The Clinical and Biological Significance of Tyrosine Kinases in Gastric Cancer

Abstract

Gastric cancer (GC) is the leading cause of cancer-related mortality, and its deadly nature can be secondary to its presentation in advanced stages. Unavailability of any gold-standard treatment and also the lack of unanimous classification schemes that can lead to inter-observer variability, lead to difficulties in the clinical reproducibility. Several classification systems have been proposed for GC. The most used classification system is Lauren classification, which classifies it into “intestinal,” “diffuse,” and “mixed” subtypes.

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© Springer Nature Singapore Pte Ltd. 2018
G. P. Nagaraju (ed.), Role of Tyrosine Kinases in Gastrointestinal Malignancies,
https://doi.org/10.1007/978-981-13-1486-5_3
N. Khetpal · S. Ali · R. Kumar · M. U. Rashid
Department of Internal Medicine, Florida Hospital, Orlando, FL, USA
e-mail: neelam.khetpal.MD@hosp.org; saeed.ali.MD@hosp.org;
ranjeet.kumar.MD@hosp.org; mammon.rashid@hosp.org
S. Ahmad (*)
FSU and UCF Colleges of Medicine, Orlando, FL, USA
Gynecologic Oncology, Florida Hospital, Orlando, FL, USA
e-mail: sarfraz.ahmad@hosp.org
3
The Clinical and Biological Significance
of Tyrosine Kinases in Gastric Cancer
Neelam Khetpal, Saeed Ali, Ranjeet Kumar,
Mamoon Ur Rashid, and Sarfraz Ahmad
Abstract
Gastric cancer (GC) is the leading cause of cancer-related mortality, and its
deadly nature can be secondary to its presentation in advanced stages.
Unavailability of any gold-standard treatment and also the lack of unanimous
classication schemes that can lead to inter-observer variability, lead to difcul-
ties in the clinical reproducibility. Several classication systems have been pro-
posed for GC. The most used classication system is Lauren classication,
which classies it into “intestinal,” “diffuse,” and “mixed” subtypes.
This chapter summarizes the characterization of GC with genomic and molec-
ular analysis to stratify the heterogeneous disease, role of tyrosine kinase (TK),
receptor tyrosine kinases (RTK) and tyrosine kinase inhibitors (TKI), targeted
therapies and ongoing clinical trials, toxicities associated with various com-
monly used agents/regimens in the disease, and future perspective of TKI in GC.
Advances in the genomic technologies have facilitated the study of key
genetic alterations in GC including gene expression, epigenetic disturbances,
chromosomal alterations, and transcriptional changes, which therefore can strat-
ify GC at the molecular levels. The characterization of GC at molecular levels
has led in developing new therapeutic targets that would potentially provide per-
sonalized prognosis and treatment. The RTKs are membrane-bound proteins that
play signicant role(s) in the pathogenesis of many cancers including GC.Of
many RTKs, epidermal growth factor (EGF), vascular endothelial growth factor
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(VEGF), platelet-derived growth factor (PDGF), and broblast growth factor
(FGF) have been found with higher frequencies in metastatic growth and pro-
gression of GC, which serve as potential target(s) for targeted therapies in
GC.Human epidermal growth factor receptor 2 (HER-2) that promotes cell pro-
liferation, adhesion, migration, and differentiation is overexpressed in 15–30%
of GC cases. The phase III ToGA (trastuzumab for gastric cancer) study evalu-
ated the role of adding trastuzumab, an anti-HER-2 monoclonal antibody, to the
chemotherapy regimen in the rst line of treatment in patients with HER-2-
positive advanced-stage GC.
VEGF is seen to be overexpressed in up to 58% of GC cases. The REGARD
study and the phase III study have shown an improved overall survival benet
with ramucirumab, a monoclonal VEGFR2 antibody. Cetuximab is used when
EGFR is overexpressed in gastric tumors, and dovinitib decreased phosphoryla-
tion of FGFR2.
Common toxicities of trastuzumab include fever, chills, hypotension, dys-
pnea, bronchospasm, and respiratory distress, but life-threatening side effects
such as cardiotoxicity and congestive heart failure can also occur. In the
RAINBOW trial, ramucirumab plus paclitaxel showed higher rates of neutrope-
nia, and other toxicities related to ramucirumab included hypertension, thrombo-
embolic disease, and hemorrhage. Cetuximab use can lead to skin disorders such
as dry skin, dermatitis acneiform/rash, and paronychia.
Taken together, these molecular and cellular mechanisms/targets and support-
ing clinical trials outcomes have a signicant impact on the overall quality of life
and the prognosis of patients with gastric cancer.
Keywords
Gastric cancer · Tyrosine kinase · Tyrosine kinase inhibitors · Receptor tyrosine
kinase · Biological functions · Tumorigenesis · Clinical trials · Anticancer drugs
· Clinical outcomes
3.1 Introduction/Background
Gastric cancer (GC) is the fth leading cause of cancer after lung, breast, colorectal,
and prostate cancers and third most common cause of cancer-related deaths in both
genders (Table3.1). The highest mortality rates are reported in East Asia, whereas
the lowest were reported in the North America [1]. Despite its gradually decreasing
incidence, ~75% mortality rate makes it one of the highest worldwide [2]. In most
countries, its 5-year survival rate is between 10% and 30%, whereas in the United
States, it ranges between 20% and 30%. The highest 5-year survival rate is found in
Japan, where it ranges from 50% to 70% for both genders [3].
Common risk factors for GC are infection by Helicobacter pylori, smoking,
pickled vegetables, and obesity. GC is generally divided into epithelial and non-
epithelial neoplasms. Most of the GCs are of epithelial origin. Non-epithelial GCs
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predominantly includes lymphomas and mesenchymal tumors. Most cases of GC
are sporadic, whereas 10% of the cases run within the families, and between 1% and
3% of the cases are due to genetic syndromes (for instance, hereditary diffuse gas-
tric cancer and gastric adenocarcinoma and proximal polyposis of the stomach).
Furthermore, gastric cancer can also develop in the setting of various other heredi-
tary cancer syndromes [4].
As depicted in Fig.3.1, gastric carcinogenesis is a complex, multifactorial, and
multistep process that progresses from normal mucosa through chronic gastritis,
atrophic gastritis, and intestinal metaplasia to dysplasia and carcinoma. This
sequence of events may take several years to complete. H. pylori has been consid-
ered as one of the most common environmental agent causing an increased risk of
gastric cancer. Per the International Agency for Research on Cancer (IARC), H.
pylori is a group 1 carcinogen for gastric cancer [5]. Two virulence factors respon-
sible for the pathogenicity of H. pylori include CagA (cytotoxin-associated gene A)
in the Cag pathogenicity island and the vacuolating cytotoxin (vacA) [6]. Besides H.
pylori, numerous dietary habits have been studied and reported that alter the risk of
gastric cancer (Table3.2).
Symptoms of GC are non-specic such as abdominal pain and dyspepsia, which
are often mistaken for indigestion or peptic ulcer disease. Patients may also present
with nausea or early satiety from the tumor mass or in cases of an aggressive form of
diffuse-type gastric cancer (linitis plastica) from poor distensibility of the stomach.
GC involving Auerbach’s (myenteric) plexus may present with dysphagia, and this
variety of dysphagia is termed as pseudoachalasia. Therefore, GC should be consid-
ered in the differential diagnosis for older patients presenting with dysphagia [7].
The threshold for suspicion of the advanced disease should be low if dyspepsia
is concurrently present with alarm symptoms like dysphagia, weight loss, gastroin-
testinal bleeding, and a palpable abdominal mass. Accumulation of alarm symp-
toms in GC is associated with a higher risk of death [8]. Like many other cancers, it
is not uncommon for gastric cancer to present in the late phases after the disease has
already reached an advanced stage. It is only possible in the early stages where a
total surgical resection of the tumor can lead to a complete cure. However, most
tumors in the early stages are often asymptomatic and even after surgical resection,
tumors can recur resulting in a relatively shorter survival times. This innate ability
of GC to present in the late stages and the lack of effective therapy for the advanced
stages unfortunately is associated with the higher recurrence and mortality rates.
