Discuss critically the current cellular and molecular level understanding of OC with a

particular focus on how we are trying to move from broad disease-focused

treatments to more personalised-focused treatments
Introduction
Oesophageal cancer (OC) is characterised by tumours in the oesophagus that can
significantly impact individual’s quality of life (Enzinger and Mayer 2003). Patients
with OC can experience dysphagia (problems with swallowing food), oesophageal
bleeding, and weight loss (Enzinger and Mayer 2003). Traditional treatments, such
as: surgery, radiotherapy and chemotherapy are not effective in all individuals with
OC and patients that undergo these treatments can have adverse side effects
(Pennathur et al 2013). Due to this, there has been a shift to personalised treatment
approaches based on an individual’s genetic profile.
Clinical studies have demonstrated these approaches to be more effective than
traditional treatments and produce less side effects (Abnet et al 2018). By
understanding the role of genetic mutations and the immune system in OC, patients
can receive personalised treatment that is more effective compared to the
effectiveness of traditional treatments.
Cellular and molecular understanding:
Subtype and origins:
The two subtypes of OC: squamous cell carcinoma (SCC) and adenocarcinoma (AC)
originate from different cellular mechanisms. SCC develops due to uncontrolled
proliferation of squamous epithelial cells that line the upper part of the oesophagus.
It is believed that this occurs due to the accumulation of gene mutations within
cellular DNA that triggers the proliferation (Lin et al 2018). Researchers believe that
certain carcinogens (tobacco and alcohol) drive the mutations of tumour suppressor
genes and oncogene (Lin et al 2018). However the other mechanisms behind these
mutations are lesser known. AC is strongly linked to Barrett’s oesophagus, which is
characterised by the transformation of squamous epithelial cells to glandular
epithelial cells in the lower part of the oesophagus due to chronic acid reflux
(Lagergren and Lagergren 2013). It is believed that the chronic inflammation from
acid reflux initiates the DNA damage, causing cells to proliferate uncontrollably.
However the mechanisms of how chronic inflammation leads to DNA damage is
lesser known (Secrier et al 2016).
Key Oncogenes, Tumour suppressors and signalling pathways:
The common tumour suppressor genes in both subtypes: TP53 and CDK2NA cause
the dysregulation of cancer protection mechanisms. Whereas common oncogenes in
the subtypes EGFR and PIK3CA drive cancer promoting mechanisms (Song et al
2014). OC patients exhibit tumour heterogeneity in terms of mutations, which leads
to the varied effectiveness of broad treatments (Weaver et al 2014). Targeted
therapies can focus on specific mutations in tumours. In addition to genetic
mutations, the ability of the immune system to recognise and kill cancer cells can
influence the formation of tumours (Mimura et al 2018). Immunotherapies can
increase immune cell recognition of cancer cells and enhance the immune system’s
response to kill cancer cells (Mimura et al 2018).
One limitation about the current understanding of OC is the mechanisms the gene
mutations is unknown and the role of inflammation in causing DNA damage.
Although, scientists know mutated oncogenes and tumour suppressor genes are
driving tumour formation, it is unclear the specific cellular and molecular interactions
within cells that lead to these mutations. In addition, scientists understand that the
acid reflux in Barrett’s oesophagus could be driving AC. However, it is unknown how
the inflammation due to acid reflux leads to DNA damage. It is suspected that it could
be oxidative stress mechanism behind this or due to the pro-inflammatory cytokines
released during inflammation (He et al 2012).
Diagnosis
Traditional methods:
The commonly used diagnostic methods to detect and stage OC are Endoscopy and
computed tomography (CT) scans (Wang et al 2008). Endoscopy is the standard
diagnosis method for OC, it involving a camera fitted tube inserted into the
oesophagus to detect for the presence of tumours. To confirm the tumours as
cancerous, a biopsy sample can be extracted and analysed under a microscope. CT
scans help identify the size and location of OC tumours, detects enlarged lymph
nodes and identify distant metastases (Vilet et al 2008). This information can be
used in staging OC which enables doctors to administer the correct treatment.
Biomarkers and Genetic profiling:
Alternative diagnostic techniques involve the detection of predictive biomarkers and
the genetic profiling in individuals. It can also be used in guiding personalised
treatment decisions (Bang et al 2010). Biomarkers are molecules in samples that
can indicate cancer when the biomarkers are present. One biomarker used in
detecting OC is high levels of human epidermal growth factor receptor 2 (HER2).
This biomarker is often overexpressed in individuals with OC and can be identified
using immunohistochemistry (IHC) (Bang et al 2010). IHC uses specific antibodies
with a fluorescent dye that complimentary bind to HER2. Genetic profiling analyses
the DNA present In biological samples and identifies typical genetic mutations
present in OC (Findlay et al 2016). One common mutation in OC is TP53, next
