Introduction
Gastric cancers are the fifth most commonly diagnosed
cancer in the world, but they are the third among the
cancer-related deaths, according to the 2018 World
Health Organization data. [] In Turkey, gastric cancer
is the second cause of cancer-related deaths in men and
third in women. []
More than 95% of gastric cancers are adenocarcinomas,
whereas 5% are lymphomas, leiomyosarcomas, and less commonly carcinoid tumors, carcinosarcomas,
and squamous cell carcinomas.[] Signet ring cell
carcinoma (SRCC), a type of gastric adenocarcinoma,
is a cohesive type of cancer and is characterized by cytoplasmic
mucin secretion and crescent-shaped cells.
SRCC is frequently diagnosed in people aged 50 and
over and its incidence increases over age 70. SRCC is
very rare to be seen under the age of thirty (0,5%). []
Compared with other types of gastric cancer, the clinicopathological
features of SRCC are reported to be different. In general, even though this type of cancer is
thought to be more aggressive and has a worse prognosis
than other types of gastric cancer, it is stated that
there are uncertainties regarding the characteristics
and survival outcomes. [,]
Since the diagnostic tools used in patients with
SRCC have low sensitivity and specificity in diagnosis
and prognosis, it is important to support diagnosis,
treatment follow-up, and relapse by molecular genetic
biomarkers.
The p53 tumor suppressor gene (17p13.1) located
in the p arm of chromosome 17 encodes a 393 amino
acid long nuclear phosphoprotein that weighs 53 kDa.
[] p53, which acts as a transcription factor, initiates a
gene expression program that will prevent proliferative
propagation of the damaged cell by being activated after
many disorders in the cell, such as DNA damage,
hypoxia, viral transformation, oncogene activation,
spindle damage, nucleotide disorder. Thus, it protects
the organism against cancer development. [] The p53
tumor suppressor gene has been studied in many cancer
studies and has been found to mutate in more than
50% of solid tumors. As with gastric cell lines and early
gastric cancers, loss of heterozygosity (LOH) and p53
gene mutation on chromosome 17p is frequently observed
in advanced gastric cancers. [] TP53 (42%) has
been reported to be the most frequently mutated gene
in SRCC. []
Many studies have shown that 350 kilobase- long
NF1, expressed as one of the largest genes in the human
genome and classified as a tumor suppressor gene,
plays an important role in many cancers, such as brain
tumors, breast cancer, sporadic colon cancer, lung cancer,
and ovarian tumors. [] However, the relationship
between gastric cancer and NF1 is not fully known. Recent
studies have shown that NF1 is a tumor suppressor
that plays a critical role in many cancers and has
been identified as a prognostic value for GC patients.
[] NF1 is caused by mutations in the Nf1 gene in the
17q11.2 region of chromosome 17 and the product of
the gene is the cytoplasmic protein "neurofibromin".
[] The region of neurofibromin which includes exon
21-27a, converts Ras-GTP to Ras-GDP and inactivates
it. Loss of neurofibromin leads to continuous cellular
activation of the mitogenic Ras signaling pathway and
cell proliferation. The Nf1 gene is classified as a tumor
suppressor gene due to its interaction with Ras. [,]
The aim of this case report is to determine the possible
mutations of TP53 and NF1 genes known as tumor
suppressors and to determine whether they can be
used as patient-specific prognostic genetic biomarkers
in the pathogenesis of SRCC at an early age.
Results
As a result of the mutation analysis, a total of 14 mutations
were identified, (Ensembl: ENSG00000141510
MIM:191170) nine in the TP53 gene known as a tumor
suppressor and (Ensembl: ENSG00000196712
MIM:613113) five in the NF1 gene. All detected
changes were previously recorded in the HGMD edatabase.
The changes detected in the TP53 and NF1
genes are shown in the electropherogram images presented
in Figure 3. All of the changes detected in the
TP53 gene (p.P72R, p.P75H, p.P77L, p.A78T, p.A79T,
p.A80G, p.P85N) caused amino acid change due to missense mutation (Table 1). All of these changes were
in the proline-rich region. This region consists of 60-90
amino acid residues (region rich in proline) located in
the N-terminal region and plays an active role in p53-
controlled apoptosis [15]. Furthermore, the change
c.1_*del detected in the splice site was found to be in
a nature that could lead to the incorrect expression of
gene transcripts.
