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  Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 56  |  Issue : 1  |  Page : 65-69
 

Cell-free circulating tumor DNA in patients with high-grade glioma as diagnostic biomarker – A guide to future directive


Department of Radiation Oncology, Kidwai Memorial Institute of Oncology, Bangalore, Karnataka, India

Date of Web Publication4-Apr-2019

Correspondence Address:
H B Govardhan
Department of Radiation Oncology, Kidwai Memorial Institute of Oncology, Bangalore, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijc.IJC_551_17

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 » Abstract 


BACKGROUND: Owing to the aggressive nature of high-grade gliomas (HGGs), its early diagnosis holds the key to a favorable prognosis. Currently, tissue biopsy is the gold standard to verify HGG's initial diagnosis and can be challenging due to its invasive nature. In this study, our objective was a noninvasive panel for timely detection of HGG and its progression using cell-free circulating tumor DNA (cfTDNA).
MATERIALS AND METHODS: Twenty-seven patients with HGG were tested with a 50-gene tumor panel. cfTDNA isolated from serum was checked for single-nucleotide variations (SNVs) or copy number alterations using targeted next-generation sequencing, with further validation of results by checking respective formalin-fixed paraffin-embedded tumor tissues for the same genetic alterations.
RESULTS: About 88.8% of the patients were detected with HGG-associated cfTDNA. Around 25% patients were detected with one, 25% patients had three, 25% patients had four, and 12.5% patients each had five and six genetic alterations. About 12 of 50 genes were detected in the serum samples. The SNVs detected included TP53 in 87.5% of patients; PIK3CA and EGFR in 50% of patients; PTEN in 37.5%; KIT and VHL in each 25% of patients; and RB1, NF2, MET, ATRX, CDK2A, and CTNNB1 each in 8.3%–16.6%. On combining EGFR, KIT, PTEN, PIK3CA, TP53, and VHL genes (Govardhan Diagnostic Genetic Module for high-grade glioma), at least one of the genetic alterations was found in 100% of patients.
Conclusion: These findings illustrate that cfTDNA is easily demonstrable and can be used as a surrogate to tissue biopsy in brain tumor.


Keywords: Biomarker, cfTDNA, high-grade glioma, next-generation sequencing


How to cite this article:
Ahmed KI, Govardhan H B, Roy M, Naveen T, Siddanna P, Sridhar P, Suma M N, Nelson N. Cell-free circulating tumor DNA in patients with high-grade glioma as diagnostic biomarker – A guide to future directive. Indian J Cancer 2019;56:65-9

How to cite this URL:
Ahmed KI, Govardhan H B, Roy M, Naveen T, Siddanna P, Sridhar P, Suma M N, Nelson N. Cell-free circulating tumor DNA in patients with high-grade glioma as diagnostic biomarker – A guide to future directive. Indian J Cancer [serial online] 2019 [cited 2019 Apr 21];56:65-9. Available from: http://www.indianjcancer.com/text.asp?2019/56/1/65/255489





 » Introduction Top


Despite high complexity in the genetic profile of high-grade gliomas (HGGs), its clinical management remains reliant on histopathology and neuroimaging studies. Intratumoral genetic heterogeneity present within the primary tumor and dynamic changes it goes with different therapies (chemotherapy and radiotherapy), hampers the proper therapeutic results.[1],[2],[3] Furthermore, it is a known fact that neuro-imaging has its own way of interpretation. A lack of positive prediction of diagnosis and monitoring therapy (for instance to differentiate between disease progression and pseudo-progression post-therapy) restricts the usage of this method.[4],[5],[6] Hence, there is a need for more sophisticated system of tumor analysis and monitoring on real-time basis.

There are several techniques available to monitor real-time tumor progression, such as neuroimaging (single-photon emission computed tomography [SPECT]and positron emission tomography [PET]) and circulating biomarkers [circulating tumor cells, cell-free circulating tumor DNA (cfTDNA)]. As we have already discussed the problems associated with neuroimaging, the new hope to confront these issues is circulating biomarkers. They can be used as real-time liquid biopsy to identify tumor burden, to monitor response to therapy, to monitor disease progression, and to monitor treatment response. Also, finding out changes in genetic profile can enable changes in management of HGG to match a constantly evolving tumor. Numerous articles advocate benefits of cfTDNA over circulating tumor cell (CTC) in terms of its specificity and ability to detect HGG.[7],[8],[9]

A number of studies have demonstrated the ability of cfTDNA to diagnose HGGs, but mainly in cerebrospinal fluid (CSF), whereas in plasma only a handful of studies exist with small sample size and poorer method of detection.[10],[11],[12],[13] Thus, in this investigation we have aimed at the use of cfTDNA as a noninvasive diagnostic tool in HGG by studying the genetic alterations in isolated cfTDNA and to generate a diagnostic genetic module from the spotted genetic alterations.


