Next Generation Sequencing for Cancer Clinical Diagnostics

What is next generation sequencing testing?
It was the year 1869 when Swiss researcher Friedrich Miescher accidentally made one of the biggest discoveries. He isolated 'Nuclein' which now is known as DNA or Deoxyribose Nucleic Acid. Watson and Crick's elucidation of the DNA structure in 1953 intrigued the science community to further study and understand the complexities around genome in general. Starting with Fredrick Sanger's dideoxy sequencing method to determine the precise order of nucleotides in a short stretch of DNA to the automations which aided in the Human Genome Project to the Next-generation Sequencing (NGS) platforms today, genomic research has undergone a massive technological overhaul.

Should next generation sequencing tests be performed on all cancer patients?
Next generation sequencing in Clinical Oncology Cancer is a highly complex and heterogeneous disease and more than 200 types have been described till date. Cancer burden in India is on rise with cases of breast cancer, cervical cancer, oral cancer and lung cancer being more commonly reported. Numerous cancer therapies are currently available depending on the cancer type and stage of the diseases , but not all patients experience a favorable prognosis, thereby affecting their life expectancy and suffering relapse. Definitive diagnosis and staging of cancer has always relied on histological evaluation of the affected tissue apart from studying certain biochemical markers. More recently focus has shifted on molecular profiling i.e. identifying prognostic and predictive genetic signatures for accurate evaluation of driver factors, cancer aggressiveness and probabilizing treatment outcome.

What is next generation sequencing for cancer?
Use of Next generation Sequencing technology in cancer management has provided treating oncologists an opportunity to understand specific mutation pattern associated with prognosis outcome (good or bad) in patients with varied types of cancers thus aiding in precision medicine i.e. providing tailor-made treatment options to patient based on mutation profile and progression of the cancer. Next generation Sequencing detects Single Nucleotide Variations (SNV), small duplications, small insertions and deletions (Indels), Structural Variations (SVs), large deletions or Copy Number Variations (CNVs) present in the genome depending on the assay design. NGS provides an edge over older-generation sequencing technologies with the ability to assess mutation status in cases with low tumor burden and also detect rare and sub-clonal variations.

What is SOLiD tumor NGS?
Next generation Sequencing in Solid Tumors Next generation Sequencing assays with its ability to assess genomic regions with up to 1000X coverage has played a key role in identifying mutations in genes associated with multiple signaling pathways including tumor suppressors which has aided in providing crucial chemotherapy outcome information for majority of solid tumors. In addition to mutations in the common TP53, EGFR and KRAS genes, use of Next generation Sequencing has also facilitated in identifying gene amplifications in CDKN2A, cell cycle driver genes (CCND1-3, CDK4), PIK3CA, FGFR-1 and MET genes in lung cancer patients. With BRCA1 and BRCA2 genes widely studied in breast and ovarian cancer cases, the advent of Next generation Sequencing has further lead to identifying pathogenic variants in cancer susceptibility genes including PALB2, CHEK2 and ATM, NF1 and mismatch repair genes (MLH1 and MSH2), and genes related to hereditary cancer syndromes (CDH1, PTEN, STK11 and TP53) that have been proven to impart an increased risk for breast and ovarian cancer.

Next generation Sequencing in Leukemia Acute Lymphoblastic Leukemia (ALL) is the most commonly reported cancer type in children with a high risk of relapse and exhibits presence of fusion genes due to chromosome translocations or inversions. Acute Myeloid Leukemia (AML), a cancer of the myeloid line of blood cells, is the most common type of leukemia among adults. Genetic mutations in signaling and kinase pathway genes (FL13, PTPN11, NF1 and KIT) and epigenetic modifications in DNMT3A, CEBPA have been shown to bear prognostic implications identified using Next generation Sequencing.

Next generation Sequencing in Cancers Of Unknown Primary Site c NGS has also played a key role in understanding the varying molecular signature behind cancers of unknown primary origin which continue to pose a major treatment challenge to oncologists. Irrespective of the origin, Next generation Sequencing by liquid biopsy helps identify underlying mutations involved in prognosis which also makes use of certain targeted and chemotherapeutics a possibility for such cases. Most patients diagnosed with a Cancer of Unknown Primary (CUP) site has a dismal prognosis and low life expectancy. Use of Next generation Sequencing data also aids in determination of Total Mutation Burden (TMB) and Microsatellite Instability (MSI) thereby assisting in providing pathway-specific (targeted) therapies in CUP.

