To characterize clinical, histological properties and SFRP2 methylation status in patients diagnosed with gastric carcinoma who underwent surgical resection at 103 Military Hospital.
Trang 1CLINICAL, HISTOLOGICAL CHARACTERISTICS AND SFRP2,
RNF180 METHYLATION STATUS IN GASTRIC CARCINOMA
PATIENTS EXPERIENCED SURGICAL RESECTION AT
103 MILITARY HOSPITAL
Nguyen Minh Phuc 1 ; Tran Van Khoa 2 ; Nguyen Thuy Vinh 3
SUMMARY
Objectives: To characterize clinical, histological properties and SFRP2 methylation status in
patients diagnosed with gastric carcinoma who underwent surgical resection at 103 Military
Hospital Subjects and methods: Case-control based, and cross-sectional study on 98 gastric
carcinoma patients whose surgical resection from December 2015 to December 2018, and 40
chronic gastritis patients diagnosed by endoscopic and histological methods Results and conclusion:
Gastric carcinoma prevalence among male patients was greater than that of female patients,
with a ratio of 2.27/1 The average age of cancer patients was 61.5 ± 10.88, with the highest
incidence was witnessed in 60 - 69 years-old group According to Lauren’s criteria, the intestinal
type was more common than the diffuse type (p > 0.05); meanwhile, in the WHO classification
system, tubular adenocarcinoma was the major histologic patterns of gastric cancers, followed
by undifferentiated carcinoma, signet ring cell carcinoma, and mucinous adenocarcinoma (9.18%)
Most of the surgery procedures were performed in II - IV stages (95.92%) SFRP2 methylation
was detected in 74.49% of gastric carcinoma group and 25.51% of control group (OR = 6.07;
95%CI: 3.28 - 11.21) SFRP2 methylation is a potential marker for gastric cancer diagnosis
* Keywords: Gastric carcinoma; SFRP2 methylation; Clinical characteristics; Histological
characteristics
INTRODUCTION
Gastric cancer (GC) is a common disease
in Vietnam and many other countries
worldwide The International Agency for
Research on Cancer (IARC) in 2018
estimated 17,527 new cases of GC
(or 10.6% of all reported cancer cases)
in Vietnam, made GC the third most
commonly diagnosed malignancy, behind
liver and lung cancers [5] Due to many
GC are diagnosed at an advanced stage,
the 5-year survival rate is very low,
at 10 - 20%
Epigenetic is heritable changes in gene expression that occurs without changes in DNA sequences, including histone modifications and promoter methylation DNA methylation is a process
by which a methyl group is covalently added into the carbon 5 position of the cytosine ring in CpG dinucleotides DNA methylation inactivates the expression of
1 Thai Binh University of Medicine and Pharmacy
2 Vietnam Military Medical University
3 Public Health University
Corresponding author: Nguyen Minh Phuc (minhphucytb@yahoo.com)
Date received: 08/10/2019 Date accepted: 22/11/2019
Trang 2tumor suppressor genes, which is considered
as a key step in carcinogenesis and cancer
development A study by Zhang X et al
(2014) revealed that SFRP2 methylation
level detected in plasma was 71.93% of
GC patients compared to 42.86% of control
group (p = 0.0036) [6]
In Vietnam, studies of DNA methylation
of genes relating to cancers, including
GC, are limited Thus, we conducted a
research with aims: To characterize the
clinical and histopathological properties
as well as to investigate methylation
status of SFRP2 among GC patients who
were performed surgical resection at
103 Military Hospital from December 2015
to December 2018
SUBJECTS AND METHODS
1 Subjects
- Case group: 98 primary GC patients
who received surgical treatment at the
Abdominal Surgery Department, 103 Military
Hospital
* Exclusion criteria: Secondary gastric
carcinomas or patients who had experienced
radio- and/or chemotherapy
- Control group: 40 chronic gastritis patients
who were diagnosed by gastrointestinal
endoscopy and histological examinations
2 Methods
- Study design: Case-control based,
cross-sectional study
- Laboratory instruments and material:
DNA Speed vac Eppendorf AG 530
concentrator (Germany); DNA Quikdrop
spectrophotometer (USA); Eppendorf
ProS PCR cycler (USA); Rotor-Gene Q
Qiagen real-time PCR cycler (Germany);
mupid-one electrophoresis system (Japan);
CV-170 video system (Japan), Qiagen DNeassy Blood & Tisiue (50) 69504, QIA quick PCR Purification Kit 28104, Methylation - Gold D5006
