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Figure 1 Overview of the therapy relevant growth factor receptors
(green box) and molecular classification criteria for intestinal GEC
(iGEC) and diffuse GEC (dGEC) (red box) in the gastroesophageal
cancer (GEC).
apoptosis resistance. In 40–80% of esophageal cancers
an EGFR overexpression was observed (Tew et al. 2005).
The EGFR pathway can be interrupted by either EGFR
antibodies (e.g., Cetuximab) or TKIs, which led in several
malignancies to good therapeutic results. In con-
to that in GEC, targeting the EGFR was rather
trast
disappointing with low improvement in overall survival
(OS) or progression free survival (PFS) for studies
including EGFR antibody or TKIs (Ilson et al. 2014).
Therefore, up to now the EGFR targeted therapy is of no
use for GE cancer treatment.
c) VEGF:
The Vascular Endothelial Growth Factor can
promote the growth of blood vessels via influencing
gene expression and therefore regulates angiogenesis
and vascular permeability in the cancer (Shibuya 2011).
It preferentially utilizes the PLCγ-PKC-MAPK pathway
for signaling and is therefore an interesting target for an
anti-angiogenic therapy. Several studies could show in
aggressive GECs a VEGF overexpression. Therefore, the
high VEGF-expression level is linked with a poor prog-
in GECs (Wei et al. 2017). Moreover, it is proposed
nosis
to use VEGF-A as a biomarker in this content.
Additionally,
several inhibitors for the VEGF were
analyzed in different studies, which included antibodies
for the VEGF receptor as competitors for the VEGF molecule (e.g., Ramucirumab), as well as TKIs (e.g., Bevacizuma) (Shen et al. 2015). Whereas the TKIs showed
ambiguous results which seem to offer little benefit all in
all, the antibody approach was more successful. Ramucirumab is a monoclonal antibody, which was tested in
several studies with esophageal and gastric cancer. As a
second line therapy after progression on chemotherapy
the antibody was approved to be used as a single agent
(Yoon et al. 2016; Wilke et al. 2014).
d) MET: The MET or hepatocyte growth factor receptor
(HGFR) is a transmembrane protein with an intracellular tyrosine kinase domain. The receptor part is
activated by the Hepatocyte growth factor (HGF) und
causes via downstream signaling the activation of several
oncogenes that are responsible for angiogenesis,
proliferation, and invasion (Cecchi et al. 2012) Among
the overexpressed proteins HER-2, EGFR and MET, the
latter showed the highest correlation with poor prognosis
(Lennerz et al. 2011). Various MET receptor inhib-
were analyzed in studies and with receptor binding
itors
molecules, but led to little success.
e) Immunotherapy:
The gene expression signature in
immune cells and the tumor microenvironment (TME)
showed a local immune resistance in the area of GECs.
The immune resistance is a complex escape mechanism
in the cancer cells which leads to a lack of cancer recognition
by the immune system. An important mecha-
as a part of the immune resistance, is the
nism,
angiogenesis and immunosuppressive responses within
the tumor tissue. This immunosuppressive microenvironment
is created by several cellular mechanisms and
soluble factors, such as growth factor receptors (e.g.,
vascular endothelial growth factor A VEGFA). Therefore,
the immunotherapies would benefit from combination
therapies with several immune checkpoint agents to
reverse the immunosuppressive microenvironment
status (Refolo et al. 2020). Immune checkpoint inhibitors as Ipilimumab (Bristol-Meyer Squibb), Nivolimab
(Bristol-Meyer Squibb), and Pembrolzumab (Merck),
which bind to the anti-programmed death 1 (PD-1)
receptor (binding also its ligand PD-L1) have been
studied independently and in combination. These
studies revealed that the combination of increased
PD-L1 expression and microsatellite instability (caused
by mismatch repair deficiency) correlate with an
increased response to the Nivolimab and Ipilimumab
combination therapy (Le et al. 2015; Muro et al. 2015).
2. The Molecular Classification of the GEC
Infection-based sporadic GECs with a previous atopic gastritis
are caused by Helicobacter pylori, or induced by a reactivation of
the latent persisting Epstein-Barr virus (EBV). In fact,
2 GENOMICS, MOLECULAR PATHOLOGY, AND PATHOLOGY OF ESOPHAGEAL AND GASTRIC CANCER 25
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Helicobacter pylori (HP) provides here the greatest risk factor for
GEC and therefore has been extensively studied. The HP infection causes global methylations of CpG islands in the promotorrelated regions of the cancer tissue and thus induces cancer. For
instance, the methylation of the MLH-1 promotor leads to a loss
of gene expression of the MLH-1 genes. The MLH-1 genes code
for the mismatch repair protein MLH-1 and dimerizes with
PMS2 to a heterodimer. A loss of this heterodimer and therefore
a mismatch repair defect in the process of the DNA replication
during cell division is one possible reason for microsatellite
instability (MSI). MSI in GEC and other cancer types, like the
colon carcinoma and a minority of esophageal cancers is associated with the Lynch syndrome. This is an inherited condition
that increases the risk of carcinoma in the gastric and colorectal
areas and therefore can only be diagnosed by a human geneticist.
Thus it is extraordinary important to make Lynch syndrome
diagnosis as early as possible.