generation sequencing (NGS) can be used to detect the presence of mutant TP53 in
the genome (Findlay et al 2016). Although, these approaches are successful in
diagnosing OC in some individuals, not all individuals have these predictive
biomarkers and genetic mutations present in them. These individuals may have rarer
genetic mutations that are not known to play a role in tumour development in OC.
This can lead to false negative diagnoses. Further research is being done to identify
new genetic mutations and predictive biomarkers.
Broad disease focused treatments
Limitations:
The traditional treatments for OC, such as surgical resection, radiation therapy and
chemotherapy have varying effectiveness in OC patients and cause unnecessary
adverse effects (Hagen et al 2012). Surgical resection can be curative for early-stage
OC patients but can cause post-surgical complication and in some cases mortality
(Mariette et al 2007). Radiation therapy can be effective at preventing the spread of
cancer cells to other areas but can cause side effects, such as: oesophagus
inflammation, narrowing of the oesophagus, fatigue and skin irritation. Similarity,
chemotherapy can cause side effects like fatigue, nausea, vomiting and hair loss.
The effectiveness of these treatments varies due to the presence of specific genetic
mutations within the DNA of cancer cells. Some mutations may be more resistant to
chemotherapy or radiation therapy, leading to inconsistent treatment outcomes of
traditional treatments (Dulak et al 2013).
Personalised treatment
Targeted therapies and immunotherapies:
Personalised patient focused treatments are therapeutic approaches prescribed
based on an individual’s genetic profile (Weaver et al 2014). NGS identify targetable
gene mutations in OC patients’ tumours (Frankell et al 2019), allowing for the
development of targeted therapies and immunotherapies.
TP53 is a commonly mutated gene in OC tumours. It is a tumour suppressor gene
that regulates cell cycle progression, DNA repair and apoptosis (Integrated genomic
characterization of oesophageal carcinoma, 2017). One targeted therapy for TP53
involves blocking the interaction between p53 and MDM2, which promotes p53
activity (Shiag et al 2022). Literature discuss the potential of using MDM2 inhibitors
in oesophageal cancer (Shiag et al 2022), however there are limited clinical studies
testing its effectiveness on OC patients.
EGFR is rarer mutated gene within OC patients (Sudo et al 2007). EGFR is an
oncogene which encodes for mutated epidermal growth factor receptor. This
receptor is a tyrosine kinase which is important in the activation of downstream
pathways: Ras/Maf/MAPK, PI3K/Akt/mTOR and JAK/STAT, which regulate cellular
proliferation and survival processes. Mutated tyrosine kinase causes the
downstream pathways to be overactivated leading to uncontrolled proliferation of
cancer cells. Tyrosine kinase inhibitors (TKIs) target mutant EGFR by binding to the
intracellular ATP binding site of the tyrosine kinase and preventing the overactivation
of the downstream pathways. Clinical studies analysing the use of TKIs on OC
patients with high levels of expressed EGFR have shown varied effectiveness of the
treatment (Huang et al 2016).
Immunotherapies induce the immune system to produce a strong immune response
against cancer cells. Some immunotherapies inhibit immune checkpoint proteins,
such as: programmed cell death protein 1 (PD-1) and programmed death ligand (PD-
L1). By inhibiting these immune checkpoint proteins, it reduces the ability of cancer
cells to evade immune cell recognition, allowing the immune system to recognise
cancer cells more easily. As a result, the immune system can produce a stronger
immune response to destroy cancer cells. Clinical studies investigating
Pembrolizumab (a monoclonal antibody inhibiting PD-1 protein on T cells) have
shown improved survival and response rates in individuals with advanced OC that
expressed high levels of PD-L1 (Kojima et al 2020).
Although clinical studies have shown the potential of using these personalised
treatments in OC patients, the clinical studies are still in early phases. Further
investigation is needed to assess the long-term safety and effectiveness of these
treatments. Some of these treatments have shown varied effectiveness in OC
patients expressing high levels of predictive biomarkers (mutations and immune
checkpoint proteins). Possible reasons for this include heterogeneity of mutated
genes and inherent resistance to the treatments within patients. More research is
needed to identify stronger predictive biomarkers and to understand why some
patients have inherent resistance to treatments.
Conclusion
The current cellular and molecular understanding of OC has led to a shift from broad
disease focused treatments to more personalised focused treatments. The
identification of tumour heterogeneity in individual OC patients has largely caused
this shift. Despite the effectiveness of personalised focused treatments, future
research needs to assess the long term safety and effectiveness of these
treatments. In addition, research needs to identify uncommon gene mutations that
play a role in OC and develop personalised treatments that target them.
References
Abnet, C.C., Arnold, M. and Wei, W.-Q. (2018). Epidemiology of Esophageal Squamous Cell
Carcinoma. Gastroenterology, [online] 154(2), pp.360–373.