Advanced-stage cases of GC manifest according to the type of tissue or organ
involvement such as jaundice, ascites, or gastrointestinal tract obstruction. Peritoneal
implant in the pelvis (Blumer’s shelf) can lead to peritoneal uid accumulation or
colorectal obstruction. Blumer’s shelf or cul-de-sac can be felt on rectal or vaginal
Table 3.1 Prevalence of the top ve types of cancers in men and women [4]
Rank 1 2 3 4 5
Cancer in men Lung Prostate Colorectal Stomach (gastric) Liver
Cancer in
women
Breast Colorectal Lung Cervix Stomach (gastric)
3 The Clinical and Biological Significance of Tyrosine Kinases in Gastric Cancer
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examination, which may indicate that a tumor has metastasized to the pouch of
Douglas. The GC involvement of the lymph nodes can easily be appreciated on the
physical examination such as supraclavicular lymph nodes (Virchow’s node) and
protuberant nodules around the umbilicus (Sister Mary Joseph nodule).
Table 3.2 Effects of key dietary factors on the risk of gastric cancer
Factors that increase the risk
of gastric cancer Factors that decrease the risk of gastric cancer
Helicobacter pylori Mediterranean diet (high consumption of fruit, vegetables,
cereals, legumes, nuts and seeds, and seafood, with olive oil)
Pickled vegetables
Smoked food
Lack of fruits and
vegetables in diet
Moderate alcohol consumption (particularly red wine)
Red meat
Processed meat Fresh fruits and vegetables
Tobaccoa
aSmoking potentiates the carcinogenic effect CagA positive H. pylori
Fig. 3.1 Schematic
presentation of the key
stages of gastric
carcinogenesis
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Clinically, the gastric neoplasm is classied as an early or advanced stage and
histologically into various subtypes (on the basis of major morphologic compo-
nents). The World Health Organization (WHO) has recognized ve major histologic
types of gastric carcinoma: tubular, papillary, mucinous, poorly cohesive (with or
without signet ring cells), and mixed [9].
3.2 Characterization of Gastric Cancer with Genomic
and Molecular Analyses to Stratify the Heterogeneous
Disease
Different classication systems exist to characterize gastric cancer. Most popular
classication systems for GC include the Lauren classication and the WHO clas-
sication (based on the predominant histological pattern) [10]. Lauren classication
further classies it into two subtypes, viz., intestinal type and diffuse type. The two
variants exhibit marked molecular and clinical heterogeneity. The intestinal type
most commonly occurs in the elderly male patients and affects gastric antrum,
which is frequently associated with intestinal metaplasia. Tumor cells exhibit adhe-
sion and lesions are scattered in the distant positions. In contrast, the diffuse type
commonly affects the relatively younger population; cells lack adhesion and forms
non-cohesive scattered tumor cell population. It frequently involves gastric body
and predominantly involves females. The diffuse type has a worse prognosis as
compared to the intestinal type and has a high propensity for intraperitoneal metas-
tasis and CDH1 (cell-cell adhesion receptor gene E-cadherin) silencing [11].
Etiologically, the intestinal type of GC is commonly caused by environmental
factors such as H. pylori, whereas the diffuse type is more genetic in etiology [12].
The carcinogenesis of intestinal GC entails H. pylori along with the diet and other
environmental factors and is a multistep process involving atrophic gastritis, intes-
tinal metaplasia, dysplasia, and ultimately cancer. The diffuse type develops directly
from the chronic active gastritis bypassing the atrophic gastritis and intestinal meta-
plasia. The 2010 WHO classication divides gastric cancer into four major histo-
logical subtypes, viz., tubular, papillary, mucinous, and poorly cohesive (including
signet ring cell carcinoma) [13].
Clinically, gastric cancer can be classied into early and advanced-stage disease.
The early GC is limited to mucosa and submucosa with and without lymph node
metastases, mostly 2–5cm in size and located along the lesser curvature. Grossly,
early GC can be divided into type 1 (protruded growth), type II (supercial growth),
type III (excavating growth), and type IV (inltrating growth with lateral spread-
ing). Histologically, most of the early gastric cancers have tubular or papillary archi-
tecture and are well-differentiated. The 5-year survival of early gastric cancer is
around 90%, depicting an excellent prognosis. The advanced-stage GC invades into
muscularis preppie and beyond. Grossly, it could be ulcerating, fungating, inltra-
tive, or combined. Pathologically, several histological patterns coexist, and there is
marked architectural and cytological heterogeneity in advanced-stage gastric can-
cer. It carries a poor prognosis with a 5-year survival of 60% or less [13].
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Histopathological classications sometimes guide therapy but are insufcient to
guide personalized treatment, which is seriously needed given the wide heterogene-
ity of gastric cancer.
The hereditary diffuse gastric cancer (HDGC) accounts for approximately 3% of
the GC cases and harbor germline mutations in CDH1 that encodes for E-cadherin
in a signicant proportion of cases. It has a penetrance rate of 80%, making it a
high-risk for developing into a diffuse gastric cancer. Additional familial syndromes
associated with GC include hereditary nonpolyposis colorectal cancer II (HNPCC/
Lynch syndrome II) with MSH2 (MutS protein homolog 2) and MLH1 (MutL
homolog 1) mutations, mostly Li-Fraumeni syndrome with TP53 (tumor protein 53)
mutations, adenomatous polyposis coli (APC) mutations, and Peutz-Jeghers syn-
drome (STK11 or serine/threonine kinase 11). Majority of the GC occurs sporadi-
cally. Some of the important risk factors include blood group A (20% increased risk
compared to other blood groups), H. pylori, and EBV (Epstein-Barr virus) infec-
tion. Clustering of H. pylori can explain the increased incidence of gastric cancer in
certain families [13].
3.2.1 Genomics of Gastric Cancer
Recent advances in genomic technology have greatly facilitated the GC biology to
be studied at molecular levels thus identifying candidate driver genomic alterations
such as gene expression, epigenetic disturbances, chromosomal alterations, and
transcriptional changes (as depicted in Fig.3.2) [10].
3.2.2 Gene Mutations in Gastric Cancer
The systemic analysis and high throughput of genetic alterations in the genome can
be facilitated by next-generation sequencing (NGS), which has identied novel
gene mutations in the GC.Mutations in the genes involved in chromatin remodel-
ing, genome integrity, cell adhesion/cytoskeleton/motility as well as Wnt and RTK
signaling pathway have been identied in GC.The genomic instability plays impor-
tant role(s) in tumorigenesis and can be caused by the mutations in “caretaker/tumor
suppressor genes” such as TP53 and BRCA2 (breast cancer susceptibility gene 2),
which are primarily involved in the DNA damage detection and repair and are fre-
quently mutated in GC.Chromatin alteration is another emerging cellular mecha-
nism of carcinogenesis which impairs DNA accessibility to transcriptional factors
and thus greatly affects gene expression. SW1-SNF (switch/sucrose non-
fermentable) is a chromatin-remodeling complex, and ARID1A (AT-rich interactive
domain 1A gene) encodes a subunit of this complex. ARID1A is a commonly
mutated chromatin-remodeling gene in GC.The genes involved in the cell adhe-
sion/motility/cytoskeleton regulate the cell-extracellular matrix and intercellular
interactions. The mutations in these genes [including CDH1, CTNNA1 (rabbit
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catenin alpha-1), and RhoA (ras homolog gene family member A)] are frequently
seen in the diffuse type of gastric cancer. While CDH1 belongs to the E-cadherin
family and regulates cell-cell adhesion, CTNNA1 encodes a protein involved in the
cell adhesion to the cytoskeleton.