 » Materials and Methods Top


Study population

A total of 27 patients (n = 27) with histopathologically confirmed HGG who underwent biopsy or near-total excision were enrolled prospectively in the study after signing the appropriate informed consent. The patients had a median age of 32.5 years (range: 24–60 years) with the majority (87.5%) of the population being male. In all the cases, a biopsy of a formalin-fixed paraffin-embedded (FFPE) tumor tissue was available. Demographical, clinicopathological features (stage, grade), courses of treatment(s), and vital status were acquired from the clinical and pathology reports.

Laboratory analysis

Sample collection and DNA extraction

A total of 27 venous blood samples were collected into BD Vacutainer® Blood Collection Tube (Becton Dickinson and Company, Franklin Lake, NJ, USA) with K3 EDTA, and 4 mL of serum was obtained by centrifugation within 3 h after blood extraction at 2500 rpm at 37°C. DNA was extracted from the serum by QIAamp Circulating Nucleic Acid Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions.

Targeted NGS, identification of SNVs, and CNAs

We designed a 50-gene panel based on our previous studies and publically available databases. The cfTDNA detected in the serum samples was sufficient to carry out NGS. Genes carrying specific somatic mutations like single-nucleotide variations (SNVs)/copy number alterations (CNAs) were screened by next-generation sequencing (NGS) using this panel.

To screen somatic mutations in tumor tissue

For the analyses of FFPE tumor samples, DNA was purified from four 5.0-mm-thick unstained FFPE sections according to the QIAamp DNA FFPE Tissue Kit (Qiagen) protocol, and the gene mutations were screened by NGS targeting 50 cancer genes [Table 1]. To rule out the germline mutations and to correlate with cfTDNA, relevant statistical test was exercised.
Table 1: List of 50 genes in the panel

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Statistical analysis

The correlation among the quantitative variables was evaluated with simple linear regression analysis. P < 0.005 was considered to be statistically significant.


 » Results Top


We report data from 27 patients (male/female: 87.5%:12.5%) with HGG who had undergone biopsy or near-total excision who were prospectively enrolled in the study. The patient characteristics of the study population are summarized in [Table 2]. The source of archival tissue sample was the FFPE blocks.
Table 2: Patient characteristics at the time of sampling

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Patients detected with cfTDNA

First, we evaluate the patients detected with cfTDNA. Overall, we detected cfTDNA in 24 patients (88.8%) out of 27 [Table 2]. Six of 24 (25%) patients had one genomic alteration indentified, 6 of 24 (25%) patients had three genetic alterations, another 6 of 24 (25%) patients had four genetic alterations, and 3 of 24 (12.5%) patients each had five and six genetic alterations.

SNVs detected

Of 50 genes, only 12 genes were detected [Table 3]. As shown in [Figure 1], SNVs detected included TP53 in 21 (87.5%) patients; PIK3CA and EGFR each in 12 (50%) patients; PTEN in 9 patients (37.5%); VHL and KIT each in 6 (25%) patients; and RB1, NF2, MET, ATRX, CDK2A, and CTNNB1 each in 2–4 patients (8.3%–16.6%) as shown in [Table 3]. Interestingly, on combination of six major genetic alterations, that is, EGFR, KIT, PTEN, PIK3CA, TP53, and VHL, at least one of these genetic alterations was found in 100% of patients at any point of time as shown in [Figure 1].
Table 3: Percentage of genetic alterations found

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Figure 1: Combination genetic alterations in EGFR, KIT, PTEN, PIK3CA, TP53, VHL genes

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There was good agreement between the results by NGS and polymerase chain reaction (PCR) for FFPE blocks. Also, additional studies showed that O6-methylguanine-DNA-methyltransferase (MGMT) gene was found to be methylated in 14 of 27 (51.85%) of the samples, whereas 13 of 27 (48.15%) of samples showed unmethylated status for MGMT. Thus, this gene panel can be used as diagnostic genetic module (Govardhan Diagnostic Genetic Module for High-Grade Glioma).


