APPLICATION OF NEXT GENERATION SEQUENCING (NGS) IN CANCER TREATMENT

1. APPLICATION OF
NEXT GENERATION SEQUENCING (NGS)
IN CANCER TREATMENT
Group 5
Waode Lornawati 0327027
Mush K Zehra 0326829
Dinie Aqilah Ahmad Fariz 0330846
Sivaranjani Thiagarajah 0331182

2. DNA SEQUENCING
Basic Concept & Principle
What is Next-Generation Sequencing?
● Massively parallel (Corless, 2016).
● Powerful platform that has enabled
the sequencing of thousands to
millions of DNA molecules
simultaneously.
● Revolutionizing fields : by offering a
high throughput option with the
capability to sequence multiple
individuals at the same time
(Bräutigam and Gowik, 2010).
● Perform sequencing of millions of
small fragments of DNA in parallel
(Bräutigam and Gowik, 2010).
● Providing high depth to deliver
accurate data and an insight into
unexpected DNA variation (Gagan
and Van Allen, 2015).
● Combination of template preparation,
sequencing and imaging, and genome
alignment and assembly methods.
● Highly scalable, rapid, affordable, and
accurate genome analysis (Corless,
2016).
Before NGS
● Tumor genotyping was performed only on specific
genomic loci that were known to be frequently mutated in
cancer, “hotspots” .(Gagan and Van Allen, 2015).
● Best suited to recurrent activating mutations in oncogenes
(Gagan and Van Allen, 2015).
● However, these approaches were insufficient to identify
alterations in tumor suppressors.
● in which an alteration anywhere in the gene may impact
its function or the increasingly complex area of “long tail”
hotspot alterations in oncogenes.
● Thus, current assay options involve approaches that may
capture known whole-genome whole-transcriptome
approaches (Bräutigam and Gowik, 2010).
Why NGS is Important in Cancer Treatment?
● Significant impact on the detection, management and treatment of disease.
● Significant improvements in the reliability, sequencing chemistry, pipeline analyses, data
interpretation and costs (Bräutigam and Gowik, 2010).
● Captures a broader spectrum of mutations than Sanger sequencing
● Genomes can be interrogated without bias (Gagan and Van Allen, 2015).
● The increased sensitivity of NGS allows detection of mosaic mutation (Gagan and Van
Allen, 2015).
Sequencing
● Roche/454 FLX
● Illumina/ Solexa
Genome
Analyzer
● Applied
Biosystems (ABI)
SOLiD Analyzer
● Polonator G.007
● Helicos
HeliScope

3. APPLICATIONS & IMPLICATIONS
Integrated Next Generation Sequencing and Avatar
Mouse Models for Personalized Cancer Treatment
First experiences to apply exome sequencing and
patients derived xenografts, developed in mice
from patient’s tumour tissues so called Avatar
mouse models, to personalizing cancer treatment
in the clinic in a real time.
Method used : Whole exome sequencing analysis
Sample : 25 patients with
advanced solid tumors
Avatar models were used as an in vivo platform to
test proposed treatment strategies.
Whole Exome Sequencing Analysis
● Genomic DNA from tumor and normal samples obtained from blood were
fragmented and used for Illumina TruSeq library construction (Illumina, San
Diego, CA).
● Exonic regions were captured in solution using the Agilent SureSelect 51 Mb
kit (version 4).
● Exome sequencing was performed at depths of 75x to >200x depending on
the tumor purity.
● The tags were aligned to the human genome reference sequence (hg18)
using the Eland algorithm of CASAVA 1.7 software (Illumina, San Diego, CA).
● The chastity filter of the BaseCall software of Illumina was used to select
sequence reads for subsequent analysis.
● The ELAND algorithm of CASAVA 1.7 software (Illumina, San Diego, CA) was
then applied to identify point mutations and small insertions and deletions.
● The resulting alterations are compared among tumor and normal sequence
data as well as to databases of known variants to distinguish common
variants, private (rare) germline changes, and potential somatic alterations.
● Potential somatic mutations were filtered and visually inspected. 3 in silico
methods (Polyphen, SIFT, SNP&GO) used in order to estimate the functional
significance of a given confirmed mutation.

4. Successful exome sequencing analyses
were obtained for 23 patients (2 patients
with pancreatic cancer progressed
rapidly and the procedures were
aborted).

