2. To monitor minimal residual disease (MRD) – MRD measurement can be very important in recognizing patients with a high chance of relapse, especially in the absence of other biomarkers
  3. Assessment of tumor mutation burden (TMB) – TMB is defined as the total number of mutations per coding region of the genome. High TMB is an emerging predictor of sensitivity to immune checkpoint such as programmed death 1 or its ligand programmed death-ligand 1
  4. For dynamic follow-up of cancer case – follow-up after surgery, early assessment of therapeutic efficacy, diagnosis of relapse, identification of treatment resistance.

Liquid biopsy especially cfDNA analysis has entered the clinic for cancer management especially for non-small cell lung, breast, and colorectal cancers. The use of liquid biopsy has exponentially increased since 2010 but the primary bottleneck remains the technical difficulty for establishing sensitive technologies to measure cfDNA circulating in the blood. The technical difficulties range from collection of specimens to storage as well as detection methodologies. More validation is required to elucidate that molecular markers detected from liquid biopsy match the markers from classical tissue tumor biopsy. New technology platforms need to be developed for reducing cost, improving quality as well as speed of detection, before liquid biopsy becomes accepted as the point-of-care testing in cancer patients. [1],[3]

The next section will deal with liquid biopsy application in central nervous system (CNS) tumors. Although not very successful till date, numerous studies are ongoing and we hope to use it to monitor CNS tumors in the near future.

Liquid biopsy in central nervous system tumors

Biopsies in the brain are very challenging as many CNS tumors are not amenable to surgical resection, either due to sensitive neuroanatomical location or the infiltrating nature of the tumor, which at times are an issue for even the most competent neurosurgeons.[4],[5] Furthermore, owing to regional heterogeneity, the sampling of a lesion by biopsy may not be able to identify mutations present across all regions of a tumor.[6] The identification of a reliable biomarker in accessible body fluids would be useful for diagnosis, prognosis, and follow-up in CNS tumors. Thus, a liquid biopsy would facilitate following up patients in a longitudinal fashion after the initial diagnosis and treatment as well as to fully capture the intratumoral heterogeneity.[4],[7]

Most commonly used liquid biopsy specimens for CNS tumors are serum and urine as these samples are easy to collect, but they have low content of ctDNA and CTC, which could mainly be due to the blood–brain barrier.[8],[9] Sampling of CSF through lumbar puncture has higher amount of nucleic acid content.[10],[11] However, it has some limitations. Many patients with brain tumors present with raised intracranial pressure wherein lumbar puncture is contraindicated.[12] Technical issues such as blood contamination in the CSF sample are also possible. A CSF-based liquid biopsy may not be appropriate for intra-axial CNS tumors, which lack any association with the CSF.[13]

Several molecular alterations of strong diagnostic, prognostic, or predictive significance have been assessed in CNS tumors using several tumor-derived circulating nucleic acids (e.g. ctDNA, cmtDNA, mRNA, noncoding RNAs), or CTCs predominantly from serum or CSF samples.[8] Advanced technologies such as droplet-based digital polymerase chain reaction (ddPCR) and NGS have significantly enhanced the sensitivity and specificity for the detection of these alterations.[14] NGS facilitates exploring a wide range of possible new mutations while ddPCR can be used for detecting known specific mutations.[15],[16] The present article reviews the important alterations analyzed for differential diagnosis, patient stratification, and follow-up in CNS tumors. Although mostly conducted in gliomas, some recent studies on other CNS tumors are also available.

  Diffuse Gliomas Top

Isocitrate dehydrogenase mutations1/2

Isocitrate dehydrogenase (IDH) mutation is present in ~80% of Grades II–IV astrocytomas and all oligodendrogliomas.[17] IDH1/2 mutations are now part of the routine diagnostic pipeline and bear prognostic relevance as IDH-mutant gliomas have better prognosis compared to IDH wild type. [18],[19] Majority of cases harbor IDH1 mutation restricted to amino acid residue 132, with >85% containing a heterozygous missense mutation of arginine to histidine (R132H).[20]

Boisselier et al., analyzed IDH R132H mutation in plasma ctDNA using a combination of coamplification and digital PCR with 100% specificity. Sensitivity was 60% for the whole series but 70% for patients with the WHO high-grade gliomas (HGGs) and 100% in HGGs with an enhancing tumor volume higher than 3.5 cm3. However, a poor sensitivity of 37.5% was observed for low-grade gliomas.[21]