Bang, Y.-J., Van Cutsem, E., Feyereislova, A., Chung, H.C., Shen, L., Sawaki, A., Lordick, F., Ohtsu, A.,
Omuro, Y., Satoh, T., Aprile, G., Kulikov, E., Hill, J., Lehle, M., Rüschoff, J. and Kang, Y.-K. (2010).
Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-
positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label,
randomised controlled trial. The Lancet, 376(9742), pp.687–697.

Dulak, A.M., Stojanov, P., Peng, S., Lawrence, M.S., Fox, C., Stewart, C., Bandla, S., Imamura, Y.,
Schumacher, S.E., Shefler, E., McKenna, A., Cibulskis, K., Sivachenko, A., Carter, S.L., Saksena, G., Voet,
D., Ramos, A.H., Auclair, D., Thompson, K. and Sougnez, C. (2013). Exome and whole genome
sequencing of esophageal adenocarcinoma identifies recurrent driver events and mutational
complexity. Nature genetics, [online] 45(5), pp.478–486.

Enzinger, P.C. and Mayer, R.J. (2003). Esophageal Cancer. New England Journal of Medicine, 349(23),
pp.2241–2252.

Findlay, J.M., Castro-Giner, F., Makino, S., Rayner, E., Kartsonaki, C., Cross, W., Kovac, M., Ulahannan,
D., Palles, C., Gillies, R.S., MacGregor, T.P., Church, D., Maynard, N.D., Buffa, F., Cazier, J.-B., Graham,
T.A., Wang, L.-M., Sharma, R.A., Middleton, M. and Tomlinson, I. (2016). Differential clonal evolution
in oesophageal cancers in response to neo-adjuvant chemotherapy. Nature Communications, 7(1).

Frankell, A.M., Jammula, S., Li, X., Contino, G., Killcoyne, S., Abbas, S., Perner, J., Bower, L.,
Devonshire, G., Ococks, E., Grehan, N., Mok, J., O’Donovan, M., MacRae, S., Eldridge, M.D., Tavaré, S.
and Fitzgerald, R.C. (2019). The landscape of selection in 551 esophageal adenocarcinomas defines
genomic biomarkers for the clinic. Nature Genetics, 51(3), pp.506–516.

He, H., Tian, D., Guo, J., Liu, M., Chen, Z., Hamdy, F.C., Helleday, T., Su, M. and Ying, S. (2012). DNA
damage response in peritumoral regions of oesophageal cancer microenvironment. Carcinogenesis,
[online] 34(1), pp.139–145.

Huang, J., Fan, Q., Lu, P., Ying, J., Ma, C., Liu, W., Liu, Y., Tan, F. and Sun, Y. (2016). Icotinib in Patients
with Pretreated Advanced Esophageal Squamous Cell Carcinoma with EGFR Overexpression or EGFR
Gene Amplification: A Single-Arm, Multicenter Phase 2 Study. Journal of Thoracic Oncology: Official
Publication of the International Association for the Study of Lung Cancer, [online] 11(6), pp.910–917.

