GENES AND TISSUE

CULTUREDNA sequencing is the process of determining the

precise order of nucleotides within a DNA molecule.

Discuss the application of next generation sequencing in

cancer treatment.

By: Chan Yanqi (0317492), Lai Woon Fui (0317553), Mandary Madiiha B (0316855), Ivy Ng (0316439), Trevor Wang (0303235), Leong Jun Yee (0317542).

OBJECTIVES

• To understand what is Next generation sequencing and how does it work.

• The differences between old generation sequencing and new generation

sequencing

• To investigates the applications of using next generation sequencing in the

cancer treatments.

• Future applications, challenges and recent developments of next generation

sequencing.

INTRODUCTION: Cancer Therapy in current context

• Cancer, in its many forms, accounts for 8.2 million deaths in 2012 (GLOBOCAN, 2012). The rapid development of DNA sequencing technologies has driven a revolution in our understanding of this highly complex and diverse group of diseases (Devita & Rosenberg 2012).

• There are a handful of cancer types, such as chronic myelogenous leukemia, that feature a single and pathognomonic DNA mutation. Working on this assumption, early methods to explore the genomic foundations of different cancers involved targeted exploration of specific variants and genes in a low-throughput fashion.

• The success of Human Genome Project, along with the increased affordability and reliability of sequencing, has led to the integration of genome science into clinical practice. The use of these data to assist in diagnosis is generally referred to as precision medicine.

HOWEVER,

Demand for revolutionary technologies

that deliver fast, inexpensive and accurate

genome information started to arise. This

challenge catalyzed the development of

next-generation sequencing (NGS)

technologies.

We are going to highlight the importance and

potential of complete or 100% genome

sequencing.

Next Generation Sequencing…

WHAT IS NEXT GENERATION

SEQUENCING?

• Commonly termed as massively parallel sequencing, it is a way of capturing a

very big amount of genomic information about a cancer.

• NGS came as a revolution in genomic research. By using NGS, a whole

human genome can be sequences in a single day as opposed to the previous

Sanger sequencing technology, that took more than 10 years to deliver the

final draft of a complete human genome (Behjati & Tarpey 2013).

HOW DOES NEXT GENERATION

SEQUENCING WORK?

• Most NGS technologies uses sequencing by synthesis whereby each DNA

fragment to be sequenced is bound to an array, and then DNA polymerase

adds labeled nucleotides sequentially.

• A high-resolution camera captures the signal from each nucleotide becoming

integrated and notes the spatial coordinates and time.

• The sequence at each spot can then be inferred by a computer program to

generate a contiguous DNA sequence, referred to as a read.

STEPS OF NEXT GENERATION

SEQUENCING.

A summary of the workflow for NGS

sequencing in oncology (Adapted from

Genome Medicine 2015)

BENEFITS OF USING NEXT

GENERATION SEQUENCING.

1. Samples no longer need to be handled in a different manner from standard diagnostic specimens.

2. New advances enabled increasingly complicated genomic data to be obtained from an individual’s blood sample only (Gagan et al. 2012).

3. Genetic mutations have long since been identified by Sanger sequencing, PCR, and microarrays. However they have limitations that don’t apply to NGS.

For example, although previous methods can detect single nucleotide variants (SNVs), they have trouble identifying larger DNA aberrations, such as large indels and structural rearrangements, which are common in cancer.

On the other hand, whole exome and whole- genome sequencing can provide a comprehensive view of the DNA aberrations, genetic recombination, and other mutations. Therefore, NGS platforms serve as a good diagnostic and prognostic tool and help clinicians identify specific characteristics in each patient, paving the road towards personalized medicine.

SANGER SEQUENCING VS NEXT

GENERATION SEQUENCING.

SANGER SEQUENCING.PROS CONS

Polymerase errors

average out (Lowest error

rate)

Can only do 1 sequence

at a time

- Only produces one

forward and one reverse

read

Long read length (~ 450

bp)

Need a lot of DNA to

start with

- High cost per base (more

expensive)

NEXT GENERATION SEQUENCING

PROS CONS

Massively parallel

- Allows millions of

fragments to be

sequenced in a single run

Fewer reads of each bases

are combined so less

accurate overall

Fast turnaround time Short read length

Cheaper – Lower cost per

sample

-

APPLICATIONS OF NGS

1. NGS has already been applied in the clinic for cancer diagnosis and prognosis.

• For example, by doing the sequencing of the whole genome, researchers were able to

identify a novel insertional fusion that makes a classic bcr3 PML-RARA fusion gene for

a patient suffering from acute myeloid leukemia (AML). The findings brought about

alterations in the treatment plan of that patient (Shyr & Liu 2013).

• Oncology studies on the identification of individualized mutational landscapes for

tumors from patients with metastatic colorectal cancer and malignant melanoma

(identifying informative mutations in a clinically relevant time frame)

APPLICATIONS OF NGS

• Used paired-end sequencing to generate a comprehensive catalog of somatic mutations

in a malignant melanoma genome.

• Sequence cancer genomes from metastasis & primary breast cancer samples to

investigate the mutational evolution of cancer cells (Lee et al. 2013).

2. By using this NGS technology, clinicians have the possibility of designing patient-

specific probes that makes use of DNA found in the patient’s blood serum to monitor the

progress of a patient’s treatment and note any signs of relapse (Shyr & Liu 2013).