The NGS has also identied key aberrant cellular pathways in GC.Two major
pathways involved are the Wnt signaling pathway and the RTK-associated path-
ways. Activating mutations in CTNNB1, inactivating mutations in APC, and ring
nger protein 43 (RNF43) are involved in the generation of dysregulated Wnt sig-
naling pathway. Similarly, for RTK-associated pathways, mutations in ErbB3 RTK
and Neuregulin 1 (NRG1)/EebB4 ligand/RTK pair have been reported in >10% of
the GC cases. The phosphatidylinositol 3-kinase (PIK3) is another pathway down-
stream of the RTK signaling and is responsible for cell growth, survival, and prolif-
eration. The PIK3 catalytic subunit alpha (PIK3CA), main catalytic component of
the PIK protein, is found to be frequently mutated in microsatellite instability (MSI)
and EBV-positive GC subtypes. The molecules targeting the Wnt signaling and
TRK signaling pathways are some of the promising targets for GC treatment [10].
Fig. 3.2 Genetic and epigenetic modications of gastric cancer (GC). The genetic alteration that
contributes to GC involves gene mutations, differential gene expression as well as somatic copy
number alterations (SCNAs). The epigenetic modications involve DNA as well as histone methyla-
tion. The subtypes highlighted in red are reported in The Cancer Genome Atlas (TCGA) study [10]
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3.2.3 Chromosomal Instability
Somatic copy number alterations (SCNAs) result from the alterations in the DNA
copy number that leads to the structural variation in DNA.Specic SCNAs are
associated with histological type in GC; for example, intestinal-type GC is associ-
ated with gain in the gene copy at 8q, 17q, and 20q, whereas the diffuse-type GC is
associated with gains at 12q and 13q. Deng etal. [24] reported that GCs exhibit
frequent focal SCNAs such as amplications in genes involved in the RTK/RAS/
MAPK signaling pathway. Many of the genes involved in this pathway such as
ERBB2, EGFR, MET, and FGFR2 can be targeted by novel medications. Medications
targeting the RTK/RAS/MAPK signaling pathway can potentially treat 37% of the
GC population [10]. Other SCNAs and amplications involved in GC include Janus
kinase 2 (JAK2), programmed death-ligand (PDL)1/2 (immune checkpoint inhibi-
tors), frequently mutated in EBV-positive subtype of The Cancer Genome Atlas
(TCGA) network, and transcription factors including Kruppel-like factor 5 (KLF5),
GATA4, and GATA6. The KLF5/GATA4/GATA6 transcription factor mutations
have been found in 30% of the GC cases. Another marker of chromosomal instabil-
ity is the loss of heterozygosity (LOH), which can result in the loss of tumor sup-
pressor genes such as APC and TP53 genes. The high-level LOH has been associated
with the intestinal or mixed-type GC and low-level LOH with diffuse-type GC [10].
Genomic instability varies in patients from the different geographical locations
indicating possible heterogeneous biological mechanisms at different locations.
3.2.4 Transcriptional Changes in Gastric Cancer
Gene expression proling using the NGS and microarrays can be used to dene the
transcriptional changes in GC.It has identied pathways involved in the cell migra-
tion, metastases, cell cycle, and the cytoskeletal organization to be upregulated in
GC that has both diagnostic and prognostic signicance. Several studies have iden-
tied expression signatures based on the gene expression proling to predict sur-
vival independent of the tumor, node, and metastasis (TNM) staging, the gold
standard for prognosis in GC.Lei etal. [14] classied GCs based on the expression
signatures into proliferative, metabolic, and mesenchymal subtypes showing the
genetic and molecular differences as well as response to the therapy offered. The
metabolic subtype has shown increased sensitivity to 5-uorouracil and phosphati-
dylinositol 3-kinase-Akt-mTOR inhibitors, respectively, whereas the proliferative
subtype has a higher rate of TP53 mutation and genomic instability [14].
3.2.5 Epigenetic Modifications in Gastric Cancer
Epigenetic dysregulation of gene expression can lead to the development of malig-
nant cell transformation. Hypermethylation of promoter regions results in transcrip-
tional silencing of the mismatch repair (MMR) and tumor suppressor genes– for
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example, hypermethylation of hMLH promoter region can cause MSI phenotype in
GC.CpG island methylation phenotype (CIMP) characterized by the genome-wide
methylation of CpG islands rather than any single gene is also demonstrated in
GC.Kim etal. [15] demonstrated that CIMP is seen in about 35% of the GC cases;
occurs in relatively younger patients; is associated with oncogene mutations includ-
ing KRAS, ERBB2, PIK3CA; and has a worse prognosis. The DNA-demethylating
drugs such as azacitidine and decitabine, which are clinically used for myelodys-
plastic syndrome (MDS), could also be promising for the epigenetic aberrations in
GC [15]. Histone modication is another mechanism responsible for the epigenetic
aberration in GC.Understanding of the epigenetic proling of the cells can help
stratify gastric cancer and also serve as potential targets for treatment [10].
3.2.6 Molecular and Genomic Stratification of Gastric Cancer
Recently, TCGA network carried out a landmark study for the genetic and molecu-
lar characterization of GC, including 295 gastric cancers samples [based on 6
molecular platforms including array-based somatic copy number analysis, whole-
exome sequencing, messenger RNA sequencing, microRNA (miRNA) sequencing,
array-based DNA methylation proling, and reverse-phase protein array (RPPA)]
[16]. Based on the integrative analysis, TCGA classies GC in four subtypes: EBV-
positive tumors, MSI tumors, genomically stable tumors, and chromosomal insta-
bility tumors [16].
The EBV subtype is suggestive of viral etiology of gastric cancer and is detected
in about 9% of the malignant cells in gastric cancer. DNA hypermethylation was the
most prevalent in the EBV-positive tumors in TCGA. The EBV-positive tumors
have a very strong predilection for PIK3 mutation, and 80% cases have non-silenced
PIK3CA mutation in this subset [16]. The rate of PIK3CA mutation ranges from 3%
to 42% in other subtypes. It also exhibits a mutation in ARID1A and Bcl6 corepres-
sor (BCOR) and has a high frequency of amplication of PD-L1/2 and JAK2 genes
[17, 18]. The MSI tumors exhibit a high prevalence of MLH1 promoter hypermeth-
ylation and occasional mutations in PIK3CA, ERBB2, ERBB3, and EGFR.Tumors
lacking higher rates of mutation or hypermethylation and aneuploidy were regarded
as genomically stable. They were predominantly present in the diffuse histological
subtype. Molecular alterations in TCGA GS subtype include abnormalities of
CDH1 and RHOA signaling pathways [11, 17]. Tumors with chromosome instabil-
ity (CIN) show marked aneuploidy and frequently exhibit amplications in RTK-
RAS pathway resulting in its activation [11, 18].
The Asian Cancer Research Group (ACRG) conducted another similar landmark
study on 300 gastric cancer samples using targeted gene sequencing, genome-wide
copy number microarrays, and gene expression proling and classied GC into four
subtypes: MSI, MS stable/epithelial to mesenchymal transition (MSS/EMT), MSS/
TP53 +ve (intact TP53 activity), and and MSS/TP53 –ve (functional loss of TP53).
The MSI tumors have the best prognosis of all the ACRG subtypes, comprise
intestinal- type tumors frequently, contain hypermutation at a molecular level, and
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are diagnosed at an early stage of the disease [17, 18]. The MSS/EMT subtypes
have the worst prognosis and highest recurrence rate of all the subtypes. It fre-
quently comprises diffuse-type cancers and harbor CDH1 and/or RHOA mutations
corresponding to the GC tumors in TCGA classication [11, 17]. The MSS/TP53
active subtype has a better prognosis than the MSS/TP53 inactive tumors. The MSS/
P53+ subtype, corresponding to TCGA EBV-positive subgroup, exhibits a higher
prevalence of the mutations in ARID1A, APC, PIK3CA, KRAS, and SMAD4 muta-
tions. Amplication of ERBB2, CDNE1, CCND1, and MDM2 is enriched in MSS/
P53 inactive subtype, corresponding to TCGA CIN subtype and could be the targets
of novel therapies currently available or are under trials such as trastuzumab, CDK2
inhibitors, CDK4/6 inhibitors, and MDM2 inhibitors, respectively [17–19].