5. The number of somatic
mutations and CNVs ranged
from 5 to 952 and from 0 to
965 respectively
The median number of
mutations was 45 and
median CNV was 6
The most relevant
alterations and the clinical
actionable genetic
alterations that could be
targeted with current drug
are extracted .
Results varied significantly
from one patient to another
with patients

6. Structural models of PI3K. Kinase domain in yellow. Mutated aa in blue.
The model on the right shows the original protein with original aa whereas
the left depicts predictive model of structural changes caused by the
mutation. Severity of the mutation estimated to be high by computational
analysis.
- Example: Patient 3
- Exome sequencing detected the p.F909C mutation in the
catalytic domain of the phosphoinositide 3-kinase protein,
leading to a volume change (from bulky F to a smaller C)
with the introduction of possible S-S bonds
- The severity is high
- Presence of GNG11 amplification suggested activation of
the Ras-Raf-MEK pathway in this tumor with wild type RAS
and RAF genes
Representative tumor growth curve of Avatar PGDX7 treated with the studied agents. PI3i: 20 mg/kg
p.o.; qd, M-F, x28d. MEKi: 4mg/kg p.o.; qd, M-F, x28d. PI3Ki + MEKi: 20 mg/kg p.o + 4 mg/kg p.o.; qd,
M-F, x28d. PI3Ki+Gemcitabine: 20 mg/kg p.o.; qd, M-F, x28d + 100 mg/kg i.p.; twice a week x28d.
Treatment with a PI3K inhibitor alone did not show evidence of
tumor control. The combination of a PI3K and MEK inhibitors is
showed to be a possible effective approach. There’s no access to
such a combination in the clinic to offer the patient. Gemcitabine
was also effective, but the patient had already failed this
treatment previously as he had initially been diagnosed as a
pancreatic adenocarcinoma in another center.

8. Liquid Biopsy Analyses
● Help early detection of tumour cells during the initial colonising process that
leads to the formation of metastatic tumours
○ Sample: CTC extracted from the plasma including a variety of cancers
○ Screening by: 54-gene-panel/NGS
○ Result: 65% of the different cancer types had detectable ctDNA
aberration and most were theoretically actionable.
● In a study on patients with colorectal cancer:
○ Sample: 68 colorectal cancer-associated genes
○ Screening by: NGS in primary tumour, metastases and CTC (circulating
tumour cells)
○ Result: CTC contained several additional mutations, comparatively (the
origin of tumour metastasis)
● Forshew et al., 2012 developed a tagged-amplicon deep sequencing (TAm-Seq)
method. NGS used to identify mutations and design primers to amplify
approximately 6000 bases that covered the selected regions of cancer-related
genes, including EGFR, TP53, and KRAS.
○ Samples: plasma
○ They showed that the method could identify mutations in TP53 at allelic
frequencies of 2% to 65%, demonstrating that it is feasible to sequence
large regions of circulating DNA by NGS.
The assessment of mutations in EGFR by CTC-NGS analysis can
be used to optimise pharmacologic treatment in patients with
non-small cell lung cancer.
High sensitivity and specificity of NGS was demonstrated in a
screening of plasma samples for lung cancer patients
Through the analysis of ctDNA in plasma samples from ovarian or
breast cancer patients, tumour mutations in TP53 and EGFR
genes were identified.

9. Cancer Somatic Mutation Analysis
● One of the first reports demonstrating the potential of NGS using the
Illumina Genome Analyser proved that NGS technology was able to
overcome the shortcomings of other technologies which were insensitive,
inaccurate and labour intensive.
● Ley et al., 2008 described the sequencing and analyses of the complete
exonic and regulatory DNA regions in one patient with acute myeloid
leukaemia using both Illumina® and Roche 454® platforms
● Performed a similar analysis on a second acute myeloid leukaemia patient in
2009 comparing the genetic mutations in tumour and matched normal skin
DNA.
○ Four of the 64 mutations identified occurred in at least one additional
patient (out of 188)
○ Somatic mutations were frequently observed in the same specific
genes (like IDH1 gene, which was mutated in more than 8% of the
samples)
○ Prove that NGS is able to identify such alterations.
Roche 454
Illumina genome analyser

10. Data Analysis &
Computing Infrastructure
● Hundreds of gigabytes of data will
be generated from NGS- difficult to
filter redundant and huge amounts
of data.
● Specialized programs must be used
for most NGS projects.
● Storing, processing, and analyzing
NGS data must be done on a high-
performance computer- impractical
for small diagnostic laboratories and
clinics (costly).
● NGS platforms (Miseq and Ion
torrent) have low throughput which
restricts their applications.
Interpretation of Variation Data
● Interpretation and clinical translation
of data collected on genetic variants
remains a bottleneck for routine
adoption of NGS.
● Difficult to accurately define clinically
significant variants or to effectively
assess disease risk based on current
research (Foretova et al., 2004 and
Maillet et al., 2006).
● For personalized cancer treatment,
filtering out tumor-promoting
mutations from passenger mutations
is also a challenge as the roles of both
may change as the tumor develops
(Ellis et al., 2012).
Ethical Issues
● Legal and ethical concerns for DNA sequencing.
● Disclosure of a patient's genetic testing result may
lead to discrimination based on genetic information
and social disorder, especially in job-hunting and
applications for health insurance.
CHALLENGES
2
3
1

