A short presentation on the applications of next generation sequencing in cancer treatment. All content displayed and shared remains the courtesy of Taylor's University. Published 17/10/18.

1. NEXT
a p p l i c a t i o n s i n c a n c e r t r e a t m e n t
GENERATION SEQUENCING
LEE WAI LEONG (0330625)
MATTHEW WONG TZE JIAN (0326319)
TIONG QI EN (0326320)
P’NG CHENG (0326594)
HAMDHA FATHMATH (0331745)

2. WHAT IS NEXT GENERATION SEQUENCING (NGS)?
• Next-generation sequencing (NGS) technology, also known
as massively parallel sequencing, includes DNA
sequencing and RNA sequencing (Wang and Xu, 2017).
• Next-generation sequencing (NGS) is a high-throughput
methodology that enables rapid sequencing of the base
pairs in DNA or RNA samples.
• Supporting a broad range of applications, including gene
expression profiling, chromosome counting, detection of
epigenetic changes, and molecular analysis, NGS is driving
discovery and enabling the future of personalized medicine
(Thermo Fisher Scientific, 2018).
• DNA sequencing includes (Corless, 2016; Serratì et al., 2016; Kamps et al.,
2017):
o Whole-genome sequencing (WGS)
o Whole-exome sequencing (WES)
o Targeted sequencing

4. • A ssDNA sequencing primer hybridizes to the end of the
strand (primer-binding region), then the four different dNTPs
are then sequentially made to flow in and out.
• When the correct dNTP is enzymatically incorporated into
the strand by polymerase enzyme, it causes release of
pyrophosphate (PPi).
• In the presence of ATP sulfurylase and adenosine, the PPi is
converted into ATP.
• This ATP molecule is used for luciferase-catalysed conversion
of luciferin to oxyluciferin, which produces light that can be
detected.
• The relative intensity of light is proportional to the amount of
base added.
• Firstly, the sequencing primers and templates are fixed
to a solid support.
• The support is exposed to each of the four DNA bases,
which have a different fluorophore attached.
• Only the correct base anneals to the target and is
subsequently ligated to the primer.
• The solid support is then imaged and nucleotides that
have not been incorporated are washed away and the
fluorescent branch is cleaved and regenerating 3’-OH.
• The cycle can be repeated
(Atdbio, 2018)
Pyrosequencing (Roche 454)
(Mason and Griffiths, 2012)
(Atdbio, 2018)
Sequencing by synthesis (Illumina
sequencing, Solexa Technology)
(Atdbio, 2018)
(Brown, 2017)

5. • A primer of length N is hybridized to the adapter (ssDNA primer-binding region), then the DNA exposed
to a library of 8-mer probes which have different fluorescent dye at the 5' end and a hydroxyl group at the
3' end.
• Bases 1 and 2 are complementary to the nucleotides to be sequenced whilst bases 3-5 are degenerate and
bases 6-8 are inosine bases.
• Only a complementary probe will hybridize to the target sequence, adjacent to the primer.
• DNA ligase is then uses to join the 8-mer probe to the primer.
• The linkage between bases 5 and 6 allows the fluorescent dye to be cleaved from the fragment which allows
fluorescence to be measured and also generates a 5’-phosphate group which can undergo further ligation.
• Once the first round of sequencing is completed, the extension product is melted off and then a second round
of sequencing is performed with a primer of length N−1.
• Many rounds of sequencing using shorter primers each time (i.e. N−2, N−3 etc) and measuring the
fluorescence ensures that the target is sequenced. (Atdbio, 2018)
Sequencing by ligation (SOLiD Platform)
(Atdbio, 2018)
Ion semiconductor sequencing (Ion Torrent sequencing)
(Voelkerding, Dames and Durtschi, 2009)
• DNA library fragment is flooded sequentially
with each nucleoside triphosphate (dNTP).
• The dNTP is then incorporated into the new
strand if complementary to the nucleotide on
the target strand.
• Each time a nucleotide is successfully added,
a hydrogen ion is released, and it detected
by the sequencer's pH sensor (semiconductor
chip).
• As in the pyrosequencing method, if more
than one of the same nucleotide is added, the
change in pH/signal intensity is
correspondingly larger.
(Atdbio, 2018)