Integrated genomic characterization of oesophageal carcinoma. (2017). Nature, 541(7636), pp.169–

175.

Kojima, T., Shah, M.A., Muro, K., Francois, E., Adenis, A., Hsu, C.-H., Doi, T., Moriwaki, T., Kim, S.-B.,
Lee, S.-H., Bennouna, J., Kato, K., Shen, L., Enzinger, P., Qin, S.-K., Ferreira, P., Chen, J., Girotto, G., de
la Fouchardiere, C. and Senellart, H. (2020). Randomized Phase III KEYNOTE-181 Study of
Pembrolizumab Versus Chemotherapy in Advanced Esophageal Cancer. Journal of Clinical Oncology,
38(35), pp.4138–4148.

Lagergren, J. and Lagergren, P. (2013). Recent developments in esophageal adenocarcinoma. CA: A

Cancer Journal for Clinicians, 63(4), pp.232–248.

Lin, D.-C., Wang, M.-R. and Koeffler, H.P. (2018). Genomic and Epigenomic Aberrations in Esophageal
Squamous Cell Carcinoma and Implications for Patients. Gastroenterology, 154(2), pp.374–389.

Mariette, C., Piessen, G. and Triboulet, J.-P. (2007). Therapeutic strategies in oesophageal carcinoma:
role of surgery and other modalities. The Lancet Oncology, 8(6), pp.545–553.

Mimura, K., Yamada, L., Ujiie, D., Hayase, S., Tada, T., Hanayama, H., Thar Min, A.K., Shibata, M.,
Momma, T., Saze, Z., Ohki, S. and Kono, K. (2018). Immunotherapy for esophageal squamous cell
carcinoma: a review. Fukushima Journal of Medical Science, [online] 64(2), pp.46–53.

Pennathur, A., Gibson, M.K., Jobe, B.A. and Luketich, J.D. (2013). Oesophageal carcinoma. The Lancet,
[online] 381(9864), pp.400–412.

Secrier, M., Li, X., de Silva, N., Eldridge, M.D., Contino, G., Bornschein, J., MacRae, S., Grehan, N.,
O’Donovan, M., Miremadi, A., Yang, T.-P., Bower, L., Chettouh, H., Crawte, J., Galeano-Dalmau, N.,
Grabowska, A., Saunders, J., Underwood, T., Waddell, N. and Barbour, A.P. (2016). Mutational
signatures in esophageal adenocarcinoma define etiologically distinct subgroups with therapeutic
relevance. Nature Genetics, [online] 48(10), pp.1131–1141.

Sihag, S., Nussenzweig, S.C., Walch, H.S., Hsu, M., Tan, K.S., De La Torre, S., Janjigian, Y.Y., Maron, S.B.,
Ku, G.Y., Tang, L.H., Shah, P.M., Wu, A., Jones, D.R., Solit, D.B., Schultz, N., Ganesh, K., Berger, M.F. and
Molena, D. (2022). The Role of the TP53 Pathway in Predicting Response to Neoadjuvant Therapy in
Esophageal Adenocarcinoma. Clinical Cancer Research: An Official Journal of the American
Association for Cancer Research, [online] 28(12), pp.2669–2678.

Song, Y., Li, L., Ou, Y., Gao, Z., Li, E., Li, X., Zhang, W., Wang, J., Xu, L., Zhou, Y., Ma, X., Liu, L., Zhao, Z.,
Huang, X., Fan, J., Dong, L., Chen, G., Ma, L., Yang, J. and Chen, L. (2014). Identification of genomic
alterations in oesophageal squamous cell cancer. Nature, 509(7498), pp.91–95.
Sudo, T., Mimori, K., Nagahara, H., Utsunomiya, T., Fujita, H., Tanaka, Y., Shirouzu, K., Inoue, H. and
Mori, M. (2007). Identification of EGFR mutations in esophageal cancer. European Journal of Surgical
Oncology: The Journal of the European Society of Surgical Oncology and the British Association of
Surgical Oncology, [online] 33(1), pp.44–48.

