Cancer Genomics and Next-Generation Sequencing

This assignment delves into the crucial role of next-generation sequencing (NGS) in the field of cancer genomics. It examines how NGS technologies are used to analyze cancer genomes, identify key driver mutations, monitor disease progression through circulating tumor DNA, and inform personalized treatment strategies. The focus is on understanding the applications of whole-exome sequencing and targeted sequencing in formalin-fixed paraffin-embedded (FFPE) tissue samples for effective cancer diagnosis and management.

1Running head: CANCER
Molecular basis of cancer
Name of student:
Name of university:
Author note:

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Table of Contents
Question 1..................................................................................................................................3
Question 2..................................................................................................................................5
a..............................................................................................................................................5
b..............................................................................................................................................6
c..............................................................................................................................................6
Question 3..................................................................................................................................7
a..............................................................................................................................................7
b..............................................................................................................................................7
c..............................................................................................................................................7
Question 4..................................................................................................................................8
a..............................................................................................................................................8
b............................................................................................................................................11
References................................................................................................................................12

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Question 1
The“hallmarks of cancer” refer to the six distinct biological features acquired by cells
within the body at the time of multistage development of tumours. These hallmarks, or
assured characteristics of cancerous cells, make up the organizing principle for understanding
the different complexities related to the neoplastic disease. Research indicates that cancer
normally develops within the human body as genetic changes and mutations accumulate. The
trademarks or hallmarks of cancer are thus the attributes that a normal cell has to acquire in
order to be distinguished as a precancerous cell. These features differentiate the normal cells
from the cancerous cells. Though each of these hallmarks contributes to cancer, it is to be
noted that a cell needs to showcase a set of these features in order to be known as a cancerous
cell. Thee six hallmarks of cancer cells are self-sufficient cell division, evading growth
suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and
activating invasion and metastasis. Genomic instability is the underlying cause of these
hallmarks, generating a genomic diversity fostering multiple functions within the body (1).
Self-sufficient cell division- Typical cells within the body are in need of molecular signalling,
such as hormones, for their proper division and growth. Cancer cells have the striking feature
of growing in the absence of such external signals. Such cells proliferate on their own
through the production of their own cells signals and signal receptors that are overactive.
Furthermore, normal cells have a tight control system for cell division. In contrast, cancer
cells have deregulated cell division due to alteration of proteins controlling these processes.
Evading growth suppressors- The cell cycle clock of cancer cells and normal cells have
completely distinct from each other. The cell cycle is regulated by a set of a protein termed as
the tumour suppressor genes that carry information between cells for ensuring that they are in
a stage to divide. In case of cancer cells, the protein is highly altered, and cell division is

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defective. The second method is the absence of contact inhibition, as exhibited by normal
cells. Unlike normal cells, that stop dividing when they come into contact with nearby cells,
cancer cells divide even if they come in contact with adjacent cells.
Resisting cell death- Apoptosis is the process by which cells die when they are either
damaged or are no longer needed by the body. This process is valuable since it limits cell
growth and discards cells that are already damaged. The cancer cells do not undergo
apoptosis and thus accumulate within the body. The cause of disruption of apoptosis signals
is mutations in tumour suppressor genes or other forms of damage.
Enabling replicative immortality- Cancer cells have the potential to replicate limitlessly. A
small section of the end of chromosomes within the body, known as a telomere, disappears
after a copy of the DNA is made. At a critical point, the loss of telomere ensures that division
of cells is not possible. Cancer cells are immortalized since they have active telomerase,
unlike normal cells.
Inducing angiogenesis- Angiogenesis refers to the process of formation of new blood vessels
in the body. Cells that are cancerous in nature are found to exhibit the capability to form new
blood cells in order to ensure that the cells are in a position to receive a continuous supply of
nutrients and oxygen. The underlying objective is the existence of these cells for a longer
period of time within the body.
Activating invasion and metastasis- The last but arresting feature is the ability to metastasize.
This refers to the ability of breaking through tissue barriers and spreading into adjacent
organs. This ability determines whether the cell assumed to be cancerous is malignant or
benign. The process involves multiple stages, each bringing about a change in the respective
tissue and organ (2).