Several molecular classifications have been proposed in
recent years. One gene expression study of 29 genes involved in
the immune response mechanism, proposed a GEC classification
in the subtypes PD-L1 positive, EBV positive and microsatellite
instable (MSI) as well as the amount of tumor infiltrating lymphocytes as a prognostic factor (Park et al. 2017). In contrary
the classification of the Asian Cancer Research Group (ACRG)
includes four subtypes, based on the gene expression profiles of
MSI, MSS/EMT (microsatellite stable/epithelial-to-mesenchymal transition), MSS/TP53+ (TP53 active) and MSS/TP53(TP53 inactive) (see Figure 2) (Rodriquenz et al. 2020).
However, The Cancer Genome Atlas (TCGA) contains a
more comprehensive overview for the genetic pathways of
GEC. Here in Figure 3 the following categorization in four subtypes is presented: Epstein-Barr virus positive (EBV), microsatellite instable (MSI), genomically stable (GS) and chromosomal
instability (CIN) (Cancer Genome Atlas Research Network
2014).
A common ground for both ACRG and TCGA is the sepa-
rate subtype for MSI GEC. In GEC the microsatellite instable
cancer is associated with a better prognosis and with an
increased benefit to immune-based therapies. Except from
immune checkpoint inhibitors also other therapeutic
approaches such as adoptive T cell transfer, oncocytic viruses,
and peptide vaccines are under investigation for MSI GECs
(Ratti et al. 2018). Among the other subgroups it is difficult to
find a lot of correspondence in the Figures 2 and 3. While the
ACRG subtyping pays more attention to the mutations in the
TP53 gene and the epithelial-to-mesenchymal transition, the
TCGA subtyping is based on more molecular features as chromosomal stability and molecular alterations. These differences
may be probably due to the geographic differences in the two
population groups (Eastern versus Western patients) and different analysis methods (Rodriquenz et al. 2020).
According to the TCGA classification the two molecular
subtypes here, the MSI GEC with promotor methylation and
the EBV positive GEC had a higher rate of 10–15% and 30–50%
respectively of PD-L1 overexpression. This makes these two
subtypes EBV positive and MSI GECs good candidates for
immunotherapy (Carneiro et al. 2019). The prognosis of these
two subtypes MSI and EBV positive is also better, than of their
counterparts (MSS and EBV negative). A histologic feature of
the EBV positive GEC is the presence of lymphoid stoma,
whereas the genomically stable (GS) subtype corresponds
(according to the Laurén histological classification) to the diffuse GEC type and the chromosomal instability (CIN) subtype
to the intestinal GEC type. Thus histological characteristics,
EBV detection, and MLH-1 promotor methylation analysis are
biomarkers of good prognosis (Carneiro et al. 2019).
Figure 2 Subtype classification according to the Asian Cancer
Research Group (ACRG) with percentage for the four subtypes:
MSI (microsatellite instable), MSS/EMT (microsatellite stable/
epithelial-to-mesenchymal transition), MSS/TP53+ (TP53 active)
and MSS/TP53- (TP53 inactive) and molecular features in boxes
(Adapted from (Rodriquenz et al. 2020)).
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- Hypermethylation
- Mutations in PIK3CA,
TP53, RTK, ARID1A
- JAK2 amplication
- Chromosomal
instability (CIN)
- Gene amplications
(coding for tyrosine
kinase receptors)
- PDL1/PLD2
overexpression
CIN
49%
EBV
9%
MSI
22%
20%
GS
- High mutationrate
- CDKN2A and MLH-1
promotor methylation
- Many genes (e.g. HLA class
1 factor) are hypermutate d
- Molecular alterations in
cell adhesion and cell
migration
- ARID1 and BCOR
mutations
Figure 3 Subtype classification according to The Cancer
Genome Atlas (TCGA) with percentage for the four
subtypes: Epstein-Barr virus positive (EBV), microsatellite
instable (MSI), genomically stable (GS) and chromosomal
instability (CIN) and molecular features in corresponding
boxes (Adapted from Rodriquenz et al. 2020).
3. Mutation Analyses for Differentiation between Diffuse
Gastroesophageal Cancer (DGEC) and Intestinal Gastroesophageal Cancer (IGEC)
a) NGS: Next Generation Sequencing (NGS) has become
the key technology for mutation analysis especially with
samples of low tumor content. Since this technique is
more widely available nowadays, it is also an important
goal to determine its feasibility and reliability in
comparison with IHC/FISH analyses of GECs. A study
in 2015 demonstrated 84% concordance rate (Mikhail et
al. 2015). Though several other attempts were under-
to test the concordance, the results are still uncer-
taken
tain.
Moreover, the identification on molecular mutation
patterns is an ongoing process of improvement.
genome sequencing (WES) made some impor-
Whole
tant
investigations for classification of diffuse and
intestinal GECs. The analysis showed several somatic
alterations in the TP53, β-catenin (CTNNB1), CDH1
and the RHOA genes for the dGEC (Wang et al. 2014).
Furthermore, the CDH1 mutation was mainly present
in the dGEC and a gain of function in the RHOA protein was more often detected in the dGEC than in iGEC
(Kakiuchi et al. 2014).