What is next generation sequencing assays?
Next generation Sequencing multiplexing can be accommodated in three different assay categories depending on need - Whole Exome Sequencing (WES), Whole Genome Sequencing (WGS) and Targeted Sequencing (TS). WGS determines the sequence of the nucleotides in the entire genome of an organism. Human genome comprises - 3 X 10 bp having both coding and non-coding regions. Coding region includes sequencing of proteins coding genes. Because the entire genome is being sequenced, changes in the non-coding regions (repetitive sequences, regulatory sequences, introns and pseudo-genes) that may affect transcription or post-translational modifications of the proteins also get recorded. Roughly about 30 - 60X coverage in WGS generates --60-350 GB of data. Sequencing depth is related to accuracy of the sequence alignment and ability to call variants as it describes the number of times a given nucleotide in the genome has been read. Use of WGS is more fruitful in research setting than in actual clinical practice which is time dependent .WES involves studying only the exomes or the protein-coding regions of the genome (-6 x 10 bp) and about 150 - 200X depth can be achieved which generates about --5-20 GB of data. TS involves use of a pre-designed panel which has a selected number of specific set of genes or gene regions known to harbor mutations that contribute to pathogenesis of diseases . Roughly about 200 - 1000X depth can be achieved in TS which generates - 100 MB-5 GB of data. Greater depth of coverage allows TS to pick out low frequency

What are some challenges of NGS?
Large data generation from Next generation Sequencing has brought along obvious and obtrusive informatics hallenges associated with data analysis and management. Integration of Artificial Intelligence (Al) approaches such as Machine Learning (ML), Deep Learning (DL), and Natural Language Processing (NLP) has assisted in data handling and their translation into clinical knowledge laying the foundation for precision medicine. In cancer genomics, NLP tools have been used for the automated extraction of genes, genetic variants, treatments, and conditions thereby reducing the time and effort required for information retrieval and speeding up curation, and provide novel opportunities for hypothesis - generation based on published iterature. ML involves training an algorithm to learn functional relationships from data using: data cleaning and pre-processing, feature engineering, model fitting and evaluation. De Na??ve Bayes (NB), k-Nearest Neighbors (k-NN), Artificial Neural Network (ANN) and Deep Leaming (DL) are few ML algorithms used in oncology. DL, a subset of machine learning, consists of multilayer neural networks that reduces or eliminates the need for feature engineering. Some of the common DL applications used in genomics are Deep Variant (variant caller in germline genomes), DeepSEA (predicting effects of non-coding variants) and DeepGene (somatic mutations based cancer type classification). Using these tools, SNVs and minor structural variation have been reliably called with error rates <1%.

Should next generation sequencing tests be performed only on cancer patients?
In complex diseases like primary immunodeficiencies numerous genes have been implicated in manifesting the clinical phenotype thus making NGS an obvious choice for investigation. Next generation Sequencing is not restricted to oncology research. Schizophrenia and other related neuropsychiatric disorders characterized by heterogeneous clinical and genetic presentations have their molecular etiology deciphered to a great extent due to the use of Next generation Sequencing assays. Use of WES and WGS aided in identifying roles of genes like DYNCIH1, GATAD2B, and CTNNB1 for intellectual disability, CHD8 and KATNAL2 variations for imparting increased risk of autism and role of de novo mutations in development of schizophrenia. In recent (hemoglobinopathies and hemostasis disorders) affecting all cell lineages derived from bone marrow. According to the ICMR-India State-Level Disease Burden Study 2016 report, Lifestyle diseases (diabetes , obesity , heart diseases) are on a rise among Indians. Expression of susceptible genes is greatly influenced by lifestyle and environmental factors.

Pharmacogenomics and Personalized Medicine
Pharmacogenomics refers to analyzing the relation between the human genetic profile (genomic variants) and individual differences in clinical drug response. The aim of pharmacogenomics is to optimize treatment outcome and lower the risk of adverse events, thereby enhancing quality of patient care. Novel and rare pharmaco-variants identified by Next generation Sequencing may facilitate personalized drug therapy. Imatinib (tyrosine kinase inhibitor) is an FDA approved drug that is used in Chronic myeloid leukemia patients positive for fusion protein BCR-ABL. Certain Next generation Sequencing panels have been made commercially available for translational purpose like the TruSight" tumor sequencing panel by Ilumina.

Next generation Sequencing in Prenatal Testing and Carrier Detection
Recently, targeted NGS is also being used for the cell free DNA-based non-invasive prenatal testing for screening the risk of trisomy 21, 18 and 13 and sex chromosome aneuploidies. It is also being used to study the risk or presence of single-gene disorders like betathalassemia and cystic fibrosis. NGS is also being used to ascertain carrier status of any individual with rare familial genetic disorders. The technology of Next generation Sequencing continues to deliver in every aspect of preventive as well as sick-care Health market and with informatics assistance, has grown to develop our understanding of complex physiological interactions in both, the human body and mind