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Question 2
a.
The contemporary literature points out the increasing use of broad cancer
susceptibility panels for germline testing for the detection of cancer susceptibility. Broad
gene panel testing is known to contribute to a better understanding of the germline mutation
status of the individual patients at the time of early-onset of cancer in any part of the body.
Application of multi-gene panels can enable patients to evaluate cancer risks, such as ovarian
cancer and breast cancer. Such panels can act as a guide for researchers when they attempt to
carry out an exploration of more tumour genes and consider classifying the genes under
different gene families. Somatic alterations can also be detected simultaneously (3).
Researchers have shown interest in coming up with novice treatment options for cancer
through targeting different biological pathways that have distinct genetic markers. With the
application of genetic panel testing, it is possible to evaluate a diverse range of genetic
markers at a single go, increasing the chances of identifying treatments targeting specific
biological pathways.
Testing with multi-gene panels can give in-depth insights into the spectrum and
prevalence of mutations such as those in mismatch repair genes MSH2, MSH6, MLH1, PMS2
and MUTYH in colorectal cancer. A report published by JAMA based on research undertaken
by the Ohio State University indicated that one in six patients with early onset colorectal
cancer had hereditary cancer susceptibility. A 25-genes hereditary cancer panel was utilized
for testing 450 patients with early onset of the disease. 75 pathogenic germline variants were
detected among 72 patients, of which 13 individuals had variation in genes that were not

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previously considered as risk genes for the cancer, such as ATM, CDKN2A CHEK2, BRCA1/2
and PALB2. This result indicated the high efficiency of the multigene panel (4).
b.
There are distinct benefits and challenges of sequencing a broad panel of genes for the
identification of genetic susceptibility to cancer. Two important benefits of this method are
time-effectiveness and cost-effectiveness in comparison to sequentially testing diverse genes.
Multigene panel has lower costs and thus are the next-generation sequencing panels. Most of
the panels used consider high penetrance genes and thus the time taken for the process to
complete is less than the conventional method. The driving force for multi panel cancer
genetic testing has wider clinical utility due to these advantages (5). New challenges have
however emerged and unveiled against the broader use of such testing systems. Firstly,
expanded panel testing might lead to findings that are unexpected. Such unexpected findings
like “off-phenotypic-target” gene mutations, coupled with the prevalence of variants of
uncertain significance (VUS), as well as pathogenic findings in low and moderate risk genes
give rise to major challenges for the established repertoire. In addition, if moderate or low-
risk genes are added on the testing panels, it is difficult to develop management guidelines
that are personalized for the patient. The rationale is that the phenotypic spectrum and
penetrance are unknown at prior stages (6).
c.
Two examples of cancer susceptible genes with high penetrance variants are BRCA1
and BRCA2 on chromosomes 17 and 13 for ovarian and breast cancer; APC on chromosome
5 for colon cancer with adenomatous polyposis coli. Two examples of cancer susceptible
genes with low penetrance variants are CYP1A1, GSTM1 for carcinogen metabolism
polymorphisms. Two examples of cancer susceptible genes with moderate penetrance
variants are ATM gene and CHEK2 1100delC gene in breast cancer (7).

