: 2018  |  Volume : 1  |  Issue : 5  |  Page : 149--154

How does one do next-generation sequencing for brain tumors in the clinical laboratories?

Kay Ka-Wai Li, Ho-Keung Ng 
 Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong; Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China

Correspondence Address:
Dr. Ho-Keung Ng
Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong
Dr. Kay Ka-Wai Li
Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong


The newly released World Health Organization Classification of Tumors of the Central Nervous System 2016 has implemented molecular information in the classification of brain tumors. A number of large-scale retrospective studies have indicated that molecular data are of diagnostic and prognostic relevance in neuro-oncology. Incorporation of molecular studies has become a prerequisite in standard-of-care practice of neuro-oncology. Next-generation sequencing (NGS) or massively parallel sequencing allows simultaneously sequencing millions of DNA fragments in an acceptable period. The technique allows examination of a number of genes and gene regions simultaneously and is capable of detecting a wide variety of molecular alterations. NGS has been rapidly adopted in cancer studies to identify mutational landscape, copy number variation, novel fusion genes, and others. With its rapidly declining cost, NGS is slowly replacing conventional molecular techniques in cancer diagnosis. In this review, we will review the development of NGS and common sequencing strategies in oncology laboratories. We will then discuss the application of NGS in detecting genetic aberrations in neuro-oncology.

How to cite this article:
Li KK, Ng HK. How does one do next-generation sequencing for brain tumors in the clinical laboratories?.Glioma 2018;1:149-154

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Li KK, Ng HK. How does one do next-generation sequencing for brain tumors in the clinical laboratories?. Glioma [serial online] 2018 [cited 2022 Aug 13 ];1:149-154
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The incidence of central nervous system (CNS) tumors has increased over the decades partly because of improvements in diagnostic technology. According to the U.S. Central Brain Tumor Registry, the annual average age-adjusted incidence rate of all primary malignant and nonmalignant brain and other CNS tumors is 22.64 cases per 100,000 population.[1] CNS tumor is the most common solid tumor among children aged between 0 and 14 years, with an annual average age-adjusted incidence rate of 5.54/100,000 population. In children, pilocytic astrocytomas, embryonal tumors, and malignant gliomas account for the majority of CNS tumors. In adult, the most common CNS tumors are malignant gliomas and meningiomas. Survival rates vary significantly depending on age, histology, and tumor behavior. For instance, 5-year overall survival for patients with pilocytic astrocytoma, a type of low-grade glioma, is over 90%. In contrast, 5-year overall survival drops to around 5% for glioblastoma.

For almost a century, the diagnosis of CNS tumors has primarily relied on histological appearance under a microscope with a grading system based on the outcome of tumors if left untreated, and tumors are classified by their resemblance to the brain cells from which they are derived.[2] For instance, “astrocytoma” is designated to a tumor derived from astrocytes. However, histologically oriented approach in diagnosis and classification has posed a number of problems. For example, patients with similar morphologies may exhibit different prognoses and responses to treatment. Furthermore, diagnosis is always challenged by significant interobserver variability.[3] In a review of 500 brain tumor cases submitted as a part of daily patient care, some degree of disagreement was present in 42.8%.[4]

In the last two decades, we have greatly enhanced our understanding of molecular alterations of CNS tumors. A number of studies have shown the identification of clinically relevant molecular signatures in CNS tumors.[5],[6],[7],[8] Importantly, molecular profile is better in prognostic classification compared with traditional histology.[5],[6],[7],[8] The newly released World Health Organization (WHO) Classification of Tumors of the CNS has integrated histological appearance and molecular parameters into the classification of CNS tumors, aiming to guide patient prognostication, patient treatment, and development of targeted treatments.[9] These changes have changed our routine clinical practice in neuro-oncology field.