Activation of the phosphatidylinositol 3-kinase/AKT
target (PIK3K-AKT) of the rapamycin pathway occurs
via cell surface receptors as: Transcription activator
STAT3, HER-2 and VEGFR2. This pathway contains
several in the pathway included genes such as PIK3A,
PTEA, AKT3, AKT2, and AKT1. Mutations in these
genes are seen in a higher percentage of patients with
dGEC.
Besides the differentiation of the two main histologic
types, squamous cell carcinoma (SCC) and adenocarcinoma
is also possible via genetic profiling with NGS,
since both show distinct mutation patterns. Most driver
mutations found in the adenocarcinoma are already
present in the Barrett’s esophagus and the one in the
squamous dysplasia also in SCC. For example, more
insertions and deletions as well as inactivating NOTCH1
mutations were found in SCC (Agrawal et al. 2012; Tay-
et al. 2013).
lor
b) Germ-line Mutations:
tion
is in the cell–cell adhesion protein E-catherin
One important germ-line muta-
(CDHI). Germline or somatic CDH1 mutations are typical
in dGEC, since inactivation of CDH1 protein is asso-
with a hereditary dGEC and shorter survival rates
ciated
in general (Cho et al. 2017). Here the signal pathways of
E-cadherin, Wnt or RHOA are altered which enables cell
adhesion and migration in GEC (Barber et al. 2008;
Hansford et al. 2015).
c) Gene Expression: Additionally, to the classifications
of the TCGA, using gene expression analyzes it was
also possible to find molecular signatures that can be
used as GEC classification criteria. Different gene
expression patterns were proposed for the iGEC and
dGEC. Here the iGEC revealed a concordance of 64%
with Laurén histopathologic subtyping (Tan et al.
2011). Furthermore, a study group suggests that a
classification into the subtypes proximal nondiffuse
gastric cancer (more common Laurén’s intestinal histology), diffuse gastric cancer (located in the body or
distal stomach and Laurén’s dGEC), and distal nondif-
2 GENOMICS, MOLECULAR PATHOLOGY, AND PATHOLOGY OF ESOPHAGEAL AND GASTRIC CANCER 27
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fuse gastric cancer (iDGEC and mixed Lauren’s histology)
was possible (Shah et al. 2011). But genetic
expression profiles are not only helpful tools for
classification issues, but can also provide genes that are
of prognostic importance or are associated with the
likelihood of relapse (Cho et al. 2011). There are even
genes as PIP5KL1 whose overexpression is correlated
with suppression of GEC development (Shi et al. 2010).
But also gene expression of certain genes as microR-
can be used as a prognostic factor for the OS of
NAs
GEC patients (Ueda et al. 2010).
d) Microsatellite
ns (CNV):
with the intestinal or mixed GEC type, suggesting
ciated
more genome instability in the iGEC type (Kim et al.
2003, Kwon et al. 2018). In the dGEC type less of somatic
and germline mutations were found compared to the
iGEC type. This indicates more genetic stability in the
dGEC type (Wong et al. 2014). In dGEC also an increased
gene amplification of the MDM2 gene and a gene fusion
of the TSC2-RMF2016 genes were reported. Additionally,
a gain in copy number variation in chromosome 13q was
also a sign for the dGEC subtype (Wu et al. 2001).
e) Epigenetic Alterations:
altered gene expression via DNA methylations and his-
modifications without altering the genetic DNA
tone
sequence. Especially promotor hypermethylation causing
transcriptional silencing of specific genes as tumor
suppressor genes, which are a well-studied cause of can-
But also silenced genes controlling the cell cycle, DNA
cer.
repair mechanisms and cellular network or apoptosis are
significant for carcinogenesis. The most common mecha-
of gene silencing is the CpG island methylations,
nism
which can be used as a prognostic and predictive biomarker.
genes in the GE cancer are for instance CDK2NA,
CDK2AP2, CDH1, MGMT, RASSF1, RUNx3,
(Kim et al. 2004). Especially Helicobacter pylori and EBV
cause a set of different hypermethylations that are associated with GE cancer (To et al. 2002). Even in early stages of
gastric carcinogenesis aberrant methylation already accumulate and lead to premalignant lesions (Kang et al. 2003).
Here the genome-wide search has identified one important methylation hotspot in GEC, the TFPI2 genes were
highly methylated (about 81 (%) and this can be used as a
prognostic indicator in GEC (Jee et al. 2009)).
Frequently hypermethylated and as such silenced
Stability and Copy Number Variatio-
The loss of heterozygosity (LOH) is ften asso-
Epigenetic mechanisms include
and DLC1
Peritoneal Metastasis
Patients with peritoneal metastasis in GEC often develop as
bowel obstruction and malignant ascites and have unsatisfactory response to chemotherapy and are associated with poor
prognosis. All in all, about 55–60% of patients with GEC
develop peritoneal metastases and the no targeted therapy or
immunotherapy is available yet. Therefore, it is necessary to
identify patients at high risk of peritoneal metastasis via molecular biomarkers. To find genes that are associated with peritoneal metastases, a group of researchers performed
comprehensive whole-genome and transcriptome sequencing
analysis of peritoneal metastasis from GEC. The found mutations are summarized in the Catalogue of Somatic Mutations in
Cancer database and are also located in eight genes that were
newly described in GEC (ARMC4, CCDC178, DAB1, DMBT1,
PDZD2, PLIN4, PKLR, and TUBB6). Analysis of gene expression showed six genes (CDH16, HOXA11, LOC100505875N,
KX2-5, NOX4, and SFRP4) which were up-regulated in the
peritoneal metastasis in GEC (Zhang et al. 2015). In patients
with peritoneal metastasis transcriptional profiling of miRNA
was also performed. Here three miRNAs (miR-30a-5p, -6593p, and -3917) were significantly overexpressed in patients with
peritoneal metastasis in GEC, which correlates with the poor
prognosis (Shimura et al. 2021). Therefore, the combination of
this miRNA signature and the Bormann macroscopic type
offers a potentially accurate approach for the detection of peritoneal metastasis in GEC (Yao et al. 2020).