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Question 3
a.
Circulating tumour DNA (ctDNA) refers to the fragmented piece of DNA derived
from tumours of a living system, present in the bloodstream with no association with the
body’s cells. DNA fragments are released into the blood from cells that are dying due to
apoptosis, cellular turnover or necrosis. The DNA normally has 180-200 base pairs. Cell-free
DNA is also released due to autophagy, and physiologic events which are commonly induced
by micro-environmental stress. The inconsistency of levels of ctDNA in cancer patients is
related directly to the burden of a tumour, vascularity and stage of the tumour, and cellular
turnover (8).
b.
Research indicates that ctDNA extraction is possible in a non-invasive manner,
making it the appealing feature of such DNA. Isolation of ctDNA can be done through a
collection of blood. The approximate amount of blood required for extracting ctDNA is 3mL,
which is to be collected into tubes coated with Ethylenediaminetetraacetic acid (EDTA).
EDTA helps in reduction of blood coagulation. The separation of serum fractions and plasma
of the blood can be done through centrifugation. Consequent extraction of ctDNAis possible
from the fractions. Isolation of ctDNA is relatively simple from the clinical standpoint.
Plasma is usually considered for ctDNA extraction owing to the lower concentration of
background wild-type DNA (9).
c.
With the advent of advanced technologies, ctDNA is now being considered as a
valuable aid for clinical applications. Reviews in the recent past have emphasized on the

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utilization of ctDNA as a biomarker with versatile abilities. ctDNA can effectively genotype
the tumour detected in the body. In addition, it can help in the identification of the precise
genetic and epigenetic alterations occurring within the whole tumour. Moreover, ctDNA has
the ability to successfully carry out the monitoring of tumour burden as well as treatment
response. This feature reduces the requirement for adjuvant chemotherapy that is harmful to
individuals and allows a faster detection of cancer relapse. The major applications of ctDNA,
when applied as a marker, include early stage successful detection of primary disease and
examination of mutations that are tumour-specific. Tumor heterogeneity in case of metastatic
disease is also possible with ctDNa. The application of ctDNA in the field of cancer research
would provide a better, illustrative and comprehensive 'screenshot' of cancer’s genetic
diversity at primary as well as metastatic sites.There are a number of challenges to be
overcome in the near future for wider adoption of the biomarker in the medical field,
encompassing optimization and large trials (10).
Question 4
a.
The past few years have witnessed increased understanding of alterations brought
about in cancer genomes that lead to disease-specific changes in the living body. The domain
of cancer genomics has seen a drastic change with the application of next-generation, or
whole genome sequencing technology. The trend in cancer genome sequencing indicates an
advancement of next-generation technology that can accelerate the whole genome sequencing
rate in a cost-effective manner. Data obtained from whole genome sequencing build up
comprehensive information pertaining to genomic alterations that are significant in the
clinical field (11).

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Cancer tissue whole genome sequencing is the process of sequencing the whole
genome of a single, heterogenous or homogenous group of cancer cells through a
biochemical laboratory process. Identification and characterization of the RNA or DNA
sequences of the cancer cells are the main aim of such sequencing. This sequencing involves
the direct method of sequencing primary tumor tissue or adjacent normal tissues. The tumor
micro-environment includes metastatic tumour cells and stromal or fibrobast cells. The
information that is gathered from such sequencing encompasses identification of nucleotide
bases (DNA or RNA), mutation status, sequence variants and copy number, and structural
changes like fusion genes and chromosomal translocations (12).
The potential benefits of whole genome sequencing primary tumour tissue are multi-
faceted. The most striking importance is probably the ability to undertake a re-sequencing
and analysis of the tumour genomes and comparing them with the normal genomes. The cost
of such method is relatively less which is justified against the increased chances of getting
immensely enhanced throughputs. It is now a simple task to sequence more than one patient
samples of a given cancer type. Cancer tissue whole genome sequencing has the power to
emerge as a remarkable aid, at the cornerstone of which lies the heterogeneity of cancers and
patients. Cancer genome sequencing is beneficial since it permits oncologists and clinicians
to detect the particular and exclusive changes taking place within the patient. It is possible to
come up with personalized therapeutic strategies meeting the needs of the patients (13). A
host of risks and challenges are involved in cancer tissue whole genome sequencing. Since
cancer cells are highly heterogenous in nature, there are high chances that important
information about the differences in expression pattern and sequence between different cells
suffers a loss. Single-cell analysis ameliorates the difficulty. Tumors have a restricted number
of properties that are clinically significant, such as drug resistance. These properties are due
to genomic rearrangements at large scale under certain circumstances. Information gathered

