Next Generation Sequencing for Cancer Clinical Diagnostics
Posted By HealthcareOnTime Team
Posted on 2021-08-25
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?
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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
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.
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,
diseases) are on a rise among Indians. Expression
of susceptible genes is greatly influenced by lifestyle and environmental factors.
Pharmacogenomics and Personalized Medicine
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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
and with informatics assistance, has grown to develop our understanding
of complex physiological interactions in both, the human body