Chen et al., validated IDH1 mRNA transcripts within CSF of patients, using BEAMing (beads, emulsion, amplification, magnetics) and ddPCR with a sensitivity of 63% and a specificity of 100%.[22] Using targeted sequencing, sensitivity of IDH mutation detection has been documented to be more than 80% for CSF while the sensitivity is far less with plasma ctDNA.[23],[24],[25],[26],[27]

Methylguanine-DNA methyltransferase promoter methylation

Epigenetic silencing of the methylguanine-DNA methyltransferase promoter (MGMT) gene through methylation of promoter CpGs has been observed in 48%–75% [wild type] IDH-mutant Grades II-IV astrocytomas and 36% IDHwt glioblastomas (GBMs).[28] MGMT promoter methylation impairs DNA repair and predicts better treatment response with alkylating agents and longer survival in patients with GBM.[29] Elderly GBM patients eligible for either radiotherapy (RT) or temozolomide (TMZ) should be tested for MGMT promoter methylation before undertaking therapeutic decision.[30],[31] Further, MGMT promoter methylation helps to differentiate true progression from pseudoprogression in patients with newly diagnosed GBM treated with surgery followed by radiochemotherapy.[32]

Using both tumor and serum DNA, Balaña et al. analyzed MGMT methylation in 28 GBM patients treated with BCNU or with TMZ plus cisplatin, by methylation-specific PCR (MS PCR). High concordance was found between cfDNA and the tumor tissue samples.[33] An average sensitivity of 65% in CSF and 1%–50% in plasma has been observed using MS PCR.[34],[35],[36],[37] Applying methylated DNA immunoprecipitation, positive rates of MGMT promoter methylation in tumor tissue, serum, and CSF were 97.0%, 71.2%, and 78.8%, respectively.[38]

Telomerase reverse transcriptase promoter mutations

Telomerase reverse transcriptase (TERT) promoter mutations (pTERTs) are among the most common genetic alterations in gliomas and are associated with dismal prognosis in IDH wild-type diffuse astrocytomas (DAs) and in GBMs irrespective of IDH status.[39] Two recurrent mutually exclusive mutations in pTERT region, C228T and C250T, are seen in 18%–40% of Grades II/III IDH-wt DAs and 70%–80% of all GBMs.[39],[40],[41],[42] Consortium to inform molecular and practical approaches to CNS tumor taxonomy (cIMPACT-NOW) Updates recommend reclassifying IDH-wt Grades II/III DAs harboring pTERT mutation as GBMs.[43] Different strategies to target pTERT activity, such as small molecule inhibitor, immunotherapy, and vaccines are being investigated.[44],[45],[46] Detecting pTERT mutations in CSF or plasma ctDNA is crucial for diagnosis and to monitor and assess response to therapy.

In a pilot study by Juratli et al., using NGS and ddPCR (for validation) platforms, pTERT mutation in the CSF-ctDNA was detected with 100% specificity and 92.1% sensitivity. However, the sensitivity in the plasma ctDNA was 7.9%.[47] An increasing pTERT mutation variant allele frequency levels in the CSF-tDNA were observed in patients with unfavorable outcomes. Similarly, Fontanilles et al., analyzed 49 glioblastomas and 3 gliosarcomas by ddPCR and observed pTERT in plasma ctDNA in only 3.8% of cases.[48] The authors suggested that the short size of the ctDNA fragments (<70 bp) was the primary factor related to failure of pTERT mutation detection in plasma.[48],[49] Interestingly, the authors documented that the cfDNA concentration varies significantly over the course of treatment and may be a biomarker of progression during the TMZ phase. Using 7-deaza-dGTP as a ddPCR additive and utilizing standardized handling strategies and technical optimization, Muralidharan et al., (2021) reported an overall sensitivity of 62.5% and specificity of 90% in ctDNA in plasma.[50]

Epidermal growth factor receptor

Epidermal growth factor receptor (EGFR) (7p12) amplification, overexpression, and/or mutations are commonly seen in IDHwt GBMs.[51] The evidence for EGFR amplification or EGFRvIII mutation as independent predictive markers for survival in GBM varies among different studies.[52],[53],[54],[55] An ongoing trial of the EGFRvIII vaccine (Rindopepimut) CDX-110 has shown longer overall survival in GBM patients treated after tumor resection.[56] Using qPCR, a sensitivity of 61% and a specificity of 98% were observed in CSF-derived extracellular vesicles (EVs) to detect EGFRvIII-positive GBMs.[57] Overall, sensitivity and specificity of semiquantitative exosome EGFRvIII PCR assay in serum were 81.58% and 79.31%.[58]