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through whole genome sequencing on single nucleotide does not have much utility thereof.
Lastly, translation of sequence information into a plan of treatment addressing the needs of
the patient is complex, and an effective plan might not be outlined (14).
In-depth detailing of the abnormalities in the gene in case of cancer cells is crucial for
the understanding of the novice methods for treatment. Massively parallel methods for
sequencing cancer genome have revolutionized in the recent past, and under this situation, it
is necessary to understand and examine the differences in the utility of fresh tumor tissues
and formalin fixed tumor tissue for whole genome sequencing. Fresh-frozen tissue samples
are poorly available, limiting the range of genomic studies. Large number of fresh tissue
samples are difficult to obtain that would contain clinical information pertaining to disease
progression and outcome. Formalin fixation and paraffin embedding (FFPE) is now the
standard technique for sample preparation. It offers a more broad resource of matched normal
and disease tissues with clinically annotated samples and patient follow-up data. Sequencing
of large archives of FFPE samples is a convenient process, and retrospective studies can be
undertaken that are more powerful. Complex genetic changes in tumor progression can be
studied with these samples. Further, FFPE tissues are important for being stable at room
temperature. Lastly, storage procedure of such samples is not complex (15).
Formalin-fixed diagnostic tissue sample is more commonly available; however, they
are not frequently used since formalin-fixation artifacts give rise to major concerns. Though
FFPE specimen might seem to be useful ones, it is still a challenge to perform whole genome
sequencing in archival samples of tumour tissues. This is both from the bioinformatic and
technical perspective. Artificial sequence alterations and poor DNA quality are the major
drawbacks of such samples (16). Sequencing analysis of DNA isolated from formalin-fixed
samples is highly challenging on the technical grounds. DNA in such cases is variable as
damage is common when the fixation is done. Formalin fixation leads to hydrolysis of

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phosphodiester bonds, which is the reason for DNA fragmentation at various degrees.
Another challenge is that FFPE samples are often in smaller quantities. DNA yields are often
insufficient for carrying out standard protocols for whole genome sequencing. There is an
urgent need of improving and advancing methods by which FFPE samples can be brought to
a level that is suitable for desirable tumour tissue sequencing (17).
b.
Tumor sequencing has helped in improving the understanding of cancer development,
treatment and progression. Clonal evolution at the cytogenetic level has been a major
contribution to the failed treatment of cancer. One such example is acute myeloid leukaemia
(AML). Ding and fellow researchers identified cellular fractions characterized by common
mutational changes that are useful for illustrating the heterogeneity of a particular tumour
before and after treatment. Sequencing methods for tumours have helped in identifying
aberrations in cells that make cancer susceptible to different drugs. This information has been
beneficial for matching patients with clinical therapies. Lastly, genetic abnormalities can be
identified with sequencing methods that can illustrate whether the cells have suffered gene
deletion, mutation or additional copies have been generated (18).

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References
1. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. cell. 2011 Mar
4;144(5):646-74.
2. Pietras K, Östman A. Hallmarks of cancer: interactions with the tumor stroma.
Experimental cell research. 2010 May 1;316(8):1324-31.
3. Slavin TP, Niell-Swiller M, Solomon I, Nehoray B, Rybak C, Blazer KR, Weitzel JN.
Clinical application of multigene panels: challenges of next-generation counseling and
cancer risk management. Frontiers in oncology. 2015;5.
4. Pearlman R, Frankel WL, Swanson B, Zhao W, Yilmaz A, Miller K, Bacher J, Bigley C,
Nelsen L, Goodfellow PJ, Goldberg RM. Prevalence and spectrum of germline cancer
susceptibility gene mutations among patients with early-onset colorectal cancer. JAMA
oncology. 2017 Apr 1;3(4):464-71.
5. Lincoln SE, Kobayashi Y, Anderson MJ, Yang S, Desmond AJ, Mills MA, Nilsen GB,
Jacobs KB, Monzon FA, Kurian AW, Ford JM. A systematic comparison of traditional
and multigene panel testing for hereditary breast and ovarian cancer genes in more than
1000 patients. The Journal of Molecular Diagnostics. 2015 Sep 30;17(5):533-44.
6. Ellisen LW, Kurian AW, Desmond AJ, Mills M, Lincoln SE, Shannon KM, Gabree M,
Tung NM, Ford JM. Clinical impact of multi-gene panel testing for hereditary breast and
ovarian cancer risk assessment.
7. Niederhuber JE, Armitage JO, Doroshow JH, Kastan MB, Tepper JE. Abeloff's Clinical
Oncology E-Book. Elsevier Health Sciences; 2013 Sep 12.
8. Bettegowda C, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N, Bartlett BR, Wang
H, Luber B, Alani RM, Antonarakis ES. Detection of circulating tumor DNA in early-
and late-stage human malignancies. Science translational medicine. 2014 Feb
19;6(224):224ra24-.