B) Germline Genetics of Gastric and Esophageal Adenocar-
cinoma
1. Gastric Adenocarcinoma
A familial clustering of gastric adenocarcinoma, here called
gastric cancer, is observed in 5–10% of all affected patients
(Zanghieri et al. 1990). Besides common environmental factors,
this implies that genetic factors are involved in disease
development. Accordingly, twin studies have estimated a
gastric cancer heritability of 28% (Lichtenstein et al. 2000).
With the development of modern molecular genetic methods,
the first genetic risk factors for gastric cancer could be identified respectively. This revealed that its genetic architecture is
heterogeneous and depends on the effect size and frequency of
the underlying genetic variants and mutations (Figure 4). Here,
monogenic forms must be distinguished from multifactorial
forms. Monogenic refers to mutations in a single gene, which
have strong effect sizes and are extremely rare in the general
population.
The only monogenic gastric cancer syndrome identified so
far is hereditary diffuse gastric cancer (HDGC). It was first
described in 1964 in three Maori families from New Zealand
(Jones 1964) and is caused by mutations in the gene CDH1
(E-cadherin) that are inherited in an autosomal dominant
fashion (Guilford et al. 1998). Gastric cancer also occurs in the
context of other hereditary cancer syndromes (Table 1).
However, other cancer types are predominant in these families.
These include Lynch syndrome, Li-Fraumeni syndrome,
familial adenomatous polyposis (FAP), harmatous polyposis
syndromes (Peutz-Jeghers syndrome and juvenile polyposis),
as well as hereditary breast and ovarian cancer (HBOC).
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Figure 4 The genetic architecture of diseases depends on the penetrance (Y-axis) and frequency of the risk-conferring mutation/variant in the population
(X-axis). At the top left are monogenic diseases with extremely rare and highly penetrant mutations. Bottom right are multifactorial diseases with common
risk variants that have small effects. In between are diseases with rare mutations that have moderate effects (adapted from (18398418)).
In contrast, the majority of all gastric cancers are multifactorial. Here, a large number of genetic variants is cumulatively
involved in cancer development, which have only small effect
sizes and are common in the general population (Figure 4).
With the development of modern sequencing technologies or
next generation sequencing (NGS) it has also become possible to identify gene variants that are rare in the general
population and have moderate effect sizes (Figure 4).
Additionally such risk variants for gastric cancer have recently
been identified.
In the following, the different genetic forms of gastric cancer will be presented in more detail. Of all hereditary cancer
syndromes listed in Table 1 this concerns HDGC, since
gastric cancer is the predominant cancer type only in this
syndrome.
a) Hereditary Diffuse Gastric Cancer (HDGC): Histopathologically, HDGC presents as diffuse adenocarcinoma and shows
an autosomal dominant inheritance (Gayther et al. 1998). However, because CDH1 mutations are not completely penetrant or
can result as de novo mutations, a negative family history for diffuse gastric cancer does not exclude HDGC. In these cases, the
age of onset is crucial for the diagnosis as HDGC is characterized
by an early age of onset (Gayther et al. 1998). Accordingly, the
majority of HDGC cases develop the disease before the age of
40 with an average age of onset of 38 years. However, the age of
onset varies even among patients within a family (Gayther et al.
1998; Guilford et al. 1998).
The clinical presentation of HDGC does not differ from
sporadic or multifactorial gastric cancer. In early stages symptoms are rather unspecific, but become characteristic in
advanced disease stages (Wanebo et al. 1993). As with sporadic
gastric cancer, the five-year survival rate of HDGC is 90% in
early stages, whereas it is less than 20% in advanced stages
(Oliveira et al. 2015). Thus, prophylactic gastroectomies are
recommended for CDH1 mutation carriers.
As with other hereditary cancer syndromes, the penetrance of CDH1 mutations is incomplete. Data from large
studies showed that around 67% of all male mutation carriers and around 83% of all female mutation carriers develop
HDGC by the age of 80 (Pharoah et al. 2001). Moreover,
female mutation carriers have also an increased risk for lobular breast cancer. Here, the prevalence is 39–52% and the
average age of onset is 53 years (Pharoah et al. 2001). Thus,
around 90% of all female mutation carriers will develop
either gastric or breast cancer. Furthermore, it remains
unknown whether colorectal cancer also occurs more frequently in patients with CDH1 mutations (Fitzgerald et al.
2010).
