Applications of NGS in cancer treatment

1. APPLICATION OF
NEXT GENERATION
SEQUENCING IN
CANCER
TREATMENT
By :Prilli Arista Fernanda , Seow Wan Xin, Tan Regine , Nur Suhaida

2. Next Generation Sequencing (NGS) ?
• Next Generation Sequencing (NGS) is a high speed
and high throughput technique for generating
millions of sequences at one time.
• This technique is used to analyze organisms at a
genomic level and allows researcher to sequence, re-
sequence and compare data.
• By using this new technology, it allow us to generate
quantitative and qualitative sequence data within
short period and lower cost compared to Sanger
sequencing.
(Behjati and Tarpey 2013 )

3. • NGS has been widely implemented for whole genome
sequencing, whole exome sequencing, and any other
sequencing, which is a great potential for NGS
application in disease management and treatment,
genetic counseling, and risk assessment.
• The technology can be used for molecular diagnosis
of genetic disease and infectious disease, prenatal
diagnosis, carrier detection, medical genetics and
pharmacogenomics, cancer molecular diagnosis and
prognosis.
(Guan et al. 2012)

4. Application of NGS

5. 1. Application of NGS in Clinical Oncology
Identification of novel cancer mutations using NGS
• NGS has been successfully utilized to
identify novel mutations in a variety of
cancers such as bladder cancer, renal cell
carcinoma, small-cell lung cancer, prostate
cancer, acute myelogenous leukaemia and
chronic lymphocytic leukaemia.
• Whole-genome or whole-exome sequencing
enables numerous novel genetic
aberrations and associated potential
therapeutic targets to be found in many
cancers.

6. • For example, PML-RARA fusion genes cause a rare
form of acute promyelocytic leukaemia.
• PML-RARA cannot be detected with standard
cytogenetic techniques but it was successfully
identified by using whole-genome sequencing with
NGS.
• NGS analysis followed by reverse transcription-
polymerase chain reaction and direct sequencing
revealed distinct breakpoints within exon and
intron.
• After identification and consolidation therapy, the
patient achieved complete remission but the
disease relapsed again (Yin CC 2016).

7. Figure 1: Model of acute promyelocytic leukaemia initiation (Gaillard et al. 2015).
- PML/RARA initially exercises subtle changes within the promyelocyte compartment.
- PML/RARA also directly represses transcription.
- The fusion protein also leads to increase in cell cycle genes, which promotes proliferation
and the expansion of the promyelocyte compartment through deregulation of a p53-
mediated axis.
- Secondary lesions need to accumulate in these cells in order to bypass the dominant
maturation program to generate an acute phenotype.

8. 2. NGS in hereditary cancer syndrome
genetic testing
• Traditional method for genetic testing of
hereditary cancer is time consuming, high
cost, and low throughput because genes
related to hereditary cancers are very large
and there is no particular mutation hot spot.
• NGS provides better solution to detect novel
and rare variations because it allows testing of
multiple genes at once.
• It is easier to find causative mutations for
hereditary cancers and it improves its
transition into clinical practice.
• It is useful in hereditary breast and ovarian
cancer (Guan et al. 2012).

9. • Every patient's cancer
contains a specific pattern of
DNA mutations and
alterations. The same
diagnosis and prescription for
cancer is unable to ensure
effective treatment to every
cancer patient.
• Personalized or targeted
treatments is more effective
in treating cancer patients
based on their individual
genetic profile.
3. NGS for personalized cancer treatment
Figure 1 : Personalized medicine recognizes that
individual patients may react in very different ways
to the same treatment given for the same problem.
The goal is to tailor therapies based on a patient's
DNA profile.