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9. Dawson SJ, Tsui DW, Murtaza M, Biggs H, Rueda OM, Chin SF, Dunning MJ, Gale D,
Forshew T, Mahler-Araujo B, Rajan S. Analysis of circulating tumor DNA to monitor
metastatic breast cancer. New England Journal of Medicine. 2013 Mar 28;368(13):1199-
209.
10. Dawson SJ, Tsui DW, Murtaza M, Biggs H, Rueda OM, Chin SF, Dunning MJ, Gale D,
Forshew T, Mahler-Araujo B, Rajan S. Analysis of circulating tumor DNA to monitor
metastatic breast cancer. New England Journal of Medicine. 2013 Mar 28;368(13):1199-
209.
11. Van El CG, Cornel MC, Borry P, Hastings RJ, Fellmann F, Hodgson SV, Howard HC,
Cambon-Thomsen A, Knoppers BM, Meijers-Heijboer H, Scheffer H. Whole-genome
sequencing in health care: recommendations of the European Society of Human
Genetics. European Journal of Human Genetics. 2013 Jun;21(6):580.
12. Mardis ER, Wilson RK. Cancer genome sequencing: a review. Human molecular
genetics. 2009 Oct 15;18(R2):R163-8.
13. Wang K, Yuen ST, Xu J, Lee SP, Yan HH, Shi ST, Siu HC, Deng S, Chu KM, Law S,
Chan KH. Whole-genome sequencing and comprehensive molecular profiling identify
new driver mutations in gastric cancer. Nature genetics. 2014 Jun 1;46(6):573-82.
14. Lohr JG, Adalsteinsson VA, Cibulskis K, Choudhury AD, Rosenberg M, Cruz-Gordillo
P, Francis JM, Zhang CZ, Shalek AK, Satija R, Trombetta JJ. Whole-exome sequencing
of circulating tumor cells provides a window into metastatic prostate cancer. Nature
biotechnology. 2014 May 1;32(5):479-84.
15. Munchel S, Hoang Y, Zhao Y, Cottrell J, Klotzle B, Godwin AK, Koestler D, Beyerlein
P, Fan JB, Bibikova M, Chien J. Targeted or whole genome sequencing of formalin
fixed tissue samples: potential applications in cancer genomics. Oncotarget. 2015 Sep
22;6(28):25943.

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16. Oh E, Choi YL, Kwon MJ, Kim RN, Kim YJ, Song JY, Jung KS, Shin YK. Comparison
of accuracy of whole-exome sequencing with formalin-fixed paraffin-embedded and
fresh frozen tissue samples. PLoS One. 2015 Dec 7;10(12):e0144162.
17. Astolfi A, Urbini M, Indio V, Nannini M, Genovese CG, Santini D, Saponara M,
Mandrioli A, Ercolani G, Brandi G, Biasco G. Whole exome sequencing (WES) on
formalin-fixed, paraffin-embedded (FFPE) tumor tissue in gastrointestinal stromal
tumors (GIST). BMC genomics. 2015 Nov 3;16(1):892.
18. Gagan J, Van Allen EM. Next-generation sequencing to guide cancer therapy. Genome
medicine. 2015 Jul 29;7(1):80.