6. COMPARISON BETWEEN NGS AND FIRST GENERATION GS
FIRST GENERATION SEQUENCING
▪ Sanger and Maxam-Gilbert sequencing
▪ Benefits
• Fast, cost effective sequencing for low number
of targets (1 – 20 targets)
▪ Challenges
• Low sensitivity (limit of detection 15% to 20%)
• Low discovery power
• Not as cost effective for the use of high number of
targets
• Low scalability due to increasing sample input
requirements
NEXT GENERATION SEQUENCING
▪ Illumina
▪ Benefits
• Higher sequencing depth enables higher sensitivity
• Higher discovery power
• Higher mutation resolution
• Larger scale of data produced with same amount
input of DNA
• Higher sample throughput
▪ Challenges
• Less cost effective for sequencing low number of
target (1 to 20 targets)
• More time consuming for sequencing low number
of targets (1 to 20 targets)
(illumina, 2018)

7. INTRODUCTION
Eradication of Large Solid Tumors by Gene Therapy with a T-cell
Receptor targeting a Single Cancer-Specific Point Mutation
(Leisegang et al., 2015)CASE STUDY 1
▪ T-cells of patients can be engineered to express antigen receptors of a chosen
specificity.
▪ T-cells were engineered to express T-cell receptors (TCRs) that recognizes shared (or
trunk) mutation.
▪ This recognition targets a single cancer-specific point mutation that eradicates a
progressively growing, genetically heterogenous cancer.
▪ This is the first study to show that under the right conditions, mutation-specific TCR
gene therapy provides an effective, truly tumour-specific cancer treatment.

8. UV irradiation caused the development of an
autochthonous tumour (8101). The tumour
was excised and 2 individual tumour
fragments were adapted to culture (Bulk) or
analysed separately using whole-exome
sequencing. Heart-lung fibroblasts (HLF)
were obtained from the same mouse as tissue
control.
Using ‘reverse immunology’, suitable neoepitopes were identified as therapeutic targets.
• 3,710 mutations were identified in Bulk using WES.
• 1,207 of the mutations were shown to be expressed using RNAseq.
• To determine which of the 1,207 mutations would be predicted as preferred epitope based on MHC
affinity, a computer algorithm was used – 1,106 potential neoepitopes were determined.
• Further analysis showed that only 194 of the mutant peptides would bind to MHC molecules at a
greater affinity.
• Upon further narrow-down, only 15 mutations were identified that would potentially be expressed as
antigen.
• Among those, mp68 was found to be a highly expressed antigen with the greatest MHC affinity.
Phylogenetic representation of somatic mutational frequency in the 8101 tumour
identifies mp68 as trunk mutation. Green represents the trunk mutation p68 that
was found in all fragments. Branches shown in blue lack the mutation p53.
Numbers on the top of each branching indicate unique mutations in 20 individual
fragments and the Bulk tumour cell culture of 8101 (Leisegang et al., 2015).

9. 1D9-engineered T-cells efficiently lyse Bulk
tumour cells. Specific lysis of Bulk and control
MC57 tumour cells was analysed in vitro using 1D9td
T-cells. One representative experiment of three is
shown (Leisegang et al., 2015).
Longitudinal confocal microscopy imaging of MC57-mp68
cancer cell in mouse and tumour vessel destruction following
adoptive 1D9 T-cell transfer.
▪ Cross-presentation of mp68 by the
tumour stroma causes rapid
destruction of cancer cells by 1D9 T-
cells entering the tumour.
▪ Day 0 is the time when the first 1D9 T-
cell was detected in the skinfold
window (see magnification, red).
▪ Viability of tumour tissue was
analysed by monitoring GFP
expression (cancer cells, green) and
blood flow (see bottom magnification,
DiD-stained erythrocytes, purple).
In Summary
▪ Established 8101
tumours are eradicated
byTCR gene therapy
when all cancer cells
express mp68 antigen
at high levels.
▪ Local tumour
irradiation followed by
adoptiveT-cell transfer
reduces relapse of 8101
tumours expressing the
autochthonous mp68
antigen.
(Leisegang et al., 2015).