* Study scheme:
- Case group: Cancer patients received
a clinical examination before the surgery procedure Tumorous specimens were sent to the Department of Pathology,
103 Military Hospital; then specimens were subjected to histological examination
and DNA methylation assessment
- Control group: Chronic gastritis patients
received gastrointestinal endoscopy and endoscopic biopsy specimens were collected and subjected to histological examination and DNA methylation assessment
- Histological examinations were conducted
at the Department of Pathology, 103 Military Hospital, including:
+ Gastric carcinoma diagnosis
+ Histologic demonstration based on Lauren criteria [7] (diffuse and intestinal adenocarcinoma) or the WHO classification system (2000) [1] (9 histologic patterns:
Tubular adenocarcinoma, mucinous adenocarcinoma, signet-ring cell carcinoma, adenosquamous carcinoma, squamous cell carcinoma, squamous cell carcinoma, small-cell carcinoma, undifferentiated carcinoma and other carcinoma) The degree
of tissue differentiation was divided into highly differentiated, moderately differentiated and poorly differentiated
- MSP-based methylation assessments
of SFRP2 gene were carried out at the
Molecular Biology Laboratory, Department
of Biology and Medical Genetics,
Trang 3Military Medical University Experiment
protocol was performed in the following steps:
+ DNA isolation: DNA was extracted by
using QIAamp DNA Mini Kit (Qiagen)
followed to the manufacturer’s instruction
Concentration and purity of isolated
DNA were evaluated by DNA Quikdrop
spectrophotometer, and stored at -20oC for
further usage
+ Bisulfite modification: DNA sample
was bisulfite modified by EZ DNA
methylation-Gold Kits (Zymo research)
followed to the manufacturer’s instruction
Ten microliters of modified DNA was
stored at -20oC for further usage
+ MSP (Methylation Specific PCR)
amplification: Specific primers were used
to analyze the methylation status of
SFRP2 promotor (table 1) Technically,
if the result of the methyl-specific primer set is positive, then the CpGs of interest are fully methylated; if the result of the unmethyl-specific primer set is positive, then the CpGs of interest are unmethylated;
if the methyl-specific and the unmethyl-specific primer sets show positive results, these CpGs are partially methylated
PCR was carried out in a 25 µL total volume containing: 12.5 µL Mastermix; 0.5 µL of each primer, 2 µL bisulfite-modified DNA; H2O (adjusted) The standardized MSP amplification consisted of 5 minutes
at 95oC, followed by 35 cycles of 30s at
95oC, 60s at 55oC, 30s at 72oC and final extension at 72oC
Table 1: Primer sequences used for MSP
+ Electrophoresis: PCR products were
run on 2% agarose gel and were applied to
100 V field, 100 mA current within 30 minutes;
gels were stained in ethidium bromide
and UV imaged, then the electrophoresis
results were analyzed
- All analysis was performed by SPSS
software version 20.0
RESULTS AND DISCUSSION
1 Gender and age characteristics
* Gender information: 68 male patients
(69.39%); 30 female patients (30.61%)
GC is associated with gender GC incidence in males was usually higher than that in females, with male/female ratio ranged between 2 - 3/1 This gender difference may be explained by the protective activity of female hormone (estrogen), and by the fact that men are more usually exposed to risk factors In the current study, male/female patient ratio was 2.27/1, which was concordant with results from studies by Le Viet Nho (2.75/1) [2] and Nguyen Quang Bo (2.5/1) [3]
Trang 4Table 2: Patients age distribution in the study
Gender
Age group
(n = 98)
Total
GC was less commonly diagnosed
under 40 years old, and the percentage
increased by age, with the highest
percentage belonged to 60 - 70 years-old
group [4] In this study, the oldest patient
was 85, while the youngest was 35,
and the average age of GC patients was
61.5 ± 10.88 Most of them were more
than 50 years old, accounted for 89.8%
of patients, with the major group was
60 - 69 years old The average age of GC
patients in our research was also in line
with that in studies by Phan Van Cuong
(2017) (59.8 - 61.9 years old) [4] and
Zhang X (2014) (61.49 ± 12.02 years old) [6]
2 Clinical and histological
characteristics.