van Hagen, P., Hulshof, M.C.C.M., van Lanschot, J.J.B., Steyerberg, E.W., Henegouwen, M.I. van B.,
Wijnhoven, B.P.L., Richel, D.J., Nieuwenhuijzen, G.A.P., Hospers, G.A.P., Bonenkamp, J.J., Cuesta,
M.A., Blaisse, R.J.B., Busch, O.R.C., ten Kate, F.J.W., Creemers, G.-J. ., Punt, C.J.A., Plukker, J.T.M.,
Verheul, H.M.W., Bilgen, E.J.S. and van Dekken, H. (2012). Preoperative Chemoradiotherapy for
Esophageal or Junctional Cancer. New England Journal of Medicine, 366(22), pp.2074–2084.

van Vliet, E.P.M., Heijenbrok-Kal, M.H., Hunink, M.G.M., Kuipers, E.J. and Siersema, P.D. (2008).
Staging investigations for oesophageal cancer: a meta-analysis. British Journal of Cancer, 98(3),
pp.547–557.

Wang, K.K., Sampliner, R.E. and Practice Parameters Committee of the American College of
Gastroenterology (2008). Updated guidelines 2008 for the diagnosis, surveillance and therapy of
Barrett’s esophagus. The American journal of gastroenterology, [online] 103(3), pp.788–97.

Weaver, J.M.J., Ross-Innes, C.S., Shannon, N., Lynch, A.G., Forshew, T., Barbera, M., Murtaza, M., Ong,
C.-A.J., Lao-Sirieix, P., Dunning, M.J., Smith, L., Smith, M.L., Anderson, C.L., Carvalho, B., O’Donovan,
M., Underwood, T.J., May, A.P., Grehan, N., Hardwick, R. and Davies, J. (2014). Ordering of mutations
in preinvasive disease stages of esophageal carcinogenesis. Nature Genetics, 46(8), pp.837–843.
3nd question:
The patient was diagnosed with OC characterised with overexpression of HER2. Trastuzumab
(immunotherapy drug) and Pertuzumab (chemotherapy drug) were used and tumour growth was
initially slowed and regressed. However, the patient acquired resistance to the drugs, and tumour
growth continued. The genetic profile taken of the tumour, showed the following additional gene
mutations: EP300, KRAS, KDM6A, DVL3, SOX2, CDKN2A, NOTCH3, MET, PIK3CA, PTEN, CREBBP,
FBXW7, CHEK1, MSH3, and AJUBA. These gene mutations need be identified as either driver
mutations (contributing to tumour growth) or passenger mutations (not contributing to tumour
growth).

There are several experimental approaches to detect passenger and driver mutations in the patient’s
tumour. One experimental approach is to study separately the effects of each mutation on cellular
behaviour. This process involves inserting vectors that contain one of the gene mutations into non-
cancerous cells and comparing the behaviour of these cells to other cells without the mutated gene.
When the specific mutated gene causes an increase in hallmarks of cancer: cellular proliferation and
survival, it indicates the mutation is a driver of tumour growth.

Another experimental approach to identify driver mutations is to introduce the wild type version of
the mutated gene into a cancerous cell line. Similarly to the previous experiment, a vector is inserted
into the cancer cells. The vector contains the wild type gene and allows the cancer cell to express the
protein the gene encodes for. When the wild type gene causes a decrease in cancer cell behaviour, it
suggests the mutated version of the gene is a driver of tumour growth.

CRISPR/Cas9 gene editing is a technology that allows genome modification by inserting mutations at
a target gene to knockout the gene and prevent its expression. This technology could be done on
cancer cells from the patient and knockout specific mutations present in the cells. CRISPR/Cas9 gene
editing requires designing guide RNAs (dRNAs) that complimentary to the DNA sequences of the
targeted mutated gene. This allows a Cas9 enzyme to recognise and introduce double strand breaks
at the target mutated gene. DNA repair mechanisms are activated at the mutated gene, introducing
insertion and deletion mutations that lead to the knockout of that gene. Cancer cell behaviour is
analysed by monitoring changes in cell proliferation and survival. When the knockout of a gene
causes reduced proliferation and survival, it indicates the mutation is driving tumorigenesis.