 » Discussion Top


To our knowledge, this is the first attempt targeting an NGS study of cfTDNA to evaluate somatic mutations in patients with HGG where minimally invasive or noninvasive methods to monitor the disease burden and to know its dynamic changes is a challenging task.

Until now, neuroimaging and surgical specimens are the available methods to know the disease burden and disease status during and after therapy. As of now, neuro-imaging and surgically obtained specimens are the available methods to know the disease burden and disease status during and post-therapy. Since imaging modalities have their own difficulties like variations in intra and inter-radiologist interpretation. Besides this, tracking the disease during and after therapy as well as its confirmation still depends on the histopathological diagnosis. Sometimes even with definitive changes/progression with neuro-imaging, we are dependent on final surgical specimen to prove and to plan for matched genedirected therapy. Therefore, there occurs a need to repeat the surgery. And in recurrent cases the surgically acquired tissue yields a necrosis scar. Thus, the associated repeated surgery and time needed to recover poses as a huge burden to the affected family and hence society.[14],[15]

There are only a few noninvasive modalities such as CTC and cfTDNA (both in plasma and CSF) available to address these issues. cfTDNA is a small component of nucleic acid material derived from tumor cell and cell fragments.[16] The CTCs are completely intact, sometimes viable, which can be isolated from blood by either physical and chemical characteristics or by cell surface molecular expressions that distinguish them from normal cells or blood cells.[17] Many studies have been conducted to compare CTC and cfTDNA, where both entities were demonstrated in advanced cases. On comparing these we have reached contrasting conclusions in the view of technical issues like isolation of the circulating nucleic acids and interpretation of the results.[18],[19],[20] A thorough research shows that only limited studies have compared cfTDNA with CTCs; some studies were limited by their isolation techniques, and some have showed that the detection rate of cfTDNA was higher than CTC detection.[19],[21],[22] An article published in Nature Communications demonstrated that both CSF-tDNA and plasma-tDNA showed enriched quantity both with primary and metastatic brain tumors, and in some cases CSF-tDNA levels correspond to a change in tumor burden. The authors have also described in some cases mutations in CSF with brain metastases that were not seen in the primary tumor.[23]

cfTDNA in CSF is a minimally invasive method and cannot be done in all patients like those with acute conditions or with heightened intracranial tension. Also, the sensitivity of CSF cfTDNA is around 70%, and plasma cfTDNA is also nearer to that value; thus, in our study we have aimed to test the plasma cfTDNA.[10] Presently, there will be definitive scarcity of literature on cfTDNA in brain tumor and utilization of cfTDNA in clinical setup. With all these, we have aimed to study the diagnostic ability of cfTDNA and its practical utility in clinical setup.

The techniques mainly used for cfTDNA detection are PCR and NGS, by which genetic alterations such as point mutations, copy number variations, and chromosomal rearrangements can be assessed.[9] So far, the available data about cfTDNA are mainly based on PCR. As we know, early evidence was raised using the data from initial tissue biopsy to inform their PCR-based approach to probe for specific and known mutations. Despite the higher sensitivity of PCR methods to define cfTDNA as a potential biomarker, it is not wise enough to assume the genetic profile of the tumor. Complete genome sequencing of plasma tumor DNA in patients with any tumor types showed that cfTDNA represents an entire tumor genome and it can reflect accurate heterogeneity.[24],[25],[26],[27]

In case if a mutational panel is used, there still arose a question of selection of list of appropriate mutations to be included in the panel. Thus, detection of platforms is broad enough for genomic coverage to fully characterize the spatial and temporal heterogeneity of tumors, but deep-sequencing approaches produce data with high levels of background noise which can obscure true genetic alterations. Newer NGS-based ctDNA detection methods such as SafeSeq, TAm-Seq, CAPP-Seq, and Ampli-Seq have improved but not entirely resolved the sensitivity-associated problems which remain difficult to optimize with broad coverage assays.[28],[29],[30],[31] Thus, in our study, to resolve all these issues we used NGS-based mutation analysis. In addition, using broad-spectrum 50-gene analyses, the genetic alterations in cfTDNA were rechecked in tumor to increase the specificity of this experiment.