Next-generation sequencing (NGS) is a revolutionary innovation capable of delineating the complexity of human cancer. NGS or massively parallel sequencing enables us to sequence thousands to millions of different DNA molecules simultaneously in a short period at an affordable cost. It is effective in detecting different molecular alterations, such as single-nucleotide variations (SNVs), insertions and deletions, and copy number variations. This powerful tool has largely changed the fields of clinical diagnostics and personalized medicine.

NGS techniques can be broadly classified into applications for examining genome, transcriptome, and epigenome. Genomic assays include whole-genome sequencing (WGS), whole-exome sequencing (WES), and target sequencing, with the latter aiming to examine unique regions of genes for the detection of alterations associated with specific diseases. Transcriptome assay such as RNA sequencing is used to study global gene expression and discover novel transcribed genes.[10],[11],[12],[13] Epigenome assays such as chromatin immunoprecipitation followed by high throughput sequencing aims to identify DNA-binding sequences for DNA-binding proteins such as transcription factors and to localize histone modifications.[14] Some of these assays are difficult to perform in routine oncology setting. For instance, WGS is expensive and time-consuming and requires sophisticated computational analysis. WES requires a matched patient blood and large amount of DNA that is difficult to obtain in small brain biopsy specimens. Target sequencing examines a panel of cancer-associated genes with relevance at a deep level. It has gained favor because of requirement of small input of nucleic acids, high coverage, short turnaround time, reduced cost, and less bioinformatics analysis.

In this review, we will first go through the development of NGS. We will then discuss common sequencing strategies and NGS workflow in the clinical laboratory. Finally, we will describe the target panel sequencing in detecting genetic aberrations in brain tumors.

 Development of Next-Generation Sequencing

The first generation of sequencing was developed by Frederick Sanger and Walter Gilbert in 1970 and was called chain termination or Sanger sequencing.[15],[16] Sequencing procedure was tedious and workforce demanding. Radiolabeled dideoxynucleotides lacked the 3' hydroxyl group that is required for extension of DNA chain are incorporated in a DNA extension reaction. This produces DNA strands of various lengths. The product is then separated on large vertical polyacrylamide gels and visualized by autoradiography. The final DNA sequence is deduced from autoradiography. In 1990, the sequencing became more convenience and partial automated when DNA extension reaction was achieved with fluorescent chain-terminating dideoxynucleotides and separation was replaced by capillary-based electrophoresis.[17],[18],[19],[20] The second-generation sequencing or NGS came into the market in 2005 when 454 Life Sciences founded by Jonathan Rothberg launched a platform called GS-20. Two years later, 454 Life Sciences was sold to Roche Applied Sciences, and the second version 454 instrument, called GS FLX, was introduced. Compared to Sanger sequencing which took over 10 years to sequence the entire human genome at the cost of ~100 million US dollars, the sequencing by 454 GS FLX took 2 months at 1/100 of the price.[21] At about the same time, another company called Solexa released another sequencing platform called genome analyzer. Solexa was then purchased by Illumina in 2007.[22] The launch of 454/Roche and Solexa/Illumina sequencers began a paradigm shift in oncology and medical science research communities. Additional NGS platforms were subsequently introduced including Ion Torrent (Life Technologies), SOLiD (Life Technologies), PacBio (Pacific Biosciences), Helicos (Helicos Biosciences), and Nanopore (Oxford Technologies).[23] At present, maximum output of sequencing platforms ranges from a few gigabases to 600 gigabases.