Table 1: Characterization of the TP53 and NF1 mutations detected in the patient
Two of the changes detected in the NF1 gene
(c.1186-5T>C, c.1186-12C>T, c.1186-26C>G, c.1186-
6C>A, c.1215 A>G) were detected as intronic variants
in the non-coding region, one of them was synonymous
change, and two of them were detected on the
splice site sequences (Table 1). In the literature, splicesite
mutations have been reported to result in transcript
ablation by causing inaccurate expression of gene transcription. The localization of the detected changes on
P53 and NF1 are given in Figure 4a-b.
Fig 3: NF1 and TP53 amplicons were sequenced and nucleotide changes were identified by Chromas software analysis.
Fig 4: (a) The structural domains of P53 Protein. Map of the nine TP53 mutations found by DNA sequencing in the
patient. The organization of the p53 coding region, including the five major functional domains is shown. (b) The
structural domains of NF1 Protein. The organization of the NF1coding region, including the four major functional
domains, is shown.
Case Presentation
A 24-year-old male patient was admitted to our center
with complaints of epigastric pain, fatigue and weight
loss for six months. The patient reported no change
in gastrointestinal complaints of nausea, vomiting,
bloating, gastrointestinal bleeding or changed bowel
movements. It was understood that the patient had a
history of a 20 kg weight loss over the past six months
before applying to our center. The patient stated that he
had not undergone any surgery and had not used any
medication, alcohol or derivatives or cigarettes. There
was no family history of cancer. Physical examination
revealed signs of epigastric tenderness and weight loss,
and pathological lymph nodes could not be palpated.
Pulmonary and cardiologic examinations were normal.
The shape of the abdomen was flat, but there was
minimal splenomegaly on palpation. Bowel sounds
were normal during auscultation. No abnormality was
observed in the rectal examination. Laboratory tests
showed microcytic anemia (Hb: 9.1 g/dL, Hct: 28.9%,
MCV: 72.6 fL).
Upper GI Endoscopy
An ulcerated mass lesion, which caused pyloric deformity
and gastric outlet obstruction, and abundant
bile secretion in the stomach were reported. The upper
abdominal CT examination revealed bronchopneumonic
infectious infiltration findings in the basal
segments of the lower lobe of both lungs and in the
lingular segment of the left upper lobe. The appearance
of the liver, gall bladder and bile ducts were normal.
The spleen was larger than normal, its long axis was
18 cm in length and parenchyma density was homogeneous.
Pancreas and bilateral kidneys were normal. No
retroperitoneal or intraabdominal LAP was detected.
There were heterogeneity and contamination in the
abdominal mesenteric adipose tissue, as well as a minimal
fluid effusion between the intestinal loops. The
stomach was larger than normal and appeared dilated
and distended. Clinical evaluation was recommended
for the narrowing in the gastric outlet (Fig. 1a-b).
Fig 1: (a) Sagittal section of abdominal CT: Dilated
stomach appearance (b) Abdominal CT axial
section: Pyloric mass appearance.
Lower Abdominal CT Examination
No gross pathology associated with bladder was observed.
Millimetric dystrophic calcifications were observed
in the central zone of the prostate gland. Pelvic
large vessels were normal and no pelvic LAP was detected.
As a result of this physical examination and laboratory
findings, subtotal gastrectomy was performed
and the material was sent to the pathology department.
Microscopic examination: In HE-stained sections,
a tumor consisting of TYH starting from mucosa and extending to the serosal surface was observed (Fig.
2a). The inflammatory response was mild, and there
was a lymphovascular and perineural invasion. A total
of 21 metastatic lymph nodes were detected. No
Helicobacter pylori were observed with Giemsa stain.
There is no intestinal metaplasia in the non-tumor
mucosa Cytoplasmic staining was observed in tumor
cells with PAS-AB and mucicarmine stains (Fig. 2b-c).
Considering the histomorphological findings, the case
was diagnosed as a signet ring cell carcinoma (SRCC)
pT4aN3bMx.
Fig 2: (a) Histopathological image of stony ring cells in
HE stained sections. (b) Histopathological image
of stony ring cells in HE stained sections. (c)
Cytoplasmic staining of tumor cells with mucicarmine
histochemical staining.
