~ AISYAH RAHMAN ~ CHRISTAL TWO ~ NURUL ALIAH ~ PREMADARSHINI ~ UMI AMIRAH ~

the application of next generation sequencing (NGS) in cancer treatment.

INTRODUCTIONFirst generation sequencing New generation sequencing

Basic principle

- Identify linear sequence

- Random separation

- Detection of nucleotide

sequences

- Sample extraction

- Template preparation

- Amplification

- Sequencing and detection

- Data analysis

METHODOLOGY4 PHASES

● Sample collections● Template generations● Sequencing reaction

and detection ● Data analysis

(Illumina n.d.)

Advantages of ngs● Massively parallel sequencing capability

● Single input of DNA/RNA

● Simultaneous screening of multiple genes in multiple

samples

● Decreased sequencing costs per gene

● Constantly improving technology

(Luthra et al. 2015)

Advantages OF NGS

cHALLENGES OF NGS● High complexity of workflow and results

● Selection of suitable target capture approach and

sequencing platforms

● Revalidation of upgrades

● Management of high volumes of data

(Luthra et al. 2015)

clinic Applications of ngs in cancer treatment❖ Whole-genome sequencing

● Provides base-pair resolution of an entire genome and identifies SNVs,

insertions and deletions (indels), and copy number variations (CNVs) in a single

run.

● Successfully used in a clinically relevant time frame to alter the treatment plan of

a patient with cancer.

● Identified a PML-RARA fusion event on a patient with acute myeloid leukemia.

● Identified and validated in just 7 weeks from biopsy and allowed for a change in

treatment plan of this patient.

(Rizzo & Buck 2012)

clinic Applications of ngs in cancer treatment❖ Transcriptome sequencing (RNA-seq)

● Sequence RNA populations to identify all of the genes that are

transcribed from that DNA - can help to determine which variants are

expressed in cancer samples.

● 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 the human

genome.

● Identified a single recurrent somatic in the FOX2 gene in GCT tumors.

● Mutation in FOXL2 is a potential driver in the pathogenesis of adult

GCTs

(Rizzo & Buck 2012)

Other clinical applications❖ Sequencing of cell-free DNA fragments.

● Use NGS to show that increased levels of cell-free DNA from a heart transplant donor's

genome. This method is used for detecting solid organ transplant rejection.

● Use NGS to detect Down syndrome and other fetal aneuploidies (trisomy 13 and 18). By

sequencing the subpopulation of cell-free DNA in a pregnant mother's bloodstream

(Rizzo & Buck 2012)

preclinical Applications of ngs in cancer treatment❖ Large-Scale Genome Sequencing Projects

● to help expedite the characterization of both normal and tumor genomes.

1) 1,000 Genomes project

● collecting WGS data from a diverse sampling of individuals to map patterns of inheritance and provide valuable insights on the genetics of complex diseases.

2) Cancer Genome Atlas

● to help identify and catalog all genetic alterations found in all cancer types using NGS methods.

(Rizzo & Buck 2012)

Current Developments of nGS in cancer treatment ❖ Immunotherapy

● With the new knowledge on that genomic methods provide, manipulation of immune response resulted in :-

➔ Promising therapies by boosting the ability of the immune system to target cancer

➔ Further advances in NGS technology have increased knowledge of the intricate pathways that regulate the

immune response.

❖ Epigenetic pathways of cancer samples● Methods for cancer epigenetics studies provide insight into tumorigenic pathways and cancer progression.

● Altered methylation often activates or silences genes, changes in the epigenome can affect gene expression and the rate

of cancer progression.

(Illumina Inc. 2016)

Columbia University Medical Centre 2015

Nestlé 2015

Illumina Inc. 2016

Future potential of ngs

● A potential future application of NGS is the evaluation of circulating

tumor cells to detect early relapse or residual cancer (Schwaederle et al.

2014).

● To improve the diagnosis of cancer (Basho & Eterovic 2015).

conclusion● NGS has a tremendous potential to transform personalized cancer medicine.

● However, further development of this field requires real-time knowledge of genome

alterations that can be used to tailor treatment plans for each individual patient.

● Thus more research has to be done to fully understand the capability and potential of

NGS in cancer treatment.

referenceBasho, RK & Eterovic, AK 2015, ‘Clinical Applications and Limitations of Next-Generation Sequencing’, The American Journal of Hematology/ Oncology, vol. 11, no. 3, pp. 17-22.

Columbia University Medical Centre 2015, Immunotherapy: New Hope for Patients with Advanced Lung Cancer, viewed 29 May 2016, <http://newsroom.cumc.columbia.edu/wp-content/uploads/2015/03/immunotherapy.jpg>.

Illumina Inc. 2016, Epigenetics, viewed 25 May 2016, < http://www.illumina.com/areas-of-interest/cancer/research/cancer-epigenetics.html>.

Illumina Inc. 2016, Immunotherapy the Next Generation of Cancer Treatment, viewed 27 May 2016, < http://www.illumina.com/content/dam/illumina-marketing/documents/products/appspotlights/ngs-immuno-oncology-application-spotlight-1170-2016-005.pdf>.

Nestlé 2014, Nestlé boosts research into cutting-edge maternal nutrition and epigenetics, viewed 1 June 2016, <http://www.nestle.com/asset-library/PublishingImages/Media/News-Features/2014-November/epigenetics-1.jpg.

Rizzo, JM & Buck, MJ 2012, ‘ Key Principles and Clinical Applications of "Next-Generation" DNA Sequencing’, Cancer Prevention Research, vol. 5, no. 7, viewed 21 May 2016, <http://cancerpreventionresearch.aacrjournals.org/content/5/7/887.full.pdf>.

Schwaederle M, Parker BA, Schwab RB, Schwaba,RB, Fantab, PT, Bolesb, SG, Danielsb, GA, Bazhenovab, LA, Subramanianb, R, Coutinhoa, AC, Ojeda-Fournierc, H, Datnowd, B, Webstere, NJ, Lippmana, SM & Kurzrocket, R 2014,‘Molecular tumor board: the University of California-San Diego Moores Cancer Center experience,’ The Oncologist, vol. 19, no. 6, pp.631-636.

Luthra, R, Chen, H, Roy-Chowdhuri, S, Singh, RR 2015, ‘Next-Generation Sequencing in Clinical Molecular Diagnostics of Cancer: Advantages and Challenges’, Cancers, vol. 7, no.4, pp. 2023-2036.