TP53 and PTEN mutations

The WHO Grades II and III, IDH-mutant astrocytic tumors frequently carry TP53 mutations (>80%).[59] Deletions and mutations in the tumor suppressor gene PTEN are frequent events (~35%) in GBMs. Loss of PTEN is associated with poor survival and therapeutic resistance in GBMs.[60],[61] In a recent study by Zhao et al., on 17 pairs of matched CSF and tumor samples from patients with gliomas, mutations of PTEN and TP53 were commonly detected in CSF ctDNA of recurrent glioma patients.[23]

H3K27M mutation

Somatic driver mutations in H3F3A or HIST1H3B/C, encoding for histone 3 variants H3.3 and H3.1, respectively, are seen dominantly in pediatric high-grade infiltrative astrocytomas arising within midline structures (thalamus, brain stem, and spinal cord) and are associated with very poor outcome (median OS: 9–11 months).[62],[63],[64] DMG, H3K27M mutant was introduced as a new diagnostic entity in the updated 2016 WHO classification of CNS tumors.[65],[66] The sensitive anatomic location and the need for specialized surgical expertise have limited the favorability of surgical biopsy, especially in recurrent cases. Although limited in sensitivity and specificity, MR imaging is still widely used to diagnose and assess response to therapy.[67],[68],[69]

In a study of 48 pediatric DMG patients, Panditharatna et al., (2018) using ddPCR detected H3K27M ctDNA in 87% of CSF samples and 90% of plasma samples analyzed from patients known to harbor oncohistones in their primary tumors. A significant decrease in H3K27M plasma ctDNA was observed in concordance with MRI assessment of tumor response to RT in 83% of patients.[70] Li et al., analyzed H3K27M mutation in DMG in ctDNA across two platforms of ddPCR (RainDance and BioRad) using CSF, plasma, and tumor specimens. The authors documented 100% sensitivity and specificity in matched tumor tissue and CSF samples. H3.3K27M mutation detection in blood specimens was most technically challenging owing to very low quantity of ctDNA. Vacuum-concentration and preamplification of ctDNA was employed to increase test sensitivity without decreasing specificity.[71]

  Medulloblastoma Top

Medulloblastoma (MB), an embryonal tumor of the CNS, is the most aggressive brain tumor in childhood that can also occur in adults, although less common.[72] It is a complex and evolving heterogeneous disease that can be divided into four molecular consensus subgroups (WNT, SHH, Group 3 and Group 4), with prognostic and therapeutic implications.[73],[74],[75],[76],[77] In a recent study by Escudero et al., on 13 pediatric MBs, ctDNA was more abundant in CSF (76.9% patients) than plasma (7.6% patients). Exome sequencing of CSF ctDNA recapitulated the tumor mutational burden and the genomic alterations, including MB common mutations (PTCH1, TP53), copy number variation (CNVs) (MYCN and GLI2 amplification), and arm-level chromosomal aberrations (chromosome 17p loss), providing diagnostic and prognostic information.[78] MB also harbors abnormal DNA methylation changes, with distinct epigenetic signatures identified across MB subtypes that can be altered during tumor progression and treatment.[74],[79] Li et al., 2020 identified reliable DNA methylation signatures in ctDNA of MB patients that have potential diagnostic and prognostic values.[80]

  Other Central Nervous System Tumors Top

Few studies have analyzed the role of liquid biopsy in other CNS tumors such as ependymoma and meningioma. However, substantial data are not available.[81],[82],[83] ctDNA has only been detected in the plasma of a minority of patients with CNS lymphoma.[84],[85] In contrast, several studies revealed that the analysis of CSF ctDNA better detects CNS disease in patients with B-cell lymphoma.[86],[87],[88],[89],[90],[91] [Table 1] elucidates the key molecular alterations in CNS tumors that have been assessed by liquid biopsy, platforms used for their identification, and sensitivity/specificity of these techniques.
Table 1: A summary of molecular alterations identified in central nervous system tumors using liquid biopsy

Click here to view

  Challenges and Future Directions Top

Liquid biopsy in CNS tumors can facilitate an early detection of relapse and identify therapeutic targets or mechanisms of resistance to adjust the therapeutic strategy at relapse. However, detection of ctDNA may be influenced by tumor burden, tumor progression, and anatomical location.[24],[82],[92] For analysis of specific mutations by sensitive techniques such as ddPCR, prior knowledge of the tumor genetic profile is required. Whole exome sequencing or targeted gene panels might provide more information. However, it is important to consider sensitivity, turnaround time, and cost-effectiveness. To determine the impact of the results and translate them into a tool for clinical practice, standardization of protocols and larger studies with more patients will be required. Altogether, liquid biopsy remains a promising strategy to improve the clinical management of patients with CNS malignancies.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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