Although there are similarities between the two classication systems, some differ-
ences exist as well in terms of the demographics and molecular mechanisms which
indicate that these are overlapping but distinct classication systems. The ACRG
classication complements the TCGA classication and uses additional incorpora-
tion of the two key molecular mechanisms (TP53 activity and EMT) to stratify
gastric cancer patients further [11, 19].
Multiple genetic and epigenetic aberrations characterize gastric cancer and are
likely responsible for the heterogeneous and complex nature of this disease.
Advancements in the genomic and molecular analyses of gastric cancer have cre-
ated a preliminary road map to stratify the heterogeneous disease and to develop
targeted therapies for the distinct group of patients with the eventual goal of improv-
ing survival in gastric cancer.
3.3 Role of Tyrosine Kinases, Receptors, and Inhibitors
in Gastric Cancer
Protein kinases are enzymes catalyzing transfer of phosphoryl group from adenos-
ine triphosphate (ATP) to proteins specically either serine/threonine or tyrosine
side chains of the protein, playing important roles in signal transduction and other
cellular cross-talk processes [20]. The RTKs are a family of 56 membrane-bound
proteins, which fall into 21 subfamilies. They are characterized by an extracellular
ligand-binding domain, a transmembrane portion, and a cytoplasmic tyrosine kinase
motif. All known RTKs, with the exception of insulin receptors, form monomers in
the cell membrane. The RTKs play key roles in the cell cycle regulation, cell prolif-
eration and differentiation, survival and metabolism, cell adhesion, and migration
[21, 22]. Mutations in the RTKs and alterations in the downstream signaling path-
ways have been associated with almost all cancers, inammation, angiogenesis, and
arteriosclerosis [22]. Overexpression and/or activation of the RTKs transforms cells
and plays signicant role(s) in the development and progression of cancers. These
causal associations led to the development of TKIs and render them the targets for
many immunological treatments such as trastuzumab (ERBB2 receptor inhibitor)
and cetuximab (EGFR2 blocker) and other TKIs such as imatinib and getinib [21].
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The ligand-induced dimerization results in autophosphorylation of cytoplasmic
tyrosine kinase moieties, and the downstream signaling proteins lead to activation
of RTKs [21, 23]. Activation mainly involves two processes with autophosphoryla-
tion being critical for both activities, amplication of intrinsic catalytic activity of
RTKs and creation of the recruitment sites for the downstream signaling proteins
[23]. Previously, ligand-induced dimerization was thought to be a simple mecha-
nism of a bivalent ligand simultaneously binding to two receptors and cross-linking
them into dimeric complex. Recent studies have provided more insights into the
four most plausible mechanisms of receptor dimerization [22, 23]: either entirely
receptor-mediated without much contribution from ligand to the dimer interface or
entirely ligand-mediated without direct contact between the two receptor molecules.
Alternatively, dimerization could involve both the ligand-mediated and receptor-
mediated components. All tyrosine kinase domains (TKDs) have a C-lobe and an
N-lobe with the crystal structure of activated TKDs being similar for most of the
TKs. In all activated TKs, “activation loop” and alpha-C helix in N-lobe exhibit a
specic conguration necessary for the phosphoryl transfer. Each TKD is cis-
autoinhibited uniquely through several molecular interactions, and the release of
cis-autoinhibition following the ligand-mediated receptor dimerization is the major
event triggering the RTK activation [22].
3.3.1 Receptor Tyrosine Kinases in Gastric Cancer
Of the 56 known RTKs falling into the 21 families, several RTKs including the
EGFR family (ErbB1 to B4), VEGFR subtypes, the FGF receptor family, and the
PDGF receptor family have been found in gastric cancer growth and progression
and thus serve as potential targets for novel therapies [21, 23]. Deng et al. [24]
reported alterations in 37% of gastric cancers with FGFR2 being the most frequently
amplied RTK (9.3%), followed by KRAS (8.8%), EGFR (7.7%), and ERBB2
(7.2%). Amplication of the RTKs has been regarded as an independent poor prog-
nostic marker in gastric cancers [24].
3.3.2 Role of EGF Receptor/HER Tyrosine Kinase Family
in Gastric Cancer
The EGF family of receptors consists of four subtypes including HER1/Erb B1, also
called EGFR, HER-2/Erb B2, HER 3/Erb B3, and HER 4/Erb B4 which are encoded
by Erb oncogenes [23]. The EGF family of receptors like other tyrosine kinase
receptors has an extracellular ligand-binding domain (I–IV), a transmembrane com-
ponent, and intracellular tyrosine kinase domain except Erb B3. The binding of
oncogenes such as EGF, transforming growth factor alpha (TGF-α) and heparin
binding EGF activates EGF receptors by either homodimerization (binding to a
similar receptor class) or heterodimerization (binding to the receptor of a different
class). This, in turn, results in autophosphorylation of the TK residues and initiation
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40
of the downstream signaling cascade. The signaling cascade is a complex pathway
with multiple passages and includes Ras/Raf/mitogen-activated protein kinase
(MAPK)/cyclin-D1 pathway, PI3K/Akt pathway, and signal transducers and activa-
tors of transcription (STAT) signaling pathways involved in the cell differentiation
and proliferation [25] (Fig.3.3).
Fig. 3.3 The EGFR signaling: EGFR is activated by ligand binding and subsequent receptor het-
erodimerization or homodimerization, which results in autophosphorylation of tyrosine residues
and binding of adaptor molecules like Shc and Gab-1 to the cytoplasmic domain. Intracellular
signaling pathways include RAS/Raf-1/MAPK, P13K/AKT, and PLC-y/PKC pathways which
require the adaptor molecules for signaling. The Src and STAT pathways are directly activated by
the phosphorylated receptors. Alternately, the activated receptors can undergo endocytosis fol-
lowed by the importin-mediated translocation to the nucleus and co-transcriptional of the key
genes like Cox-2, iNOS, aurora kinase-A, and cyclin-D1. All these pathways lead to changes in the
gene expression and stimulation of cell proliferation, survival, invasion, and metastasis. The EGFR
manipulation can be approached extracellularly by monoclonal antibodies through inhibition of
the ligand binding and intracellularly by the TKIs, which compete with the ATO binding to recep-
tor kinase for activation [25]. Abbreviations: EGFR endothelial growth factor receptor, Shc Src
homology-2 domain containing transforming protein-1, Grb2 growth factor receptor bound pro-
tein- 2, Sos son of sevenless, Ras rat sarcoma, Raf-1 rapidly accelerated brosarcoma, MAPK
mitogen-activated protein kinase, Gab-1 Grb2-associated binding protein-1, P13K phosphoinosit-
ide 3 kinase, AKT protein kinase B, Src sarcoma gene, FA K focal adhesion kinase, S TAT signal
transducer and activator of transcription, TKI tyrosine kinase inhibitor, PLC-y phospholipase C-y,
PIP2 phosphatidylinositol 4,2 biphosphate, IP3 1,3,5-triphosphate, DAG 1,2-diacylglycerol, PKC
protein kinase C, and ER endoplasmic reticulum
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The EGF receptors also promote tumor growth independent of the abovemen-
tioned pathways. The EGF receptor is internalized after the activation and is trans-
ported to the nucleus by cytoplasmic importin-ß where it activates many gene
promoters [25]. Of all the EGF family of receptor tyrosine kinases, the HER-2 tar-
geted therapy has been most successful and well-studied treatment for advanced
gastric cancer. HER-2/Erb B2 is overexpressed in 10–38% of gastric cancers [23].