Systematic monitoring of the cases with cfTDNA in parallel with therapy showed a burden of the tumor, effect of the therapy or progression during therapy, or appearance of new lesion. In a study involving 18 patients with colorectal carcinoma (CRC), who underwent surgical resection, cfTDNA levels corresponded to the extent of resection.[32] Preliminary data in CRC also demonstrated that ctDNA may be useful in predicting tumor recurrence.[33] Even with different carcinoma such as breast, colon, and ovarian cancer and melanoma, cfTDNA levels showed clinically functional tool in monitoring the treatment outcome. Besides this, cfTDNA level correlated without survival.[23],[27],[34],[35],[36]

cfTDNA can be exploited in monitoring the molecular basis of acquired drug resistance all through the course of treatment via tumor genetic profiles.[27],[31],[35] Treatment allied genetic resistance can be sensed with cfTDNA at least 6 months sooner than it can be discovered by clinically available techniques or imaging.[37],[38] It is a hypothesis that in gliomas, the blood–brain barrier can become an obstruction both in treatment delivery and in detection of an effective biomarker. According to some studies, the frequency of detectable cfTDNA for gliomas was <10%, whereas in another study done by Piccioni et al.[36] the detectibility of cfTDNA in glioblastma was 37%; but in our study, we managed to detect cfTDNA in all the above cases. The reason for low detectability may be the inferior technique used to isolate DNA.[27] Though cfTDNA has showed diagnostic, prognostic, and predictive ability as well as utility in glioma, further large scale studies are required to prove its utility in outcome. The cost-effectiveness of the cfTDNA in the clinical setting remains another issue. Currently, the cost of NGS has been condensed over time and is improbable to be a limiting factor for this sort of work.

Many minor studies have illustrated significant genetic alterations in EGRF, MGMT methylation, and 1p19q loss of heterozygosity, which can be detected in cfTDNA from patients with gliomas.[39],[40],[41],[42],[43],[44] As most frequently mentioned, genetic alterations are used daily in clinical practices, and thus in our study we are aiming at detecting a broad spectrum of genetic alterations more important and relevant to less important relevant ones. Thus, we used a 50-gene panel to resolve the aforementioned issue. Twelve genes of the 50 in the panel were detected. The detection rate (24/27, 88.8%) was similar to another of our team's study that included patients suffering from cervical cancer where the detection rate was shown to be 84%.[44] Six of 24 (25%) patients had one genomic alteration detected, 25% of patients had three genetic alterations, another 25% patients had four genetic alterations, and 3 of 24 (12.5%) patients had five and six genetic alteration each. As shown in [Figure 1], SNVs detected included TP53 in 21 of 24 (87.5%) patients; PIK3CA and EGFR in 50% patients each; PTEN in 37.5%; KIT and VHL in 25% patients each; RB1, NF2, MET, ATRX, CDK2A, and CTNNB1 in 2–4 patients (8.3%–16.6%) each as shown in [Table 3].

On combination of six major genetic alterations, that is, EGFR, KIT, PTEN, PIK3CA, TP53, and VHL, it was revealed that at least one genetic alteration among the combinations was found in 100% of patients at any point of time. Thus, it can be used as diagnostic genetic module (Govardhan Diagnostic and Screening Genetic Module for High-Grade Glioma). Using this model can guide further utilization of this module for initial screening will aid in rapid diagnosis of brain tumor as it is more sensitive and specific than imaging in this study. Additionally, the employment of this study holds a significant benefit in differentiating posttreatment progression and pseudoprogression but it is uncertain at this point on account of unavailability of recurrent cases genetic profile. Furthermore, follow-up data regarding correlation of these genetic alterations with prognosis will follow in future.

The minute numbers of cases involved and limited known genetic[45] alterations used in the panel are the drawbacks of this study. As time progresses, we may find new methods of detection and more validated and clinically important genetic panels.


 » Conclusion Top


cfTDNA is easily demonstrable and can be used as a minimally invasive diagnostic tool in brain tumors. Combination of six genetic alterations, that is, EGFR, KIT, PTEN, PIK3CA, TP53, VHL, is the main alteration, and on combining these genetic alterations (Govardhan Diagnostic and Screening Genetic Module for High-Grade Glioma), at least any one variation among the combination was found in 100% of patients at any point of time. That can be a future guide toward creating a feasible, repeatable, faster, cost-effective, noninvasive, and highly predictable diagnostic tool for early diagnosis/screening. Added to this, it can also become a route for detecting early recurrence and early response assessment in HGGs.

Continuation of this study on a large-scale basis with different race and ethnicity is needed to create a proper genetic module to aid as a diagnostic and prognostic marker in brain tumors as well as to guide for a genomically matched therapy. Further follow-up to correlate the genetic relationship with overall survival and disease-free survival is awaited.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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    Tables

  [Table 1], [Table 2], [Table 3]



 

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