 Advantages of Next-Generation Sequencing

NGS technology possesses a few distinct advantages over single-gene studies that are relevant in clinical applications. First, it is capable of massively parallel reading of millions of short nucleic acid fragments, and thus, it allows examination of multiple target regions in a single run and generation of a diagnostic report in a short period. The assay provides an opportunity for precision medicine in oncology patients. In multiple randomized clinical trials, patients treated with target therapy showed improved response rates and progression-free survival compared to those given standard chemotherapy.[24],[25] Second, the amount of genomic DNA required for NGS assay is lower as compared to traditional sequencing, and this feature has allowed detection of genomic changes in a small biopsy or fine-needle aspiration sample. Third, NGS is versatile. It can comprehensively study the entire genome and screen different types of genomic changes.[26] In contrast, in the traditional molecular test which employs “one-gene-one-test” approach, only a single type of molecular alteration in a given gene can be tested. Furthermore, NGS has a relatively high sensitivity to detect genomic aberrations at low frequency, and it can be beneficial in routine surgical specimens which are mixed with normal tissues. This is particularly true for target sequencing that offers high depth of coverage. Very often, target sequencing achieves an average depth of coverage of ×500 or more, and such magnitude surpasses other NGS strategies, such as WGS or WES. Coverage refers to the number of times a unique base has been sequenced. In summary, improved capacity and turnaround time, low sample input, and greater sensitivity are characteristics of NGS that make it superior to conventional molecular tests.

Sequencing types

There are a few types of sequencing strategies in the oncology setting. The scope of sequencing types ranges from target gene panels encompassing 20–500 “cancer” genes to WES of the ∼25,000 human protein-coding genes to WGS of the entire 3.3 billion bases of the human genome. Among the sequencing strategies, WGS is the most comprehensive. It can uncover almost all types of molecular aberrations in coding and noncoding regions (untranslated region, promoters, and enhancers). WGS was employed in the identification of TERT promoter mutation associated with pathogenesis in glioblastoma.[27] Chromosomal rearrangement is surprisingly common in brain tumors. With WGS, several fusion genes were found in pilocytic astrocytoma and pediatric high-grade glioma.[28],[29] However, due to generation of massive data in WGS, interpretation of WGS result demands complex computational analysis. WES targets exonic region of the human genome which constitutes approximately 1% of the whole genome. Given that most cancer-relevant mutations are found in protein-coding regions, WES can detect up to 85% of cancer-causing mutations.[30],[31] Collaborating groups in Canada and Germany sequenced the exomes of 48 pediatric glioblastomas and identified driver mutations in histone H3.3 and chromatin remodeling genes.[32] With exome sequencing, novel somatic mutations and subtype-specific mutations were found in medulloblastomas.[33] A major limitation of WES is the inability to comprehensively evaluate structural variants and large rearrangement.[34] Target sequencing selectively sequences regions of interest or genes with relevance to cancer and has become a popular alternative in clinical laboratories. It runs at low cost with a short turnaround time and incurs manageable computational analysis. Furthermore, the deep coverage offered by target sequencing enables the opportunity for rare variant detection and increases sensitivity for variant detection in low purity tumors. However, the application of target sequencing for cancer gene discovery is limited. Gene panel does not help in uncovering the exact role of mutation in carcinogenesis even though most genes in a gene panel are of known function. Gene panel is also of limited value for detecting variants outside predesigned panel.

 Target Sequencing Workflow in Clinical Laboratory

NGS involves two major processes: wet bench steps and bioinformatics interpretation of raw data. Regardless of the NGS platform used, common wet bench steps include isolation of nucleic acid from tissue specimen, library preparation, target enrichment, and run on platform to generate sequence reads.

DNA isolation can be achieved by various extraction methods. Assessment of tumor content in formalin-fixed paraffin-embedded (FFPE) tissues is crucial before DNA isolation. The proportion of nonneoplastic cells would reduce the frequency of mutation and lead to difficulty in distinguishing between a true mutation at low frequency and sequencing artifacts. Thus, it is important that a pathologist conducts histological assessment of the FFPE slides and marks an area enriched with tumor cells and devoid of heavy inflammatory cell, nonneoplastic cells, and necrotic cell. Microdissection may be undertaken to increase tumor purity. The tumor purity should be assessed and documented to assist data interpretation. It should be noted that tumor purity is indicated by the number of tumor cell nuclei rather than the proportion of tumor area.[35] In standard NGS, the quality and quantity of DNA should be assessed by Qubit or Picogreen instead of usual spectrophotometry.