The clinical criteria for a CDH1 mutation analysis have
been established by the International Gastric Cancer Linkage
Consortium (IGCLC) (Pharoah et al. 2001) (Table 2). In
addition to a detailed medical history of the index patient
(including tumor histopathology), data from a three-generation pedigree should be considered. Depending on the
2 GENOMICS, MOLECULAR PATHOLOGY, AND PATHOLOGY OF ESOPHAGEAL AND GASTRIC CANCER 29
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Table 1 Hereditary cancer syndromes with higher risk to develop gastric cancer (adapted from (van Nistelrooij et al. 2018)).
Gastrointestinal cancer syndromes Multiorgan cancer syndromes
Familial
adenomatous
Lynch syndrome
(
HNPCC)
Gene(s)
Inheritance
Predominant
cancer type
Prevalence
Gastric
cancer risk
1 HNPCC = hereditary non-polyposis colon cancer
2 including GAPPS (gastric adenocarcinoma and proximal polyposis of the stomach)
DNA mismatch
repair genes
(MLH1, MSH2,
MSH6, PMS1,
PMS2), EPCAM
Autosomal
dominant
Colorectal cancer Colorectal cancer Colorectal and gastric
1:370 to 1:2,000 1:8,300 1.9:1,000,000 1:100,000 1:400 to 1:500 1:20,0000
0.2–13% 2.1–4.2% 29% 21% 2.6–5.5% 3.1–4.9%
polyposis (
(Jankowski
etal. 1999)
APC STK11 SMAD4,
Autosomal
dominant
FAP)
Peutz-Jeghers
syndrome
Autosomal dominant Autosomal
cancer, pancreatic
cancer, breast and
ovarian cancer
Juvenile
polyposis
BMPR1A,
ENG
dominant
Colorectal and
gastric
cancer,
pancreatic
cancer
Hereditary
Breast and
Ovarian
Cancer
(
HBOC)
BRCA1, BRCA2 TP53
Autosomal
dominant
Breast and
ovarian
cancer
Li-Fraumeni
syndrome
Autosomal
dominant
Broad cancer
spectrum (including
sarcomas, breast
cancer, brain
tumors)
inclusion criteria used for molecular genetic testing, the
mutation detection rate in CDH1 is 30–50% (Oliveira et
al. 2015). Accordingly, no CDH1 mutation is identified in
50–70% of all clinically defined HDGC cases. Here, it must
be assumed that the mutations are localized in CDH1 regions
that cannot be analyzed with the sequencing techniques
available to date. Alternatively, so far unknown disease genes
for HDGC might exist.
CDH1 on chromosome 16q22 consists of 16 exons and
covers a genomic region of > 100 kb. The gene product
Table 2
Criteria for the diagnosis of HDGC according to the IGCLC guidelines (only one criterion must be fulfilled).
• Family criteria (first or second degree relatives of each other)
– ≥ two cases of gastric cancer in family regardless of age, with at least one diffuse gastric cancer
– ≥ 1 case of diffuse gastric cancer any age and ≥ 1 case of lobular breast cancer < 70 years in different family members
– ≥ 2 cases of lobular breast cancer in family members < 50 years
• Individual criteria
– Diffuse gastric cancer < 50 years
– Diffuse gastric cancer at any age in individuals of Maori ethnicity
– Diffuse gastric cancer at any age in individuals with a personal of family history (first degree) of cleft lip/cleft palate
– History of diffuse gastric cancer and lobular breast cancer, both diagnosed < 70 years
– Bilateral lobular breast cancer, diagnosed < 70 years
– Gastric in situ signet ring cells and/or pagetoid spread (bottom to top) of signet ring cells in individuals < 50 years
represents a transmembrane protein that is ‒ among others ‒
involved in cell‒cell adhesion processes (Oliveira et al. 2015).
So far, more than 120 different CDH1 mutations have been
identified in HDGC cases, the vast majority of them result in a
truncated gene product at the protein level (Oliveira et al.
2015).
If a CDH1 mutation is detected in a patient with diffuse
gastric cancer, a total gastrectomy is recommended unless
alternative therapeutic procedures are more appropriate
because of the disease stage or other clinical reasons. In
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addition, regular gynecologic and colonoscopic screenings are
recommended for mutation carriers. Moreover, if a predictive
diagnostic lead to the detection of a familial CDH1 mutation in
healthy family members, a prophylactic gastrectomy is recommended from the age of 20 (Fitzgerald et al. 2010; Oliveira et al.
2013; Syngal et al. 2015).
b) Sporadic or Multifactorial Gastric Cancer: The majority
of all gastric cancers is multifactorial. Here, single nucleotide
polymorphisms (SNPs), which represent the most frequent
class of common variants in the genome, contribute to disease
development in a cumulative fashion (Figure 4). SNPs are
characterized by the substitution of a single nucleotide and
can be analyzed through genome-wide association studies
(GWAS) since around 15 years. In GWAS, all common SNPs
of the genome are systematically tested for disease association
in large case-control cohorts. If an allele of a SNP is more frequent in patients than in controls and this difference exceeds
a certain significance threshold, the variant is disease-associated.