10. CASE STUDY 2
▪ The Siteman Cancer Center in partnership with
Washington University’s genomics and pathology services
(GPS) is taking what was learned from decoding the human
genome and translating it into life-changing outcomes for
cancer patients.
▪ The GPS team is using NGS to help doctors in making
personalized medicine for their cancer patients.
▪ Cancer happens due to genetic changes in the DNA
sequence over time that cause the cells to lose their normal
function. As a result, they grow and divide uncontrollably
and form a tumour.
(Siteman Cancer Center, 2015)
NGS CAN BE USED: (Siteman Cancer Center, 2015)
NON-SMALL-CELL
LUNG CANCER (NSCLC)
▪ Includes adenocarcinoma
(gland-forming)
▪ Squamous cell carcinoma
▪ Large-cell carcinoma
histosubtypes
▪ Represents
approximately 85% of all
new lung cancer cases
(Travis, Brambilla and Riely, 2013)

11. DIAGNOSIS ANDTREATMENT:
▪ Sue was diagnosed with stage 4 non-small-cell-
lung cancer and benefited from chemotherapy
for some time but then developed worsening
shortness of breath and cough due to spread of
her cancer.
▪ Targeted NGS was done and revealed a gene
mutation.
▪ Sue volunteered received clinical treatment as
part of the clinical trial.
OUTCOME:
▪ The metastasized tumour disappeared right
away.
▪ Original tumour shrank in size (Complete
response).
▪ Cancer patients can be treated individually with
drugs that specifically target their tumour cells
based on its genetic code.
WHY PERSONALIZED MEDICINE?
(Siteman Cancer Center, 2015)
(Siteman Cancer Center, 2015)
I n S u m m a r y :
▪ NGS is an important
tool to design
personalized medicine
for more personalized
treatments by using
information from the
genetic make up of the
patient.
▪ With NGS, cases
like non-small-cell-
lung cancer had been
targeted to reveal the
gene mutation where
personalized medicine
is applied and
successfully treated the
patient.
(Siteman Cancer Center, 2015)

12. FUTURE PROSPECTS(Gonzalez-Garay, 2014) (Guan et al., 2012)
ADVANTAGE & CHALLENGES
▪ More comprehensive analysis of
variation type
• More single-nucleotide variants
and small insertions/deletions
compared to Sanger's
• NGS able to fully detect all
mutation types of target genes
and even chromosomal
abnormalities
▪ Less DNA is required
• In this case Sanger sequencing
for BRCA1 and BRCA2 requires
approximately 3 µg of DNA,
whereas in chip-captured NGS
sequencing requires about 500 ng
ADVANTAGES
▪ Data analysis and computing infrastructure
▪ Specialized programs must be used for NGS
projects as hundreds of gigabytes of data will
be generated from NGS which requires a high-
end personalized computer.
▪ It is impractical for small diagnostic
laboratories and clinics to satisfy these
essential requirements especially the cost
▪ Interpretation of variation data
▪ A difficulty in interpreting and accurately
define clinically significant variants as few
variants contributes to a disease
▪ 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
▪ Ethical issues
▪ By using NGS it is ease to patient's genetic
results which may lead to discrimination based
on genetic information and social disorder,
especially in job-hunting and applications for
health insurance.
CHALLENGES

13. IN A NUTSHELL
▪ Next-generation sequencing is a high-throughput method used to rapidly sequence the base
pairs in DNA or RNA samples.
▪ NGS has been applied in gene expression profiling, chromosome counting, detection of epigenetic
changes, molecular analysis, and personalized medicine.
▪ NGS has led to the development of genetically engineered T-cells that successfully eradicated a
progressively growing, genetically heterogenous cancer.
▪ NGS has led to the development of highly personalized patient-specific medication that
specifically targeted the specific genetic mutations found in the tumour of the patient.
▪ NGS can detect almost all types of mutations in targeted genes and requires significantly less
DNA or RNA sample. The downside of NGS is that proprietary software and high-end
equipment are needed. Besides that, an individual’s privacy would be at great risk and
corporations can use the ‘weak points’ in their genes to discriminate.
▪ Apart from becoming the standard set of newborn screening tests, NGS will soon be used to
establish a large database that houses disease-causing mutations and pharmacogenomics
markers. NGS will also be adapted by oncologists in the near future for personalized cancer
treatment and other genetic disorders.

14. REFERENCES
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sequencing [Accessed 30 Sep. 2018].
Brown, S. (2017). Sequencing-by-Synthesis: Explaining the Illumina SequencingTechnology. [online] BitesizeBio.Available
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[Accessed 30 Sep. 2018].
Corless, C. (2016). Next-Generation Sequencing in Cancer Diagnostics. TheJournal of Molecular Diagnostics, 18(6),
pp.813-816.
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15. REFERENCES
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Nakamura,Y. and Schreiber, H. (2015). Eradication of Large SolidTumors by GeneTherapy with aT-Cell Receptor
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Mason, J. and Griffiths, M. (2012). Molecular diagnosis of leukemia. Expert Review of Molecular Diagnostics, 12(5), pp.511-526.
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