Library construction involves DNA fragmentation and adaptor ligation so that nucleic acid is prepared into a form that is compatible with the sequencing system to be used.[36] DNA is fragmented either by enzymatical or mechanical methods. Fragmented DNA has overhangs that have to be enzymatically repaired to blunt ends. Insertion of A-tail at the 3' ends of DNA to facilitate sequencing adaptor ligation is then completed by either Taq polymerase or Klenow fragment.

The resulting library undergoes enrichment either by hybridization-based (hybrid capture) or by amplification-based approach. In hybrid capture approach, target nucleic acids are hybridized to probes with sequences complementary to the regions of interest on the targets, either in solution or on a solid support.[37] After hybridization, unbound sequences are washed away, and the targeted sequences are eluted for further process. Polymerase chain reaction (PCR)-based approach relies on primer-driven amplification of the region of interest on fragmented nucleic acids.[38]

Before running the sample on a sequencing platform, DNA is subjected to clonal amplification for generation of cluster of identical DNA molecules for signal detection. Single DNA fragment is either bound to beads, ion surfaces, or flow cell and objected to amplification by either emulsion PCR or bridge PCR method, resulting in millions of DNA strands for sequencing.[39],[40]

Finally, DNA fragments are subjected to sequencing. In general, Illumina and Ion Torrent are two common sequencing platforms in the clinical laboratory, and they are different in their chemistry and detection methods.[41],[42] Illumina sequencers utilize a method called sequencing by synthesis in which a fluorescent nucleotide containing a 3' end terminator is added to a DNA strand in a step-by-step manner. In each round, all four fluorescence tagged nucleotides are added along with DNA polymerase to a flow cell. Once a tagged nucleotide is incorporated to a growing DNA fragment, further extension of growing strand is prevented by the 3' terminator, unincorporated nucleotides and DNA polymerase are washed away, and emitted fluorescence is recorded by a detector. The 3' blocking group is then chemically removed, and the process is repeated.

Ion Torrent uses semiconductor sequencing method. During nucleotide incorporation, a hydrogen ion is released. This causes a change in pH that can be detected by an integrated complementary metal-oxide-semiconductor and an ion-sensitive field-effect transistor.[41],[43]

In general, postsequencing bioinformatic analysis involves conversion of sequence image to base sequences, change sequence file to a readable file, sequence alignment with a reference genome, and annotation and variant calling.[44]

 Clinical Utility of Next-Generation Sequencing in Brain Tumor Diagnostic and Management

At present, a few CNS-specific or glioma-specific target panels have been developed for clinical use in routine neuropathology. Nikiforova et al.[45] established an amplification-based GlioSeq panel that detected mutations, gene fusions, and gene copy number changes known to be found in adult and pediatric CNS tumors. This panel was designed to detect >1360 CNS tumor-related mutational hot spots in 30 genes, 24 copy number alterations, and 14 fusion types, involving BRAF, FGFR3, and EGFRvIII using FFPE or snap-frozen tissues with a turnaround time of 7 days.[45] With the GlioSeq panel, clinically relevant genetic alterations were found in 52/54 examined brain tumors. For example, detection of an H3F3A point mutation would strongly suggest an infiltrative glioma over a noninfiltrative tumor.[45] Similarly, a large research group at Heidelberg, Germany, demonstrated the value of NGS assay in facilitating tumor diagnosis. The group led by Sahm et al.[46] reported an establishment of a customized, hybrid capture-based target sequencing panel that detected SNV, fusions, and copy number aberrations from FFPE tissues. The panel was designed to study the entire coding and selected intronic and promoter regions of 130 genes recurrently altered in brain tumors.[46] Using stringent criteria of at least 60% of cancer cells in the tissue samples, the NGS assay reached 100% specificity and 99% sensitivity in SNV detection. The concordance between target panel and methylation array in detection of copy number alterations reached 100%.[46] Importantly, the molecular alterations detected by NGS assay alone could infer molecular subgroups of diffuse glioma in the updated 2016 WHO Classification. The NGS assay would also detect potential actionable aberrations in over three-quarter of glioblastomas. The application of target sequencing in routine diagnosis was further illustrated by Zacher et al.,[47] who developed a glioma-tailored, 20-gene sequencing panel at high sensitivity for molecular diagnostic tool in gliomas. They showed that incorporation of NGS panel findings would lead to diagnostic changes from a histological-based WHO 2007 Classification to an integrated-based WHO 2016 Classification.