The EGFR overexpression has been detected in 27–64% of gastric cancers. Elevated
levels have been detected in the advanced-stage gastric cancer with poor prognostic
factors (T3/T4, G3, lymph node +ve, diffuse subtype) [21, 23]. Treatments targeting
EGFR/Erb B involve small molecule kinase inhibitors and monoclonal antibodies.
In contrast to the kinase inhibitors, monoclonal antibodies against EGFR/ErbB2
have inherent ability to recruit inammatory cells via binding of antibody Fc domain
to the tumor cell-specic receptors [21].
Trastuzumab (Herceptin®) is a humanized IgG1 monoclonal antibody against
the extracellular domain IV of HER-2/neu receptor. It prevents activation of its
intracellular tyrosine kinase domain and downstream signaling pathways, thus
interrupting cell cycle progression and growth of tumor cells. Postulated mecha-
nisms include activation of natural killer cells, antibody-dependent cellular cytotox-
icity (ADCC) and destruction of the tumor cells bound to the Fc domain of
trastuzumab [25, 26]. The ToGA (trastuzumab for gastric cancer) trial was a phase
III, international, randomized controlled, open-label trial which evaluated the role
of trastuzumab in the treatment of advanced gastric cancer. Results from ToGA trial
demonstrated that trastuzumab in combination with standard chemotherapy
improves the overall survival by more than 1 year in advanced gastric cancer and
has proven to be the landmark study for advanced gastric cancer [26, 27]. Ado-
trastuzumab emtansine (T-DM1) is an antibody-drug conjugate of trastuzumab with
microtubule polymerization inhibitor, derivative of maytansine. It undergoes
receptor- mediated internalization after the binding to HER-2 and releases DM1 into
cytoplasm, which can induce apoptosis and ADCC.An ongoing phase II study of
T-DM 1, the trastuzumab-emtansine conjugate, in advanced gastric cancer is being
conducted as this conjugate drug has shown to inhibit the gastric cancer cells invitro
and invivo [25].
Cetuximab is a chimeric monoclonal antibody against EGFR, which blocks EGF
and TGF-α ligand binding to EGFR and activation of TKR.In gastric cancer cell
lines, it inhibits EGF-induced EGFR and HER-2 phosphorylation, EGFR homodi-
merization, EGFR and HER-2 heterodimerization, and signal transduction via the
MAPK and Akt pathways. In contrast to cetuximab, which is a chimeric antibody,
panitumumab is a full humanized monoclonal antibody against EGFR and has mini-
mal immunogenicity and is unable to stimulate ADCC.
Pertuzumab is another fully humanized monoclonal antibody binding to HER-2
domain II instead of domain IV by trastuzumab. It inhibits HER-2 heterodimeriza-
tion with other EGFR receptors and activates ADCC.Combination of trastuzumab
and pertuzumab has been studied to be more effective than therapy alone in the
xenograft models, and currently clinical trials are being done to evaluate this com-
bination in advanced gastroesophageal cancers [25].
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Getinib and erlotinib are the EGFR receptor tyrosine kinase inhibitors inhibit-
ing phosphorylation step that follows the EGFR receptor dimerization and thus hin-
ders the downstream signaling proteins [25]. These drugs have already been
approved for the treatment of non-small cell lung cancer (NSCLC) when the rst-
line therapy failed but have not shown promising results yet in a phase II trial for
gastric cancer [4].
Lapatinib is a dual reversible tyrosine kinase inhibitor blocking both HER-2 and
EGFR.In contrast to erlotinib and getinib, it has slower receptor dissociation rates
and binds to the inactive EGFR conformation. Lapatinib produces its effect through
survivin, an apoptosis inhibitor protein. It causes growth inhibition in HER-2 ampli-
ed gastric cell lines in both invitro and in vivo studies. Currently, two phase III
trials are evaluating its role as a rst-line and second-line therapy for advanced
gastric cancers [23, 25].
3.3.3 Vascular Endothelial Growth Factor Receptor
Abnormal angiogenesis is ubiquitous in all the malignant tumors with VEGF
secreted by the tumor cells being the key mediator of angiogenesis. VEGFR pro-
motes cell growth and metastases and is frequently overexpressed in the gastric
cancer cell lines [21, 26]. Kinase insert domain receptor (KDR) is one of the main
VEGF receptors involved in physiological and pathological angiogenesis, and
VEGF-KDR signaling pathway is a potential therapeutic target for cancer treatment
since VEGF expression is associated with high recurrence of gastric cancer.
Bevacizumab is a monoclonal antibody against VEGF-A and interrupts the signal-
ing pathways by neutralizing the VEGF ligand instead of directly binding to the
tyrosine kinase receptor [21]. Ramucirumab is a humanized monoclonal antibody
against VEGFR-2 and interrupts downstream pathways for angiogenesis. Apatinib
is a small molecule TKI that targets VEGF-2, which has been shown to improve the
overall survival in heavily pre-treated metastatic gastric cancer which has failed two
or more chemotherapy agents in phase II and III trials. It is the rst VEGFR tyrosine
kinase inhibitor to have a small but clinically signicant effect on advanced gastric
cancer among the Asian patients [26].
3.3.4 Fibroblast Growth Factor Receptor
Growth factors of FGF family, which are secreted by broblasts, result in tumor
proliferation in scirrhous gastric cancer. Gastric cancers expressing high levels of
FGF2 mRNA (basic FGF) have demonstrated higher microvascular density, tumor
progression, and worse outcomes. Orally active inhibitor of FGF autophosphoryla-
tion has shown efcacy in animal models, but additional clinical/translational stud-
ies in humans are pending [28].
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3.3.5 Platelet-Derived Growth Factor Receptor
PDGF family members are often expressed at high levels in malignant tumors. They
promote tumor growth by either directly stimulating certain cell lines, stimulating
angiogenesis, recruiting pericytes, or controlling stromal interstitial uid pressure
inuencing trans-vascular transport and lymph node metastases. Kodama etal. [29]
showed that high expression of tumor cell-secreted PDGF-B and stromal cell-
secreted PDGF-B receptor is associated with lymphatic metastases in gastric carci-
noma [29]. Imatinib mesylate, an inhibitor of PDGFR tyrosine kinase, has not
shown efcacy by itself in gastric cancer cells but may serve as an important chemo-
sensitizer with antitumor drugs targeting PDGF/PDGFR pathway in tumorigenesis
and angiogenesis [30].
3.3.6 Combined VEGFR and PDGFR Tyrosine Kinase Inhibitors
Sorafenib is a multi-target inhibitor of BRAF, VEGF, PDGFR, and the Ras/Raf/
MERK/ERK pathways. A phase II trial determined safety of sorafenib with oxali-
platin in advanced gastric cancer but did not support phase III study. Sunitinib sup-
presses PDGFR, Kit, rearranged during transfection (RET), Flt-3, and
VEGFR.Sunitinib has not shown efcacy as a second-line agent in a phase II trial
for gastric cancer [23].
3.3.7 Hepatocyte Growth Factor Receptor Tyrosine Kinase
HGF receptor tyrosine kinase, also known as c-Met receptor tyrosine kinase, is a
receptor for HGF/c-Met oncogene, which has an extracellular alpha subunit and a
transmembrane beta subunit. The binding of Met oncogene to extracellular domain
results in phosphorylation of intracellular tyrosine kinase domains. The phosphory-
lated MET (p-MET) recruits various downstream proteins and activates signaling
pathways such as PI3K/AKT and extracellular signal-regulated kinase (ERK)/
mitogen- activated protein kinase (MAPK) pathways and plays signicant role(s) in
the tumor growth, survival, angiogenesis, and metastasis [31]. Amplication of
c-Met and co-amplication of c-Met and c-Myc have been implicated in gastric
cancers. Co-expression of c-Met and Erb-B2 has been associated with poorer sur-
vival in gastric cancer as compared to the overexpression of either one [32].