The genetic profile of the patient’s tumour showed mutations: DVL3 and FBXW7 in the cancer cell
DNA. Both genes affects the Wnt/Beta caternin signalling pathway in different ways. DVL3 affects the
pathway directly by encoding for a scaffold protein that relays a signal to downstream signalling
targets. Mutations to the DVL3 gene can lead an overactivation of the Wnt/Beta caternin leading to
increased cell proliferation and survival. FBXW7 affects the pathway indirectly. FBXW7 is an enzyme
that degrades c-Myc, mutations to this gene prevents the degradation of c-myc and increase levels of
c-myc. C-myc is known to work with the Wnt/Beta-caternin pathway to transcribe target genes that
express proteins which regulate cell proliferation and survival. The effects of these mutations on the
Wnt/beta caternin pathway can be investigated by measuring the levels of beta-caternin and the
expression of target genes associated with the pathway. This can be done using a qRT-PCR analysis to
calculate the expression of the target genes of the pathway compared the expression of the target
genes of the pathway in cells with the wild type version of the genes. Higher expression levels of
target genes indicate the pathway is being overactivated due to the mutation.

Other acquired mutations found in the tumour were: PIK3CA and PTEN. PIK3CA gene encodes for a
catalytic subunit for a protein needed for the activation of downstream signalling proteins. PTEN is a
tumour suppressor gene and is a negative regulator of the pathway by inhibiting downstream
proteins. Akt is a serine/threonine kinase and is responsible for activating downstream proteins that
induce cell proliferation and survival. Mutations of these genes can cause the overactivation of this
pathway, stimulating uncontrolled proliferation of cells. The effects of these mutations on the
pathway can be measured the phosphorylation status of key components in the pathway, such as:
Akt and mTOR. Phosphorylation activity of these proteins can be measured using western blot
analysis to measure expression and activation status of proteins downstream the pathway. A higher
expression and activation of Akt and mTOR in cancer cells compared to normal cells with the wild
type genes, indicate the pathway is being overactivated.

The patient’s tumour also included KRAS mutations. KRAS is a gene that encodes for K-Ras which
functions as a molecular switch and cycles between an inactive GDP state to an active GTP state. The
activation of K-Ras is triggered by a guanine exchange factor and stimulates the activation of different
pathways, including the Ras/MAPK pathway. Mutations in this gene can lead to the overactivation of
the pathway leading to uncontrolled proliferation of cells and promoting the survival of cancer cells.
To identify the effect of the mutated gene on the pathway, western blotting analysis can be used to
measure the level of expressed and activation status downstream signalling proteins. The level of
expression and activation status of RAS, MEK and ERK proteins can be measured in the cancer cell
from the patient and compare it to the level of expression and activation status in cells with the wild
type gene. Significantly higher expression and activation of these proteins, indicate the pathway is
being overactivated by the mutated KRAS gene.

4th question:
The patient has a mutation in the NOTCH3 gene. This gene encodes for the
NOTCH3 receptor. A gain of function mutation of this gene can cause the NOTCH3
receptor to be continually activated, even when there is no ligand binding to it. It
leads to overactivation of the NOTCH signalling pathway, responsible for cellular
processes, such as: proliferation and survival. The effects on the signalling pathway
by the mutated NOTCH3 gene could be inhibited using a gamma secretase inhibitor
(nirogacestat). This inhibitor prevents the cleavage of the NOTCH receptor causing
inactivation of the receptor. Consequently, suppressing tumour growth driven by
overactivation of the NOTCH signalling pathway.
Additionally, the patient was given the MEK inhibitor (PD198306) which reduced the
phosphorylation of ERK1/2, RSK, mTOR and S6K. Whilst, the AKT inhibitor reduced
the phosphorylation of mTOR, S6K and GSK3. As the phosphorylation level of the
signalling proteins decreased, it means that the activation of the RAS/MEK/ERK and
PI3K/Akt/mTOR pathways is also reduced. These pathways are key regulators of the
cellular processes that drive cancer. The phosphorylation of proteins involved in the
pathways by the inhibitors highlight the potentiality of combining the inhibitors to
produce a synergistic inhibition of the pathways that drive cancer.