11. Single Molecule Real-Time (SMRT) Sequencing
by Pacific Biosciences (PacBio)
PacBio data differs from short read sequencing technologies in several aspects:
● Fewer but longer reads.
● Permit sequencing/assembly through repetitive elements, direct variant
phasing, and even direct detection of epigenetic modifications (Flusberg
et al., 2010 and Jain et al, 2016).
● Sequencing only lasts several hours.
● Biases such as GC-skewing are near absent.
● Error types (more indels than mismatches).
● Higher abundance (∼13–15%) though they are spread randomly across the
reads. This randomness enables
● Highly accurate consensuses (>99%) to be build up rapidly by sequencing
multiple times the same molecule (CCS reads) (Eid, 2009) or by
combining different CLRs derived from the same locus
- Eliminates the need for fragmentation, instead sequencing cDNAs from
the 5 end of transcripts to the poly-A tail, termed Iso-Seq.
- Can be used to determine the repeat size and the detection of the
number of interrupting AGG units- unambiguous separation of the two
CGG repeats on the different X chromosomes of females.
- Detection of TKI resistance mutations down to a level of 1%, a
significantly lower detection threshold (compared to the 15–20%
reached by Sanger sequencing.
Figure 1: Overview of SMRT Sequencing Technology.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5861413/

12. Bräutigam, A. and Gowik, U. (2010). What can next generation sequencing do for you? Next generation sequencing as a valuable tool in plant research. Plant Biology, [online] 12(6), pp.831-
841. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3841808/ [Accessed 18 Oct. 2018].
Corless, C. (2016). Next-Generation Sequencing in Cancer Diagnostics. The Journal of Molecular Diagnostics, 18(6), pp.813-816.
Eid,J., Fehr,A., Gray,J., Luong,K., Lyle,J., Otto,G., Peluso,P., Rank,D., Baybayan,P., Bettman,B. et al. (2009) Real-time DNA sequencing from single polymerase molecules. Science, 323, 133–
138.
Ellis MJ, Ding L, Shen D, et al. Whole-genome analysis informs breast cancer response to aromatase inhibition. Nature. 2012;486:353–360. [PMC free article] [PubMed]
Flusberg,B.A., Webster,D.R., Lee,J.H., Travers,K.J., Olivares,E.C., Clark,T.A., Korlach,J. and Turner,S.W. (2010) Direct detection of DNA methylation during single-molecule, real-time
sequencing. Nat. Methods, 7, 461–465.
Foretova L, Machackova E, Navratilova M, et al. BRCA1 and BRCA2 mutations in women with familial or early-onset breast/ovarian cancer in the Czech Republic. Hum Mutat. 2004;23:397–
398.[PubMed]
Forshew, T., Murtaza, M., Parkinson, C., Gale, D., Tsui, D., Kaper, F., Dawson, S., Piskorz, A., Jimenez-Linan, M., Bentley, D., Hadfield, J., May, A., Caldas, C., Brenton, J. and Rosenfeld, N.
(2012). Noninvasive Identification and Monitoring of Cancer Mutations by Targeted Deep Sequencing of Plasma DNA. Science Translational Medicine, 4(136), pp.136ra68-136ra68.
Gagan, J. and Van Allen, E. (2015). Next-generation sequencing to guide cancer therapy. Genome Medicine, 7(1).
Garralda, E., Paz, K., Lopez-Casas, P., Jones, S., Katz, A., Kann, L., Lopez-Rios, F., Sarno, F., Al-Shahrour, F., Vasquez, D., Bruckheimer, E., Angiuoli, S., Calles, A., Diaz, L., Velculescu, V., Valencia,
A., Sidransky, D. and Hidalgo, M. (2014). Integrated Next-Generation Sequencing and Avatar Mouse Models for Personalized Cancer Treatment. Clinical Cancer Research, 20(9), pp.2476-
2484.
Jain,M., Olsen,H.E., Paten,B. and Akeson,M. (2016) The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community. Genome Biol., 17, 239.
Ley, T., Mardis, E., Ding, L., Fulton, B., McLellan, M., Chen, K., Dooling, D., Dunford-Shore, B., McGrath, S., Hickenbotham, M., Cook, L., Abbott, R., Larson, D., Koboldt, D., Pohl, C., Smith, S.,
Hawkins, A., Abbott, S., Locke, D., Hillier, L., Miner, T., Fulton, L., Magrini, V., Wylie, T., Glasscock, J., Conyers, J., Sander, N., Shi, X., Osborne, J., Minx, P., Gordon, D., Chinwalla, A., Zhao, Y.,
Ries, R., Payton, J., Westervelt, P., Tomasson, M., Watson, M., Baty, J., Ivanovich, J., Heath, S., Shannon, W., Nagarajan, R., Walter, M., Link, D., Graubert, T., DiPersio, J. and Wilson, R.
(2008). DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome. Nature, 456(7218), pp.66-72.
Lupski JR, Reid JG, Gonzaga-Jauregui C, Rio Deiros D, Chen DC, Nazareth L, et al. Whole-genome sequencing in a patient with Charcot-Marie-Tooth neuropathy. N Engl J Med. 2010;362:1181–
91.
REFERENCES