Thus, various RTKs are implicated in the pathogenesis of gastric cancer and are
potential targets for ongoing therapy against gastric cancer in addition to the cur-
rently available chemotherapy agents. Figure3.4 summaries the targets for various
RTK therapies in gastric cancer.
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3.4 Targeted Therapies and Ongoing Clinical Trials
Gastric cancer is a not uncommon cancer; however, there are limited treatment
options, and therefore the prognosis is often poor. Patients diagnosed with stages III
and IV gastric cancer have 5-year overall survival rates of only 9.2–19.8% and
4.0%, respectively [33]. GC is one of the most common causes of cancer-related
mortality, and its deadly nature can be accounted for various reasons. First, like
many other neoplasms, GC typically presents with advanced-stage disease, and in
most counties, there is no population-based screening. Second, there are no gold-
standard treatment regimens available; therefore, the treatment varies from center to
center. Third, GCs are notorious for a high degree of heterogeneity at the histologic
and molecular level, which plays a vital role in terms of tumor behavior and response
to therapy. Finally, the lack of unanimous classication schemes can lead to signi-
cant inter-observer variability between the pathologists, thereby leading to difcul-
ties in reproducibility. Several classication systems have been proposed for GC,
and the most widely used GC classication system is the Lauren classication
which classies GCs into “intestinal,” “diffuse,” and “mixed” subtypes. The other
GC classication schemes include the WHO classication, which subdivides DC
into papillary, tubular, mucinous, and poorly cohesive subtypes [34].
Cetuximab
Trastuzumab Bevacizumab Figitumumab
Receptor tyrosine kinase inhibitors
Gefitinib
Erlotinib
HER-1
Lapatinib
Ras
MEk
ERK
Cell proliferation and survival
mTOR
Akt
P13K
PTEN
Raf
Sorafenib Sunitinib GSK089
HER-2 VEGFR PDGFR Met IGF-1R FGFR
Panitumumab
Fig. 3.4 Schematic representation of the targets for various receptor tyrosine kinase inhibitors.
Abbreviations: VEGFR vascular endothelial growth factor receptor, PDGFR platelet-derived
growth factor receptor, IGF-1R insulin-like growth factor 1 receptor, FGFR broblast growth fac-
tor receptor, HER human epidermal growth factor receptor, PTEN phosphatase and tensin homo-
log, mTOR mammalian target of rapamycin, PI3K phosphoinositide 3-kinase, and Akt a serine/
threonine-specic protein kinase B, also known as Akt
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In recent times, increased availability of molecular sequencing has paved the
way for newer classication based on the molecular characteristics of the tumors
and with the identications of the molecular targets, creation of new therapeutic
targets will potentially provide personalized (precision) prognosis and treatment.
The TCGA study performed sequencing of 295 gastric cancer samples and clus-
tered GC into 4 groups: EBV positive (9%), tumors with MSI (22%), genomically
stable tumors (20%), and tumors with chromosomal instability (50%) [16].
As noted earlier, the RTKs play pivotal roles for the targeted therapies in gastric
cancer. Deng etal. [24], with the use of high-resolution single-nucleotide polymor-
phism (SNP) arrays, proled the copy number alterations in a panel of 233 gastric
cancers and found enormic alterations in RTKSs in at least 37% of cases that are
potentially treatable by the RTK/RAS directed therapies. Those genomic alterations
included FGFR2 (9% of tumors), KRAS (9%), EGFR (8%), ERBB2 (7%), and
MET (4%) [24].
3.4.1 Targeted Therapies for HER-2 Overexpressed Gastric
Cancers
HER-2 is a transmembrane tyrosine kinase and a member of the EGFR family,
which promotes cell proliferation, adhesion, migration, and differentiation. It begins
with heterodimerization with other members of the HER family causing activation
of the RAS-MAPK and PI3K/AKT pathways. The HER-2 gene is located on chro-
mosome 17q21 [35]. HER-2 overexpression is seen in 15–30% of gastric cancers,
and its prevalence is based on the histology and location of the tumor. It is more
common in the intestinal-type and in gastroesophageal junction tumors [27].
HER-2-positive gastric cancers have been targeted successfully by the anti-
HER- 2 monoclonal antibody trastuzumab. It causes inhibition of the MAPK and
PI3K/Akt pathways causing suppression of cell growth and proliferation. Other
anti-HER-2 agents that have been studied in the treatment of HER-2-positive
advanced gastric cancer include lapatinib, a dual anti-EGFR and anti-HER-2 tyro-
sine kinase inhibitor, and pertuzumab, a monoclonal antibody that binds the extra-
cellular dimerization domain of HER-2 preventing its dimerization. Currently, a
double-blind, placebo-controlled, randomized, multicenter, international, parallel-
arm study is underway to evaluate the efcacy of pertuzumab combined with trastu-
zumab and chemotherapy (cisplatin plus a uoropyrimidine) (NCT01774786).
3.4.2 Ongoing Trials for HER-2 Targeted Therapies
As noted earlier, the phase III ToGA studied addition of trastuzumab in the rst line
of chemotherapy treatment in patients with HER-2-positive advanced gastric can-
cer. Patients were randomized to uorouracil-based chemotherapy and cisplatin
with or without trastuzumab. Patients in the trastuzumab arm had an overall survival
benet [median overall survival (OS) 13.8 vs. 11.1months (HR 0.74, CI 0.60–0.91;
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46
p=0.0046)] without any increase in grade 3 or 4 adverse events [27]. The objective
response rate was also higher with trastuzumab, at 47% vs. 35% in the standard arm.
In the ToGA population, there was a proportion of patients (22%) who were uores-
cence in situ hybridization (FISH) positive but immunohistochemistry (IHC) 0-1-;
this subset of patients did not benet from the addition of trastuzumab [27].
In the TRIO-013/LOGIC trial, patients with advanced or metastatic HER-2-
positive gastric cancer were randomized to capecitabine and oxaliplatin with lapa-
tinib or placebo [36]. The primary endpoint of the overall survival benet was not
reached. Median overall survival was 12.2 months in the lapatinib group and
10.5 months in the placebo group (HR 0.91, 95% CI 0.73–1.12, p = 0.35).
Progression-free survival (PFS) was also not signicantly improved [median PFS
6.0 vs. 5.4months (HR 0.86, 95% CI 0.71–1.04, p=0.10) [36]. TyTAN trial com-
pared paclitaxel alone with lapatinib plus paclitaxel and again did not show any
signicant benets in such outcomes such as the median overall survival, PFS, or
time to progression (TTP) [37].
3.4.3 Targeted Therapies for the VEGF-Positive Gastric Cancers
VEGF stimulates the formation of blood vessels and promotes carcinogenesis by
angiogenesis and neovascularization. Therefore, the VEFGR signaling pathway is
deemed a strategic therapeutic target. The VEGF overexpression is a common fea-
ture in gastric cancers, seen in up to 58% of the cases with this disease [38].
Ramucirumab is a human IgG1 monoclonal antibody against VEGFR2 and has
been recognized as a second-line treatment of metastatic gastric cancer. Another
VEGF-directed monoclonal antibody for GC is bevacizumab (Avastin®), but less
promising results have been observed. In the AVAGAST study, cisplatin and
capecitabine were given with bevacizumab or placebo, and there was no overall
survival benet from the addition of bevacizumab [median OS 12.1 months vs.
10.1months (HR 0.87, 95% CI 0.73–1.03, p=0.1002)], although PFS and objective
response rates (ORR) were clinically improved [39].
3.4.4 Trials for VEGF Targeted Therapies
The REGARD study was a phase III trial, which compared ramucirumab with the
best supportive care in the second-line advanced gastric cancer. The results revealed
an overall survival benet of 1.4months (5.2 vs. 3.8months, HR 0.776, 95% CI
0.603–0.998, p=0.047) [40].