Aside from diagnostic utility, target sequencing can also be a robust approach for prognostic evaluation in brain tumors. Dubbink et al.[48] demonstrated the application of target sequencing to identify molecular groups of diffuse glioma with very different outcomes. They found that NGS approach is more powerful in prognostication compared to the classical histological assessment. Furthermore, target sequencing is useful in selecting patients who can benefit from target therapy and those who can be harmed by inappropriate target therapy.[49] For instance, Cole et al.[50] showed that target sequencing panel could be used in rare pediatric brain tumors to identify driver mutations for target therapy. In the future, it is anticipated that NGS assay can be employed to detect known resistance mutations in patients treated with target therapy.

Our research group has also designed a PCR-based target sequencing panel for detecting a wide range of genetic alterations in CNS tumors. The customized NGS panel encompasses 1879 amplicons covering 47 genes relevance in CNS tumors, and it can detect various types of mutations such as point mutations and small insertion/deletions as well as copy number changes with an input as little as 10 ng nucleic acid. The gene panel is listed at the website: Similar to the two panels described above, our assay can be employed on FFPE tissue, which is a main routine clinical material and yields DNA in limited quantity due to degradation and cross-linking. Although FFPE is known to introduce sequence artifacts, such as uracil and thymine deriving from cytosine deamination, the problem can be minimized through biochemical methods, as in the case of our assay.[51],[52]

An overview of the assay is shown in [Figure 1]; the overall workflow takes about seven working days. In brief, FFPE sample is first assessed by pathologists under microscope for quality evaluation. Purified genomic DNA is then quantified and qualified before NGS library preparation. In DNA library preparation, DNA is subjected to fragmentation, end repair, and A-tailed in a multi-enzyme reaction. Treated nucleic acids are then ligated at their 5' ends with sequence-specific adapter containing sample index. Targeted enrichment and amplification are conducted to yield final DNA library. Quality control of the constructed library is performed before the library is sequenced on MiSeq® Personal Sequencer platform. Data analysis is achieved with an automated, integrated bioinformatics pipeline to generate variant calling for further interpretation and reporting.{Figure 1}

As an example, we examined molecular aberrations with our target panel in a congenital high-grade glioma, which is a very rare entity. The test achieved an average of 500-fold coverage and at least 100-fold coverage for 95% of target bases. Our panel correctly detected SNV for ATRX, EGFR, GLI2, MET, NF1, PIK3R1, PTCH1, SETD2, and TP53 at an allele frequency >10%. Sanger sequencing confirmed the genetic changes [examples are shown in [Figure 2].{Figure 2}

 Future of Next-Generation Sequencing Assay in Neuro-Oncology

As the cost of NGS is falling and the development of NGS is accelerating, it is expected that NGS will become more important in the clinical setting. In the very advanced centers, NGS data are already included in the reports. It is anticipated that neuro-oncologists soon have to interpret NGS data and translate them into useful information or actions at the clinics. It remains to be seen how comprehensive molecular profiling through NGS technologies will allow the refinement of tumor classification. Finally, in the era of precision medicine, NGS can guide new clinical trial designs, such as umbrella or basket trials, and patients will be allocated to therapy based on their tumor molecular profiles.[53]

Financial support and sponsorship

The study of target panel sequencing was supported by the National Natural Science Foundation of China (NSFC, grant number 81472373) and Shenzhen Science Technology and Innovation Commission (reference number JCYJ20170307165432612).

Conflicts of interest

There are no conflicts of interest.


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