10. • Through NGS technology :
- It is possible to generate a comprehensive molecular profile of a patient.
- Allows cancer genomes to be profiled very quickly and with great sensitivity.
- Able to analyze more types of genetic abnormalities than conventional DNA sequencing
technologies.
- Allow analysis of 341 of the most important cancer genes that play a role in the
development or behavior of tumors. These genes represent all “actionable targets” —
genes that can be targeted with drugs (Kiesler, 2014).
- Able to identify patient-specific therapeutic strategies and potential treatment
approaches, including current clinical trials.
- Improve rationally designed individualized medicine.
• Example :
- Targeted sequencing of 25 cancer-related genes identified a codon deletion in
KIT that has been associated with imatinib sensitivity, and subsequent treatment
with imatinib resulted in stabilization of disease (Kidd et al. 2015).

11. Figure 2: The anticipated work flow of individualized cancer treatment
based on the unique molecular prolife of a patient (Guan et al. 2012).
For a given patient, the
normal genome and tumor
genome is sequenced by
using next-generation
sequencing. The genetic
information is analyzed,
validated, and clinically
interpreted by a panel of
multidisciplinary experts. A
personalized treatment
regimen is designed based
on the unique genetics of
the tumor and the patient's
normal genome (Guan et al.
2012).

12. 4. Detection of circulating tumor DNA
(ctDNA) by NGS
• Circulating tumor DNA (ctDNA) is a promising biomarker for noninvasive
assessment of cancer burden.
• The existing ctDNA detection methods have insufficient sensitivity or
patient coverage for broad clinical applicability (Newman et al., 2014).
• Using NGS based approaches, it has high
sensitivity in detecting ctDNA and it is
possible to detect ctDNA in a large
number of advanced and localized
malignancies (Takai et al., 2015).

13. Advantages
• Applied to the clinic in many areas including prenatal diagnostics, pathogen detection,
genetic mutations, and more .
- whole exome and whole-genome sequencing can provide the clinician a comprehensive view
of the DNA aberrations, genetic recombination, and other mutations. ( in cancer diagnostic and
prognostic tool )
• Improved existing technologies such as chromatin immune precipitation (ChIP) assays –
where bound DNA was previously hybridised to microarrays (ChIP-chip)
- Now can be sequenced to determine the exact genomic sequence of the captured DNA and
more sensitive expression measurements
• Less DNA is required
eg : in Sanger sequence, BRCA1 & BRCA2 requires approximately 3 ug of DNA, whereas 500 ng is
enough for chip-captured NGS sequencing (Upadhyay et al. 2014).
Applications of NGS that are currently under development :
 Evaluation of free plasma DNA that harboring tumor-specific genome alterations to
detect early relapse or residual cancer.
- using liquid biopsy , determine the genetic landscape of solid cancer from circulation.
 Identify molecular aberrations that cause tumors which is very sensitive to certain
therapies, resulting in exceptional responses.
- Improve understanding of molecular features that can predict response to certain drugs.
(Basho & Eterovic 2015)

14. Challenges
• Data analysis and computing infrastructure
- Hundreds of gigabytes of data will be generated from NGS. It is a difficult
and complicated task for bioinformatics staff to filter redundant and huge
amounts of data ( Guan et al. 2012)
- Dealing with tumour genome , twice amount of data to be generated
( Upadhyay et al. 2014)
• Interpretation of variation data
- Because few variants contribute to disease pathogenesis, it is difficult to
accurately or to effectively assess disease risk based on current research.
- For personalized cancer treatment, filtering out tumor promoting
mutations from passenger mutations is also a challenge, especially
considering that the roles of both may change as the tumor develops
(Guan et al. 2012)

15. References
• Basho, R.K, Eterovic, A.K , Bernstam, F 2015, ‘Clinical Applications and Limitations
• of Next-Generation Sequencing, The American Journal of Hematology/Onclogy, vol.11, no.3, pp 17-22.
• Behjati, S and Tarpey, PS 2013. What is next generation sequencing?. Arch Dis Child Educ Pract Ed, 98(6), pp. 236–
238.
• Dana-Farber Cancer Institute 2016, Profile & Personalized Cancer Treatment & Research - Dana-Farber Cancer
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• Guan, Y, Li, G, Wang, R, Yi, Y, Yang, L, Jiang, D, Zhang, X & Peng, Y 2012, ‘Application of next-generation sequencing
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