Next generation sequencing is a technology used for determining the sequence of genetic material (DNA or RNA) to gain insight into the genetic variation associated with diseases such as cancer. Next generation sequencing finds its application in detection of cancer mutation, genetic testing for hereditary cancer, personalised cancer treatment, detection of circulating cancer DNA. Next generation sequencing can be used to test multiple tumor specific markers simultaneously, thus saving money, time and patient samples.
What is NGS?
Next-generation sequencing (NGS) is a technology used for determining the sequence of genetic material (DNA or RNA) to gain insight into the genetic variation associated with various diseases and biological phenomena. Before the advent of NGS, the Sanger sequencing technique was used to determine the DNA sequence. However, due to the limitations of Sanger sequencing in throughput and relatively high costs, it was difficult to sequence a large DNA/RNA sample. The NGS technology was developed to overcome the shortcomings of Sanger sequencing. NGS technologies can parallelly sequence thousands of DNA molecules simultaneously with high throughput and speed (Sanger et al., 1977).
Oncologists often search for specific mutations during cancer biopsy as they can help identify important targets for targeted therapy and specific treatment options among cancer patients. Various types of cancer-specific mutations are observed for different cancer types. Different cancer types also differ in the number of potential target mutations. Previously, multiple tests had to be performed for the detection of a few such targeted mutations in cancer which was time-consuming as well as costly. Further research in personalised medicine and cancer-specific treatment has led to the identification of potentially important target mutations for different cancer types. In this scenario, NGS is the technology that allows clinicians to test many cancer specific genes simultaneously. NGS can be performed from various types of samples, which includes tumor and blood samples of cancer patients. NGS can also be performed from a very small amount of DNA isolated from circulating cancer cells in the patient’s blood sample (Ewalt et al., 2019).
Different NGS platforms
Currently, there are various NGS platforms commercially available: the Illumina Miseq and Hiseq, the Roche 454 GS, Ion torrent, and the Life Technologies SOLiD (Guan et al., 2012). Some NGS platforms, such as Ion torrent and Illumina Miseq, are more favourable for clinical use due to their shorter run time and increased flexible throughput.
Types of NGS genetic testing
- Whole-genome sequencing
Whole-genome sequencing involves sequencing the patient’s entire genome and mapping it back to the human genome database to detect mutations. The main advantage of whole-genome sequencing involves sequencing the entire genome, including both coding (regions that translate into proteins) and non-coding regions (regions that do not translate into protein but have regulatory functions) of the DNA sequence. Therefore, whole-genome sequencing is mostly used to identify rare and novel mutations (Guan et al., 2012).
- Whole-exome sequencing
In whole-exome sequencing, the exons (regions that translate into mRNA) of all the known genes are sequenced simultaneously. The significant advantage of exome sequencing over whole-genome sequencing is that it is cost-effective and less time-consuming. Whole-exome sequencing is frequently used for the identification of cancer-associated genes (Shoubridge et al., 2012).
- Transcriptome sequencing
Transcriptome sequencing, also referred to as RNA-seq, involves sequencing cDNA (complementary DNA) fragments generated by reverse transcription of RNA. Clinicians can determine the RNA expression profile and splicing profile based on transcriptome sequencing results.
- Epigenetic Analysis
Epigenetic analysis is an emerging application of NGS used to characterize epigenetics in cancer. The potential diagnostic and prognostic application of DNA methylation and protein-DNA binding profiles have been found to understand the development of various cancer types (Esteller, 2008).
Advantages and Disadvantages of NGS
There are many advantages and disadvantages of NGS based genetic testing in comparison to the traditional genetic testing approach for individual markers.
Advantages of NGS
- NGS can be used to test many genes simultaneously, thus saving money, time and patient samples.
- NGS can help detect mutations that are needed specifically and help identify new markers that may provide additional treatment options.
- Genetic testing using NGS offers a broad collection of markers for researchers and clinicians, which may help identify new patterns of patients’ responses.
Disadvantages of NGS
- NGS is not suited for the study of a single marker as it takes more time in comparison to techniques designed for the analysis of individual markers.
- NGS is significantly costlier for studying single or few markers compared to techniques designed for individual marker study.
- NGS results are usually difficult to interpret. Therefore, misinterpretation of the NGS result might lead to wrong treatment decisions that could harm the patient (Ewalt et al., 2019).
Who should get NGS testing?
Before going for any test, the importance of the test in the treatment regime has to be looked into carefully. If the test does not have any significant role in the treatment outcome, it is not advisable to conduct the tests, especially if the concerned test is time consuming, invasive and comes at a high cost. NGS genetic testing is more appropriate for certain cancer types where several molecular markers are commonly observed. For cancer types, NGS may help identify mutations that are associated with a new treatment option for the patient; however, the odds of this are usually very low (Ewalt et al., 2019).
Application of NGS in Clinical Oncology
- Identification of novel cancer mutations
NGS technologies have facilitated the accurate and efficient detection of rare and novel somatic mutations. The use of NGS in cancer research has successfully helped to identify novel mutations in various cancer types, including renal cell carcinoma, bladder cancer, small-cell lung cancer, acute myelogenous leukaemia, prostate cancer, and chronic lymphocytic leukaemia. Whole-genome and whole-exome sequencing has helped to identify various novel and rare genetic mutations and the associated potential therapeutic targets for cancer types (Tran et al., 2012).