The phase III RAINBOW study compared the use of ramucirumab vs. placebo,
in combination with paclitaxel, in patients with advanced gastric cancer that had
progressed on the rst-line chemotherapy of uoropyrimidine/platinum with or
without an anthracycline [41]. The results revealed signicant overall survival ben-
et with ramucirumab of 9.6 months vs. 7.4 months (HR 0.807, 95% CI
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47
0.678–0.962, p = 0.017), with an increase of 1-year overall survival from 30% to
40%. The PFS, ORR, and the disease control rates (DCR) were also improved [41].
3.4.5 Anti-EGFR-Targeted Therapies and Trials
Epidermal growth factor receptor (EGFR) is a protein that spans across the cell
membrane and is activated when epidermal growth factor binds on it. After the
EGFR is activated, it then phosphorylates and triggers the intracellular protein-
tyrosine kinase activity, which in turn, causes activation and signaling of further
intracellular proteins. Subsequently, several signal transduction cascades, predomi-
nantly the MAPK, AKT, and JNK pathways, are initiated, resulting mainly in DNA
synthesis and cell multiplication but also partake in the cell migration, adhesion,
and proliferation [42].
Waddell etal. [44] in the REAL3 study, a randomized open-label phase III trial,
evaluated the addition of panitumumab (an anti-EGFR antibody) to the regimen of
epirubicin, oxaliplatin, and capecitabine (EOC). The REAL3 study found that the
addition of panitumumab to EOC does not increase survival [43]. Currently, a phase
III study (NCT01813253) is evaluating the addition of monoclonal antibody nimo-
tuzumab in combination with irinotecan and then comparing the effects of irinote-
can alone in patients with advanced gastric and gastroesophageal cancers [44].
3.4.6 Targeted Therapies for Gastric Cancers with FGFR2
Amplification
FGFR2 (broblast growth factor receptor type 2) with its ligand broblast growth
factor, promotes mitogenesis, cell proliferation, and angiogenesis. Various studies
have shown FGFR2 amplication in 3–16% of gastric neoplasms. FGFR2 is found
to be associated with the diffuse-type gastric cancers and advanced stages and car-
ries poor prognosis [45].
Drugs that target the FGF receptors have shown promising results in some pre-
clinical studies. For instance, dovitinib (TKI258), which is a multi-targeted RTK
inhibitor of FLT-/c-Kit, FGFR, VEGFR, and colony-stimulating factor, is being
actively studied. Niantao Deng etal. [24] in their study found that dovitinib has
potent inhibitory activity against gastric cancers that overexpress FGFR2. They also
observed that dovitinib reduced the mean tumor size in an FGFR2-amplied human
by inducing apoptosis by inhibiting several intracellular proto-oncogenes [24].
3.4.7 Clinical Trials for FGFR2-Targeted Therapies
Dovitinib alone or in combination is being tested currently by few clinical trials of
gastric neoplasms overexpressing FGFR2. In one phase II study (NCT01719549),
the efcacy and safety of dovitinib monotherapy is being evaluated when the
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48
rst- line chemotherapy failed to control the growth of gastric cancers with FGFR2
amplication. Another phase I/II study is investigating the combined effects of
docetaxel with dovitinib in patients with gastric neoplasms (NCT01921673).
The SHINE study assessed the efcacy and safety of AZD4547, a selective
FGFR1-3 inhibitor, and compared the results with paclitaxel treatment in patients
with advanced gastric cancers [47]. A total of 960 patients were enrolled in the
SHINE study, among which the prevalence of the FGFR2 amplication was 9%.
The results of this study showed 1.8months of overall median PFS on the AZD4547
arm and 3.5months for paclitaxel. On the other hand, the PFS was 1.5months in the
9% of patients with FGFR amplication on AZD4547 arm and 2.3months for pacli-
taxel (NCT01457846) [46].
3.4.8 Targeted Therapies for the PI3K Pathway and Clinical
Trials
The PI3K/Akt/mTOR pathways are crucial for the cell growth and survival pertain-
ing to both the physiological as well as in pathological conditions. During the time
of cellular stress, for instance, the tumors have stressful intrinsic environment owing
to the limited supply of nutrients and oxygen. The PI3K/Akt pathway plays key
regulatory role(s) in the survival of cancer cells during the cellular stress [47]. In the
TCGA study, the rate of the PIK3CA mutations has been reported from 0.8% to
20% overall.
Although currently various trials are studying the PI3K inhibitors in gastric can-
cer, but denitive clinical results are still lacking. LY294002 is a commonly used
inhibitor of PI3K/Akt pathway when used with vincristine showed synergistic inhi-
bition of the tumor cell growth by inducing apoptosis. BEZ235 and BKM120in
another study by Mueller etal. have also shown potential pro-apoptotic effects in
the cells with PI3KCA mutation [48, 49]. Other currently ongoing clinical trials of
the PI3K pathway inhibition include PI3K inhibitor BYL719in combination with
HSP90 inhibitor AUY 922in advanced or metastatic gastric cancer, (NCT01613950)
and LDE225in combination with BKM120 in cases with advanced solid tumors
(NCT01576666).
Everolimus, an mTOR inhibitor, in one multicenter phase II trial has shown
promising results such as the disease control rate (DCR) of 56% and median PFS of
2.7months [50]. The GRANITE-1, a randomized, double-blind, phase III study,
subsequently investigated the effects of everolimus vs. placebo in patients with
advanced gastric neoplasms. A total of 646 patients were enrolled in this study, and
the median OS with everolimus was 5.4months vs. 4.3months with placebo. With
everolimus, the median PFS was 1.7months and 1.4months with placebo. Looking
at the above results, the GRANITE-1 study showed that everolimus do not signi-
cantly improve overall survival in cases of advanced gastric cancers [51].
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3.4.9 Targeted Therapies and Clinical Trials for MET-Amplified
Gastric Tumors
The c-MET is a RTK and HGF is its ligand, which after binding with its ligand
activates a wide range of intracellular signaling pathways, including those involved
in the cell proliferation, motility, migration, and invasion. The overexpression of
MET is found in 0–23% of gastric neoplasms; therefore, it can be used as a target
by the MET inhibitors [52]. AMG 337, rilotumumab, and onartuzumab are a few
prominent MET inhibitors that are currently being tested in various clinical trials (as
summarized below).
NCT02016534 is a multicenter phase II trial evaluating the role of AMG 337, a
c-MET inhibitor, in patients with gastric and gastroesophageal cancers with MET
overexpression. Rilotumumab, a human monoclonal antibody to HGF (hepatocyte
growth factor), was tested in the RILOMET-1 study, but the study was halted pre-
maturely as it did not meet its primary endpoints, and risk of death was higher with
the test drug [53].
Onartuzumab is an antibody against the MET-amplied gastric neoplasms and
was assessed in a phase III study (METGastric trial) in combination with
mFOLFOX6, but this study was also terminated after the study revealed that the test
drug is associated with serious toxicities such as neutropenia [54].
3.4.10 Immunotherapy for Gastric Cancer
Due to the limited availability and success of the targeted chemotherapy for gastric
cancer, immunotherapy is being developed as one of the newer approaches for can-
cer treatment. Tumor inltrated cells usually escape from the immune detections
and destruction by involving the pathway of PD-1/PD-L1 (programmed cell death
protein-1/programmed death-ligand-1). Some tumors cells also interact with cyto-
toxic T-lymphocyte-associated antigen 4 (CTLA4) and its ligands. The binding of
the PD-L1 to its receptor transmits an inhibitory signal, which in turn, suppresses
T-lymphocyte proliferation causing apoptosis of the tumor-specic T-cells. As per
the TCGA study, PD-L1 and PD-L2 are specially overamplied in the EBV-positive
gastric cancers; hence, anti-PD-1 immunotherapy would prove to be crucial in treat-
ing the EBV-positive gastric cancers [24].
During the past several decades, the use of monoclonal antibodies (mAbs) to
target cancer cells has been well tested. The mechanisms by which mAb works
include blocking the growth factor/receptor interactions, downregulating proteins
required for tumor growth, and activating effector mechanisms of the immune sys-
tem including complement-dependent cytotoxicity (CDC) and antibody-dependent
cell-mediated cytotoxicity [55].