Mutation Analysis
For mutation analysis, DNA was isolated from the tissue
sample obtained from the patient using a commercial
DNA isolation kit. Approximately 200 ng of DNA
was used as a template for PCR amplification. Total
volume of reaction mix was 25 µL, which comprised of
16.75 µL sterile water, 2.5 µL reaction buffer (750 mM
Tris-HCl pH 8.8 at 25° C, 200 mM (NH4)2SO4, 1µL
dNTP mix (A, C, G, T 200 mM), 1.25 µL MgCl2 (25
mM), 1 µL forward and reverse primers (10 pmol), 0.5 µL Taq polymerase (500 U, Fermentas) and 1 µL template
DNA Amplification reactions were performed
using Applied Biosystems Veriti VR 96-Well Thermal
Cycler and started with an initial denaturation step at
94 oC for 5 min, followed by 40 cycles of denaturation
for 45 s at 94 ºC, primer annealing for 1.5 min at 56-58
oC, extension for 1.5 min at 72 ºC and final extension
for 10 min at 72 ºC. After amplification, PCR products
were run on 2% agarose gel containing GelRed in
1xTAE for 40 minutes at 90 V. Thermo Scientific Gene
Ruler 100 bp DNA Ladder was used as a marker. Subsequently,
all gels were visualized using KODAK Gel
Logic 100 System. PCR products were sequenced using
the ABI 3500 XL DNA Sequencer. Data were analyzed
using Data Collection Software and Chromas
2.6.5 (Technelysium) to determine sequence variations
in comparison to reference TP53 (Exon 4) and NF1
(Exon 21) sequences from Ensemble genome browser.
Discussion
Gastric cancer, which is the third most common cause
of cancer-related deaths, is an adenocarcinoma, the
genomic basis of which has not been fully elucidated,
and underlying genetic heterogeneity has been still
unclear. Gastric cancer is more common in middleaged
and older individuals compared to the young
population. Diffuse type gastric carcinoma is known
to be more common in young patients, and such cancers
are known to have a worse prognosis. On the
other hand, intestinal-type gastric carcinoma is more
common in the elderly. [] Gastric cancers tend to be more aggressive in young people and women,
which suggests a genetic predisposition in this age
group.[] Furthermore, the majority of cases are
asymptomatic. Thus it is difficult to diagnose. Nakamura
et al. reported in their study published in 2011
that 54.9% of the young patients with gastric cancer
had no complaints, and the diagnosis was made incidentally
during examinations. The patient included
in our study stated that he only had rapid weight loss
and that he did not have any other complaints.[]
Progression and tumor stage affect survival in patients
with gastric cancer, and the sensitivity and specificity
of the existing noninvasive serological biomarkers
are insufficient for both the diagnosis and prognosis.
Therefore, early diagnosis and follow-up are very
necessary and important in gastric cancer cases. [,] In some studies, the relationship between apoptosis
and TP53 changes was evaluated, and it was
suggested that the balance between cell proliferation
rate and programmed cell death, also called apoptosis,
in gastric epithelium maintained homeostasis and
that the disruption of the balance between these two
processes leading to proliferation of gastric epithelial
cells could increase the effects of carcinogens on DNA
and the risk of mutational changes. As a result, it
might cause gastric cancer development.[21] Recent
studies have shown that NF1 is a tumor suppressor
that plays a critical role in many types of cancer and
has been identified as a prognostic value for patients
with gastric cancer. In a study conducted by Liu et
al. [] (2017), immunohistochemical staining and
qRT-PCR analysis have revealed that NF1 protein and
mRNA levels decrease significantly in cancer tissues
compared to healthy tissues. In the present case, mutations
causing the formation of the abnormal transcript
were detected in the splice region in both TP53
and NF1 genes. Given that splice sites are conserved
sequences, point mutations in these sequences may
cause false exon and intron recognition, and mutated
gene may cause the formation of the abnormal transcript,
mutations detected herein can be considered as
important. [,] The mutations identified through
genetic analysis performed in the present study have
been found to have the potential of causing structural
changes in the end-products of the genes. In particular,
P53 and NF1 can be predictive factors in the
pathogenesis of SRCC seen at a young age.
Conclusion
In light of all this data, it is possible to say that molecular
analyses may contribute to the diagnostic process, considering the inadequacy of clinical findings and
analyses to help diagnose.
Informed consent: A written informed consent was provided
by the patient.
Peer-review: Externally peer-reviewed.
Conflict of Interest: No potential conflict of interest was reported
by the author.
Financial Disclosure: There is no financial support from
any foundation.
Authorship contributions: Concept - M.E., D.F.A.B.; Design
- M.E., D.F.A.B., S.H.A.; Supervision - M.E; Materials -
M.E.; Data collection &/or processing - M.E., D.F.A.B.; Analysis
and/or interpretation - M.E., D.F.A.B., S.H.A.; Literature
search - D.F.A.B., S.H.A., T.K.; Writing - M.E., D.F.A.B.,
S.H.A.; Critical review ? M.E., D.F.A.B., S.H.A., T.K.
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