APPLICATIONS OF NGS

3. NGS is utilized by zooming in deeply in sequence

target regions (Target sequencing).

- Target multiple candidate genes in providing a

comprehensive view of the genetic makeup of cancer

patients.

(Rizzo & Buck 2012)

APPLICATIONS OF NGS

4. NGS on Transcriptome sequencing (RNA sequence).

• simultaneously analyzed to provide insight into the level of gene expression, the structure of genomic loci, and sequence variation present at loci.

- Adult granulose cell tumors (GCT) using paired end RNA sequencing and compared their sequencing results with the transcriptomes of 11 epithelial ovarian tumors and published sequences of human genomes.

- FOXL2 gene involved in granulose cell development

- Mutation in FOXL2 is a potential driver in the pathogenesis of adult GCTs.

(Rizzo & Buck 2012)

TREATMENT USING NGS

• Standard Clinical Practices in Guiding Cancer Therapy

• Personalized Treatment of Cancer in Clinical Oncology

• Pancreatic Cancer Treatment

• Non-small Lung Cancer

Standard Clinical Practices for Cancer Therapy

• NGS, a result of multiple technological and practical advances

• Earlier protocols require outside of typical pathology workflows to harvest/obtainthe starting material.

• Standard formalin-fixed/paraffin-embedded specimens used.

• Knowledge bases of NGS are being massed

• Protocols for analysis and NGS data interpretation

• Allow clinicians easily obtain information for patients care (Gargan & Eliezer 2015)

Personalized Treatment of Cancer

• Low costs, shorter turnaround time,

• Due to the ability to fully sequence large number of mutations genes of all types in single test.

• More comprehensive analysis of variation

• Able to detect all mutation types of targets gene and even chromosomal abnormalities. Compare with Sanger sequencing.

• DNA required less

• Small amount from tumour tissue, as not always possible to obtain large quantity (Guan et al. 2012)

PANCREATIC CANCER TREATMENT

• Using somatic alterations of pancreatic cancer genomes in NGS.

• Able to identify therapeutic options for patients. (Mardis 2012)

NON SMALL LUNG CANCER.

• Exon 19 micro-deletions been revealed by NGS as the most frequent genetic alterations affecting Epidermal Growth Factor Receptor.

• Able to response to treatment with tyrosine kinase inhibitor. (Marchetti et al. 2012)

RECENT DEVELOPMENTS OF NGS

Sequencing of cell-free DNA fragments (cffDNAs) Reducing invasive medical procedures:

Definition : Fetal DNA circulating freely in the maternal blood circulation

Detecting Down syndrome and other fetal aneuploidies (trisomy 13 and 18) with high sensitivityand specificity

E.g. - By sequencing the subpopulation of cffDNAs in a pregnant mother's bloodstream belonging to herfetus Next Generation Sequencing plasma-based DNA test

Detecting solid organ transplant rejection

E.g. - increased levels of cffDNAs from a heart transplant donor's genome found in a recipient’sbloodstream when a transplant recipient is undergoing an acute cellular rejection deep sequencing ofcffDNAs to detect changes in a small fraction of that DNA population which belonged to the organdonor.

(Rizzo & Buck 2012)

RECENT DEVELOPMENTS OF NGS

Microbial diversification (Preclinical application)

Used to characterize microbial populations in a culture-independent fashion,

by probing for the presence and abundance of microbe-specific DNA

sequences in both tumor and normal human cell environments. (Rizzo &

Buck 2012)

RECENT DEVELOPMENTS OF NGS

Forensic molecular biology

Genetic markers on the Y chromosome Y-short tandem repeats (Y-STRs)

To unambiguously resolve the male component of DNA mixtures when a high female

background is present

To reconstruct paternal relationships between male individuals

NGS comparing more than 10 million nucleotides of the Y chromosome between two male

individuals who shared the same ancestor 13 generations ago four genetic differences detected

Helps solves the problem of distinguishing between mixed male samples from the same parent.

(Yang, Xie & Yan 2014)

CHALLENGES OF NGS

• Presence of repetitive DNA

• To differentiate rare mutations in tumour from sequencing and alignment artefacts

• Tumour complexity and heterogeneity

• Deluge of data derive from NGS might take a long time to be translated into clinical relevantinformation.

• Prediction of the functional effect and the identification of disease-causal variants are still inan early phase

• Require high-performance computing and bio-informatics support.

• Quality control and standardisation of NGS experiments and data reporting.

(Reis-Filho, 2009)

FUTURE STUDIES OF NGS

• Forensic studies

• To move from the current descriptive and prognostic classification systems to a

functional genomic taxonomy (cancer research)

• To improve diagnosis of cancer

• To identify molecular aberrations that render tumours exquisitely sensitive to certain

therapies, resulting in exceptional responses.

• To facilitate clinical diagnosis and personalized disease-risk profiling

• Use as a diagnostic tools for higher detection rate in early onset diseases (Shyr & Liu 2013).

CONCLUSION

• From this presentation, we can conclude that today’s complex genomic

research questions demands a depth of information beyond normal capacity

of traditionally DNA sequencing technology. With its unprecedented

throughput, scalability and speed, NGS enables researchers to study

biological system at a level never before possible.

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