- NGS in hereditary cancer syndrome genetic testing
Almost 5%–10% of all cancer types are hereditary. In the US and Europe, for more than ten years, genetic testing has been routinely used for hereditary cancer patients (Garber & Offit, 2005). The development of NGS opened many opportunities for genetic testing. NGS provides a promising solution for the detection of rare genetic aberrations. NGS significantly improves the variation detection rate due to its ability to check for multiple genes simultaneously. Many hereditary cancer patients were reported negative for genetic abnormalities, but with NGS, it was easier to identify the causative mutations. The use of NGS for hereditary cancer syndromes may soon find its use in clinical practice. Data obtained using whole-genome or whole-exome sequencing of various malignant tumors has been used in various personalised cancer therapy clinical trials.
- NGS for personalised cancer treatment
Apart from identifying somatic and novel genetic mutations, NGS can also improve personalised medicine for cancer treatment. Many studies have used NGS for developing personalised cancer treatment. For example, NGS was used to treat pancreatic cancer. It was also used in non-small cell lung cancer to detect epidermal growth factor receptor (EGFR), which was found to have significant clinical and pathogenetic implications for non-small cell lung cancer patients. In addition, NGS was also used to detect PML-RARA fusion gene in patients with acute promyelocytic leukemia, which helped in modifying the therapeutic schedule of cancer patients. Studies have demonstrated the practical clinical applications of NGS in cancer treatment.
- Detection of circulating cancer DNA
NGS has been long used to detect somatic mutation and genetic variation in the circulating DNA of cancer patients’ blood for effective management and diagnosis of cancer. Tumor suppressor genes like TP53 are highly mutated in cancer patients, and rare mutations in this gene are very difficult to detect and identify. NGS serves as a cost-effective method to detect such mutations in the TP53 gene.
When should an NGS test be considered clinically?
Many researchers support a conservative approach to genetic testing of cancer. They have suggested using NGS testing only in clinical trials or controlled research environments. In contrast, many others have proposed the routine use of NGS for genetic testing for patients with metastatic cancer. The following are the areas where the use of NGS can be attributed in clinical settings.
For certain cancer types, such as advanced non-small cell lung cancer, the first line of cancer treatment depends on the genetic status of multiple molecular markers. NGS can be used in such a context to detect and identify multiple genetic alterations and to obtain information on patient personalised cancer therapy. The fact that most available samples for the treatment of advanced lung cancer have low tumor cell content (patients are usually diagnosed with small biopsies) is of great importance in this case. NGS may also find its application in other types of tumors for which multiple screening is essential, such as melanoma (BRAF, KIT) or colorectal cancer (RAS, BRAF).
- Infrequent molecular alterations
Another reason to support the use of NGS genetic testing in lung cancer is that rare molecular alterations occurring in other cancers may also be present in a small proportion of NSCLC, such as those suitable for tumor agnostic or transversal treatment with NTRK TKIs, transversal or microsatellite instability, NGS facilitates the testing of these additional and rare biomarkers. Therefore, the integration of genomic markers is becoming increasingly important for the treatment of cancer patients. NGS could also be very useful in identifying genetic responses to anticancer drugs or personalised therapies.
- Rare cancers
Rare cancer types are often seen in a small number of patients, and often no standard second-line treatment is prescribed. Because of their rarity, these malignancies are often not studied in conventional phase 3 clinical trials to determine the value of new treatments. Some examples are sarcomas, biliary tract cancers, mesothelioma, and cancer of unidentified primary origin. In rare malignancies, the majority of mutations detected using NGS confers no clinical benefit to patients. However, clinical trials based on NGS data may provide treatment alternatives to investigational drugs for patients with limited treatment options.
- Clinical trial networks
The use of NGS and precision medicine might help patients to get personalised cancer treatment. Of course, this requires new clinical trial designs and organised clinical trial networks. There is currently unprecedented progress in the development of early-stage cancer clinical trials in the form of adaptive studies using NGS based clinical trial designs to optimise biomarkers and drug co-development processes.
- Clinical judgement-oriented testing
Because NGS testing is often not indicative, clinical judgement should therefore precede molecular testing in cancer patients. In general, NGS testing should not be recommended to patients unless genomic testing results have an important effect on the clinical management of cancer patients. For example, somatic gene panels are usually not required in early-stage patients receiving final treatment. They will not have effective NGS-mediated changes beyond what can be determined from standard assessments (ER, PR, HER2 for breast cancer). Patients with rapidly growing cancer, poor performance status, or patients with a life expectancy of less than three months should not be molecularly profiled. These patients are most likely to be referred for palliative care (Guan et al., 2012).
Future of NGS testing in cancer
NGS is inextricably linked with the implementation of precision medicine in oncology. In its current state, it is unlikely to preclude conventional prognosis, but it provides a complete picture of the pathogenesis of cancer. RNA sequencing using NGS can provide information about the relative expression of mutated genes. Furthermore, immunotherapy is increasingly finding its importance in cancer therapy, particularly in melanoma (Robert et al., 2015). NGS sequencing may be of great importance for predicting response to immunotherapy. Exome sequencing can be combined with mass spectrometry to determine which neoantigens are effectively presented by the major histocompatibility complex (MHC) (Yadav et al., 2014).