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3.4.11 Clinical Trials on Immunotherapy for Gastric Cancer
Pembrolizumab (Keytruda®) was evaluated in the KEYNOTE-012 phase 1b trial.
The results of the KEYNOTE-012 study have shown the overall response rate
(ORR) of 22% by the central review and 33% by the investigator review. Noticeable
antitumor activity and manageable toxicities also made pembrolizumab a promising
immunotherapeutic agent (NCT01848834) [56].
Nivolumab (Opdivo®), a human anti-PD-1 monoclonal antibody, is another
exciting immunotherapeutic agent that works as a checkpoint inhibitor, blocking a
negative regulatory signal of the T-cell activation, thus allowing the immune system
to identify and destroy the tumor cells (NCT02267343). Another trial is currently
testing the effects of nivolumab alone and in combination with anti-CTLA-4 anti-
body ipilimumab (Yervoy®) for the patients with advanced solid tumors
(NCT01928394). There is one other ongoing multicenter study by Jose Lutzky etal.
evaluating a human IgG1 monoclonal antibody against PD-L1 durvalumab
(MEDI4736) in patients with advanced solid tumors. Preliminary results have
shown acceptable safety prole and measurable tumor shrinkage [57, 58].
3.5 Toxicities Associated with Various Commonly Used
Agents/Regimens in Gastric Cancer
Below we provide a summary of the toxicities associated with some of the key
agents/regimens that have been investigated in patients with gastric cancers in
regard to tyrosine kinases.
3.5.1 Trastuzumab
As noted earlier, trastuzumab is a monoclonal antibody used for the treatment of
HER-2 receptor-positive breast cancer and gastric cancer. Common toxicities of
trastuzumab mainly include the involvement of cardiorespiratory systems.
Respiratory toxicities include dyspnea, bronchospasm, and reduced oxygen satura-
tion, whereas a decrease in the left ventricular ejection fraction or congestive heart
failure is an important cardiac toxicity associated with trastuzumab treatment. A
meta-analysis of the randomized clinical trials in patients treated with anthracycline-
based chemotherapy and trastuzumab in the adjuvant setting showed higher risks of
congestive heart failure (CHF) and asymptomatic cardiotoxicity [59]. Limited data
are available on the effect of trastuzumab during pregnancy and lactation. Few cases
of trastuzumab treatment during pregnancy and development of oligohydramnios
and reversible fatal renal failure have also been reported [60].
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3.5.2 Lapatinib
Lapatinib is a dual TKI targeting the EGFR and HER-2. Gastrointestinal disorders
such as nausea, vomiting, and diarrhea and skin conditions such as rash, hand-foot
syndrome, and dry skin are the most frequently reported adverse events for lapa-
tinib. The adverse events largely are not lethal; however, failure to recognize and
prompt the treatment can potentially lead to decrease treatment adherence, treat-
ment discontinuation, and thus poor quality of life [61].
3.5.3 Ramucirumab
Ramucirumab is an FDA-approved monoclonal antibody that binds to the VEGFR-2
and thus blocks its anti-angiogenic activity in such cancers as gastric or GE junction
adenocarcinoma. Ramucirumab in combination regimens is associated with higher
toxicity. For example, in the RAINBOW trial, ramucirumab plus paclitaxel was
reported to be associated with an increased chances of developing neutropenia than
paclitaxel with placebo [62]. Specic toxicities related to ramucirumab include
hypertension, hemorrhage, and thromboembolic disease. In the REVEL trial, hyper-
tension was found in about 6% of the cases treated with ramucirumab [63].
3.5.4 Bevacizumab
Bevacizumab, like ramucirumab, is a monoclonal antibody targeting the
VEGF.Preliminary results of some phase III clinical trials have noted somewhat
greater degrees of bleeding, arterial thromboembolic events, gastrointestinal perfo-
ration, altered wound healing, proteinuria, and hypertension with bevacizumab as
compared with placebo [64].
3.5.5 Cetuximab
Cetuximab is a monoclonal antibody against the EGFR.The more common adverse
reactions associated with cetuximab are skin disorders such as dermatitis acne-
iform/rash, dry skin, and paronychia. Other drug-related toxicities include electro-
lyte abnormalities (e.g., hypomagnesemia, hypocalcemia, and hyperkalemia),
cardiotoxicity (e.g., myocardial infarction, cardiac failure, right cardiac failure, and
coronary spastic angina), diarrhea, and leukopenia [65].
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3.5.6 Dovitinib
Dovitinib is a multi-targeted RTK inhibitor that includes potent inhibitory effects to
the FGF receptors as well. The most common adverse reactions of dovitinib are
gastrointestinal such as nausea, diarrhea, vomiting, decreased appetite, and fatigue.
Skin and subcutaneous tissue toxicities were also commonly described in the
patients taking dovitinib, and other less common events such as hypertension,
hypertriglyceridemia, non-cardiac chest pain, pulmonary embolism, hemiparesis,
neutropenia, and cerebrovascular accident are also reported [66].
3.5.7 Rilotumumab
Rilotumumab is a human monoclonal antibody against the human HGF, and it func-
tions by blocking the signaling via the MET receptors. Early phase II clinical stud-
ies reported fatigue, nausea, peripheral edema, and constipation as some of the main
treatment-related side effects. Other rare serious adverse reactions reported include
edema, deep vein thrombosis, pulmonary embolism, and diarrhea [67].
3.5.8 Pembrolizumab
Pembrolizumab is a humanized antibody used in cancer immunotherapy, which tar-
gets the PD-1 receptor of lymphocytes and thereby allows the immune system to
destroy cancer inltrated cells. The most common adverse events were fatigue, pru-
ritus, and decreased appetite. The inammatory or immune-mediated toxicities
noted were infusion-related reactions, hypothyroidism, and pneumonitis [68].
3.6 Conclusions and Future Perspectives
Gastric cancer ranks fth worldwide as the most frequent malignancy, and the
majority of patients are diagnosed at the advanced stages, which along with the rela-
tively limited treatment options makes this a poor prognostic cancer. Recent
advances in the gastric cancer genomics and sequencing have discovered many
potentially targetable mutations, which are being used for the creation of promising
therapies– and with the development of these therapies, our hopes in improving the
patients’ survival have also increased.
Amid advancement in the eld of molecular therapies, immunotherapies have
also gained wider recognition in the treatment of gastric cancer due to the presence
of high levels of somatic mutations in gastric cancer. Though molecular therapies
and immunotherapies are being developed, we still should succeed various chal-
lenges before any gold-standard treatment become available for the treatment of
advanced gastric cancer. Some of those challenges include genomically complex
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53
nature of gastric tumors, tumor heterogeneity, and the development of molecular
classication system for gastric cancer.
Molecular therapeutic agents targeting RTKs are constantly being developed and
have been effective in various clinical trials in patients with advanced gastric carci-
noma. For example, trastuzumab, a TKI targeting EebB2, for the treatment of
human ERBB2-positive advanced gastric cancer patients has already been approved.
Activation of other RTKs has been associated with gastric carcinoma, and these
include EGFR, VEGF, PDGFR, c-Met, IGF-1R, and FGFR2.
Various trials of TKIs are currently underway, which can have a greater impact
(positive outcomes) on the treatment of gastric carcinoma soon. A trial examining
bevacizumab, a TKI inhibiting VEGF, showed longer PFS in gastric carcinoma
patients, although it did not seem to meet its primary goal of increasing the OS dura-
tion. Other clinical studies, especially phase III trials that have tested the drugs tar-
geting RTKs such as EGFR, combined targeting of HER-2 and EGFR, VEGFR, and
combined targeting of VEGFR and PDGFR, have shown modest effects against
gastric cancer.
Conict of Interest None of the authors has any potential nancial or commercial conict of
interest associated with this research manuscript.
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