In gastric cancer, seven GWAS in the Asian population and
one GWAS in the European population have been carried out
so far (Helgason et al. 2015; Hu et al. 2016; Jin et al. 2012;
Sakamoto et al. 2008; Shi et al. 2011; Syngal et al. 2015; Wang
et al. 2017; Yan et al. 2020). The size of the studied samples
ranged from 937 to 2,240 patients (Abnet et al. 2010; Sakamoto
et al. 2008). In total, 10 genomic loci could be identified that
showed genome-wide significant association to gastric cancer. At present, for most of these loci it is rather unclear what
genes and cellular mechanisms are involved in disease
pathology. However, the functional characterization of the
identified risk loci will be of major importance in future as
this represents the prerequisite for developing novel and biologically based therapy and prevention strategies. At one
locus, however, the cellular pathomechanism is already
known. It involves the gene ATM (ataxia telangiectasia
mutated) on chromosome 11q22, which encodes a serineprotein kinase involved in the repair of DNA double-strand
breaks. The ATM variants that showed disease association
(Helgason et al. 2015) are located in the coding region and
lead to a premature termination of the protein. The risk SNPs
are also relatively rare in the general population (< 1%) and
compared to most GWAS show relatively strong effect sizes
(odds ratio (OR) > 4). Therefore, many scientists consider
ATM to be rather a moderately penetrant disease gene for
gastric cancer (see following section) then a multifactorial
risk gene.
consortium study, which analyzed 33 different cancer types,
including WES data from 443 patients with gastric cancer
(Huang et al. 2018). Overall, pathogenic or likely pathogenic
mutations in various genes could be identified in 13% of all
patients. Surprisingly, the age of onset was 61 years in the group
of mutation carriers and thus not significantly lower than in the
overall cohort. Mutations were found significantly frequently
in two tumor suppressor genes, namely PALB2 (partner and
localizer of BRCA2) on chromosome 16p12 and ATM on
chromosome 11q22 (see above). In both genes mutations were
present in patients with both intestinal and diffuse gastric cancer. PALB2 mutations were also identified among patients with
other cancer types, but showed no significant enrichment in
these cases. In contrast, mutations in ATM were also observed
significantly frequently in patients with breast, lung, pancreas,
and prostate cancer.
Other NGS-based studies primarily analyzed CDH1-negative
patients with diffuse gastric cancer, some of whom met the
clinical HDGC criteria. The size of the study cohorts ranged
from 22 to 183 patients (Gaston et al. 2014; Hansford et al.
2015; Majewski et al. 2013). Although pathogenic mutations in
the gene CTNNA1 (catenin alpha 1) on chromosome 5q31
were found in some cases, no significant enrichment of mutations was observed (Reid et al. 2010). However, CTNNA1
interacts at the cellular level with CDH1, the disease gene for
HDGC. This can be considered as further evidence that
CTNNA1 is a disease gene for diffuse gastric cancer.
2. Esophageal Adenocarcinoma
Also in esophageal adenocarcinoma, here called esophageal
cancer or Barrett’s carcinoma, genetic factors are involved in
disease development. Accordingly, the heritability estimates
range from 30‒40% (Reid et al. 2010). However, as with gastric
cancer, a spectrum of genetic risk factors appears to contribute
to esophageal cancer. The majority of these cases are sporadic
and of multifactorial etiology. Here, a large number of common
genetic variants, each with small effect size, contribute cumulatively to disease risk (Figure 4). In contrast, esophageal cancer
and its precursor lesion Barrett’s esophagus do not appear sporadically in some patients. This is called familial Barrett’s
esophagus/carcinoma, which affects about 7% of cases, and is
defined by three or more affected relatives (To et al. 2016). In
these patients, it is assumed that gene variants, which are rare
in the general population and have moderate effect sizes, are
contributed to disease risk (Figure 4). However, in contrast to
gastric cancer, esophageal cancer does not develop in the context of a monogenic or hereditary tumor syndrome.
c) Moderately Penetrant Disease Genes for Gastric Cancer:
In recent years, NGS or whole-exome sequencing (WES) has
led to the identification of the first disease genes for gastric
cancer with moderate effect sizes (Figure 4). The largest of
these studies represents The Cancer Genome Atlas (TCGA)
a) Sporadic or Multifactorial Esophageal Cancer: In 2016,
a GWAS meta-analysis was published that jointly analyzed
all previous GWAS cohorts (Gharahkhani et al. 2016; Su et
al. 2012). The sample comprised of > 6,000 patients with Barrett’s esophagus, > 4,000 patients with Barrett’s carcinoma and
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> 17,000 controls, all of European descent. In total, 14 risk
loci for Barrett’s esophagus/carcinoma were identified by this
GWAS. Of note, one of these loci is located on chromosome
3q27 (SNP rs9823696) and was associated specifically with
Barrett’s carcinoma (p = 1.6× 10
-08
), but not with Barrett’s
esophagus (p = 0.45). On the single marker level, this genetic
variant represents the first variant that predicts the progression from Barrett’s esophagus to esophageal cancer. However, through increasing GWAS sample sizes further genetic
risk and progression factors will be identified in future. The
GWAS data also revealed that sex-specific genetic effects are
involved in the development of Barrett’s esophagus/carcinoma
(Dong et al. 2013). Accordingly, one SNP on chromosome
6q11 (rs112894788) showed disease-association only in
males, whereas one SNP on chromosome 8p23 (rs13259457)
showed disease-association only in females. For most of the
identified GWAS loci it is rather unclear what genes and cellular mechanisms are involved in disease pathology. However,
their functional characterization will be of major importance
in future for the development of novel and biologically based
therapy and prevention strategies.
b) Moderately Penetrant Disease Genes for Esophageal Cancer:
Familial Barrett’s esophagus/carcinoma affects about 7% of
cases and is defined by three or more affected relatives (To et
al. 2016). In addition to the familial clustering, an earlier age at
onset has been observed in these cases when compared with
sporadic forms (To et al. 2016). Examples of published reports
of familial Barrett’s esophagus/carcinoma are listed in Table 3.
