NGS in cancer treatment

APPLICATION OF NEXT GENERATION SEQUENCING IN CANCER TREATMENT

By :Prilli Arista Fernanda , Seow Wan Xin, Tan Regine , Nur Suhaida

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 )

• 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)

Application of NGS

1. Application of NGS in Clinical OncologyIdentification 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.

• 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).

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.

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).

• 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.

• 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).

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).

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).

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)

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)

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 Institute

| Boston, MA. Viewed 20 May 2016, <http://www.dana-farber.org/Research/Featured-Research/Profile-Somatic-Genotyping-Study.aspx>.

• Guan, Y, Li, G, Wang, R, Yi, Y, Yang, L, Jiang, D, Zhang, X & Peng, Y 2012, ‘Application of next-generation sequencing in clinical oncology to advance personalized treatment of cancer’, Chin J Cancer, vol.31, no.10, pp.463-470.

• Kidd, B, Readhead, B, Eden, C, Parekh, S & Dudley, J 2015, ‘Integrative network modeling approaches to personalized cancer medicine’, Personalized Medicine, vol.12, no.3, pp.245-257.

• Kiesler, E 2014, Tumor Sequencing Test Brings Personalized Treatment Options to More Patients | Memorial Sloan Kettering Cancer Center. Memorial Sloan Kettering Cancer Center. Viewed 19 May 2016, <https://www.mskcc.org/blog/new-tumor-sequencing-test-will-bring-personalized-treatment-options-more-patients>.

• Newman, A, Bratman, S, To, J, Wynne, J, Eclov, N, Modlin, L, Liu, C, Neal, J, Wakelee, H, Merritt, R, Shrager, J, Loo, B, Alizadeh, A &Diehn, M 2014,’An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage’, Nature Medicine, vol.20,no.5, pp.548-554.

• Shibata, T 2015 ‘Current and future molecular profiling of cancer• by next-generation sequencing’ , Japanese Journal Of Clinical Oncology, vol.45, no.10 , pp 895-899.• Takai, E, Totoki, Y, Nakamura, H, Morizane, C, Nara, S, Hama, N, Suzuki, M, Furukawa, E, Kato, M, Hayashi, H, Kohno, T,

Ueno, H, Shimada, K, Okusaka, T, Nakagama, H, Shibata, T &Yachida, S 2015, ‘Clinical utility of circulating tumor DNA for molecular assessment in pancreatic cancer’, Sci. Rep., vol 5, pp.18425.

• Upadhyay, P, Dwivedi, R, Dutt, A 2014, ‘Applications of next-generation sequencing in cancer’ , Current Science, vol.107, no. 5, pp 795-802

• Yin CC, e 2016. Identification of a novel fusion gene, IRF2BP2-RARA, in acute promyelocytic leukemia. - PubMed – NCBI, viewed 17 May 2016, <http://www.ncbi.nlm.nih.gov/pubmed/25583766>