It is assumed that gene variants, which are rare in the general
population and have moderate effect sizes, are contributing to
familial Barrett’s esophagus/carcinoma (Figure 1). Accordingly,
first WES studies have been carried out on single families with
several affected individuals. This led to the identification of the
gene VSIG10L (V-set and immunoglobulin domain containing
10 like) on chromosome 19q13, where the mutation p.S631G
segregated with disease status in a family (Fecteau et al. 2016).
Furthermore, the mutation p.V120G in the gene MSX1 (Msh
homeobox 1) on chromosome 4p16 segregated with disease
status in another pedigree with familial Barrett’s esophagus/
carcinoma (van Nistelrooij et al. 2018). In the pre-WES era, the
gene MSR1 (Macrophage scavenger receptor 1) on chromosome
8p22 was identified as another potential disease gene for
familial Barrett’s esophagus/carcinoma (with the mutations
p.R293X and p.L254V). In this study, a linkage analysis was
performed on 21 concordant-affected sib-pairs and 11 discordant sib-pairs. Subsequently, genes in linkage regions were
sequenced in 176 further patients and 200 controls (To et al.
2016). However, the relevance of the mentioned genes for
esophageal cancer development has so far not been confirmed
by studies on independent cases.
C)
Pathology of Esophageal and Gastric Adenocarcinoma
1. General Differences
Cancer of the distal esophagus and stomach share a lot of the
histological criteria when to diagnose neoplasia. Both entities
are adenocarcinomas whereas esophageal cancer on normal is
a squamous cell carcinoma with different criteria, etiology, and
Table 3 Selection of published case reports and case series on familial Barrett’s esophagus (BE) and esophageal adenocarcinoma (EAC) ordered according
the number of included patients. The average age at diagnosis (AAD) is listed for BE and EAC. In all studies, the observed inheritance pattern was
autosomal dominant (AD) (adapted from (To et al. 2016)).
References Families (n) BE (n) EAC (n) Mean BE AAD Mean EAC AAD Inheritance
(Drovdlic et al. 2003) 70 21 2 51.0 60.5 AD
(Sappati Biyyani et al. 2007) 20 7 7 60.3 60.8 AD
(Poynton et al. 1996) 3 6 8 62.0 60.0 AD
(Fahmy and King 1993) 4 8 2 62.0 74.0 AD
(Groves et al. 2005) 1 7 3 48.1 74.0 AD
(Munitiz et al. 2008) 1 4 6 46.2 60.5 AD
(Jochem et al. 1992) 1 6 3 43.6 74.0 AD
(Eng et al. 1993) 1 7 2 54.3 68.0 AD
(Crabb et al. 1985) 1 4 0 59.5 - AD
(Everhart et al. 1983) 1 3 0 23.6 - AD
(Melzer et al. 2006) 1 0 3 - 65.5 AD
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molecular background. Squamous cell carcinomas of the
esophagus are etiologically linked to alcohol and tobacco consumption but also chronic inflammatory conditions like Lichen
planus can be seen as a precancerous condition.
Dysplasia is thought to be the first visible step of malignant
transformation in the gastro-intestinal tract. The term dysplasia was first used by E. Krompecher, Budapest in 1924
(Krompecher 1924) describing cells that look abnormal under
a microscope but are not yet cancer. In fact, we have no better
description nowadays and all our attempts for a better risk
stratification in subgrading dysplasia in low and high grade
cases are partly very subjective since criteria are still ill-defined
and there may be a considerable overlap between the stages
(Figures 5 and 6).
Carcinomas of the distal esophagus are adenocarcinomas most
often based on metaplastic changes of the squamous epithelium
into columnar epithelium based on chronic damage due to gastro-esophageal reflux disease called Barrett’s esophagus (esophagitis-metaplasia-dysplasia-adenocarcinoma sequence) (3). Gastric
carcinomas are mainly based on chronic active inflammation due
to infection with Helicobacter pylori. Therefore, the WHO is
regarding Helicobacter as a category I carcinogen. It is also a good
example that antibiotic treatment has the potential to cure a precancerous condition. In case of low grade gastric MALT
Lymphoma which is Helicobacter-induced in most cases, antibiotic treatment is even capable to cure a malignant disease.
Concerning the aggressiveness of cancer, squamous cell carcinoma is more aggressive than Barrett’s associated adenocarcinoma and gastric adenocarcinoma. The rate of lymph node
metastasis is higher in squamous cell carcinoma compared
with adenocarcinoma. This is the reason that the depth of infiltration still acceptable for local endoscopic removal/ablation
Figure 5 Steps in the cascade of malignant transformation
with overlaps. It has to be noted that the time one stage takes
to the next stage up may vary considerably in length and in
some cases it even looks like whether stages are so short in time
that lesions advance very fast. It is not foreseeable at the
moment which neoplasm will behave more or less aggressive.
This is the reason why cases with dysplasia should not be
watched but removed today. (LGD = low grade dysplasia; HGD
= high grade dysplasia).
should not exceed 200 microns into the submucosal layer for
squamous cancer (Figure 7) and 500 microns for Barrett’s adenocarcinoma or gastric adenocarcinoma (Figure 8). In
comparison colonic carcinoma is even less aggressive then
gastric adenocarcinoma and thus the border for endoscopic
removal in the colon is 1000 microns into the submucosal layer,
in case there are no further risk factors such as lymphatic vessel
permeation, poor differentiation or high budding (single tumor
cells at the invasive front).
2. Diagnostic Dilemma
Besides squamous cell neoplasia, diagnostic criteria for neoplasia of columnar mucosa are partly ill defined, non-validated,
nor are they accepted worldwide. These criteria vary internationally and institutionally depending on the place where people have been trained. This is the reason for a lower carcinoma
threshold in Japan and a higher threshold for a carcinoma diagnosis in the United States. Europe is located somewhere in between. Pathologists’ abilities are different but not poor. In their
differences all the colleagues are very consistent and can even
predict that a certain lesion diagnosed in Japan would be called
carcinoma whereas the colleagues in the United States would
prefer the diagnosis of high-grade dysplasia/intraepithelial
neoplasia.
It has been shown during the Vienna consensus that the differences are mainly found in biopsies but not in resection
specimen (Figures 9 and 10).
A carcinoma diagnosis in Japan is mostly based on the
cytology of the epithelial cells whereas in Western countries
architecture and secondary effects like desmoplastic stromal
reaction or single tumor cells play an important diagnostic role.
It is known that desmoplastic stromal reaction can be detected
in deeply submucosal tumors but not in early mucosal carcinomas. Also, well differentiated adenocarcinomas are capable
of building their own basal membrane and thus no single
tumor cells can be expected in early carcinomas. The diagnostic dilemma is that it is not clear who is wrong or who is
right. The ultimate proof would be the presence of vessel or
perineural permeations or metastases. Unfortunately, these
sequels of a carcinoma may not be present in early lesions.
Colleagues (Sakurai et al. 2014) from Japan were able to document these issues by asking Japanese and Western pathologists to make a biopsy diagnosis and a final diagnosis on the
resection specimen of the same lesion. It turned out that
Western pathologists tended to diagnose clear cut submucosal
cancer as high grade dysplasia and signed out cases with additional vessel permeations as high grade dysplasia in biopsies as
well. On the resection specimen the Japanese and Western
pathologists all agreed upon the carcinoma diagnosis. Due to
the different medical systems and the different history of the
evolvement of these systems, there is an alternative for both
sides to overcome these issues on biopsy specimen. Takahashi
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Figure 6 Criteria for differentiating High grade intraepithelial neoplasia/dysplasia from invasive carcinoma within the gastrointestinal tract. Most
important is the epithelial expansion underneath the surface to diagnose cancer in combination with malignant cytology (Vieth and Stolte 2005/
Springer Nature).
Figure 7 Esophageal m2 squamous cell carcinoma invading the tunica
propria, reaching the muscularis mucosae but without invasion of the
muscle layer. The upper third of the submucosal layer is defined as a
depth of invasion less than 200 microns (CIS: carcinoma in situ).
and Iwama (Takahashi and Iwama 1985) evaluated the expansion patterns of neoplasia within the gastro-intestinal tract,
independently with different approaches. In the end, the earliest sign of invasion is the so-called lateral expansion of tubules
or intertubular fusions that are believed to represent nothing
else but invasion into the tunica propria. (Figures 11–12).
In an unpublished series we were able to show with a
Japanese colleague that lateral expansion plus cytology is a
very strong marker for a carcinoma diagnosis and works on
biopsies and resection specimens as well. There was 100% concordance with the Japanese cytological criteria of a carcinoma
diagnosis. At this very moment one can state that the lateral
Figure 8 Barrett’s mucosa with characteristic double muscularis
mucosa (a probably reactive sequel of chronic inflammation). There
are two proposals available. Unfortunately, studies often never state
what system is used and this harbors a lot of issues in respect of
interpreting results. The m1-m3 proposal (Westerterp et al. 2005)
needs to be seen as an attempt to harmonize squamous and
columnar epithelial neoplasia. Unfortunately, this means that HGD
(high grade dysplasia) is now called an m1 carcinoma, m2 means a
carcinoma that is confined to the upper mucosal lamina propria and
m3 invasion of the upper layer of the muscularis, the space in
between and the lower layer of muscularis mucosae. The upper third
of the submucosal layer is defined as less than 500 microns. The
competing proposal (Vieth and Stolte 2005) is based on the
anatomical structures. Here, high grade dysplasia still exists as an
entity, m1 names a carcinoma that is confined to the upper mucosal
level, m2 invasion into the upper muscularis mucosae, m3 into the
layer in between the muscle layers, and m4 invasion into the lower
muscularis mucosae. One or the other system should be used to force
the pathologists to clearly identify layers and to avoid of
overdiagnosing an m3 carcinoma as being submucosal since this
would have major therapeutic consequences compared to a lesion
that is eligible for local endoscopic ablation.
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