* Clinical characteristics:
- The reasons of hospitalization of GC
patients: Epigastric pain: 76 patients (77.56%);
gastrointestinal bleeding: 11 patients
(11.22%); vomiting/nausea: 7 patients
(7.14%); weight loss: 1 patient (1.02%);
other reasons: 3 patients (3.06%)
In our study, none of the GC patients
was diagnosed by a regular medical
checkup Our result was therefore similar
to many other studies, in which the
reason for hospitalizing was mainly due to
epigastric pain [2, 3]
- Systemic and functional symptoms: Epigastric pain: 92 patients (93.87%); weight loss: 50 patients (51.02%); vomiting/nausea: 41 patients (41.84%); gastrointestinal bleeding: 17 patients (17.35%); indigestion, belching, heartburn:
33 patients (33.67%); anemia: 46 patients (46.94%)
There were no specific symptoms of
GC among patients Meanwhile, in our study, epigastric pain, weight loss, vomiting, nausea was common symptoms; remaining symptoms were less widespread Our research results were in line with other studies [2, 3]
- Physical symptoms:
In our research, the most common physical symptom amongst gastric carcinoma patients was epigastric pain while palpating (73 patients = 74.49%) and a very limited number of cancer patients had palpable epigastric tumors (11 patients = 11.22%) or enlarged liver (3 patients = 3.06%) There was no patient with lymphadenopathy or ascites Our data thus in concordance with the result of Nguyen Quang Bo’s study,
in which most GC patients experienced epigastric pain with palpation but rarely encountered other physical symptoms [3]
Trang 5* Histological characteristics:
- Histological classification relying on Lauren’s criteria: Intestinal gastric carcinoma (52 patients = 53.06%) was diagnosed more frequently than diffuse gastric carcinoma (46 patients = 46.94%) This result was similar to those of Le Viet Nho’s study [2] and Zhang Zh’s study [8]
Table 3: Histological classification relying on the WHO classification system (2000)
Histologic subtypes:
Differentiation degree:
Based on WHO classification system (2000), we noticed that the tubular adenocarcinoma accounted for the largest proportion of total histologic subtypes None of the other subtypes was detected in our studies These indicated the similarity between Le Viet Nho’s and our study, wherein the major percentage belonged to the tubular subtype [2] With regard to differentiation degree, poorly differentiated cancer was the major proportion
Table 4: Cancer staging according to AJCC/UICC (2010)
T
N
Trang 6M0 94 95.92
M
GC overall stage
All of the gastric carcinoma patients in this research performed surgical resection when the tumors had invaded via the submucosal route, thus classified into T2 to T4 stages Most of the patients (93.88%) also had lymph node metastases, which belonged to N2 - N4 stages and others were in the status of distant metastasises Hence, nearly almost gastric patients (95.92%) were in advanced stages, who could be sorted into stages II to IV in overall
3 Methylation assessment result
Table 5: SFRP2 gene methylation status
Methylation status
OR (95%CI)
6.06 (3.28 - 11.21)
DNA methylation resulted in inactivating genes that have roles in cancers, especially
tumor suppressor genes, which leads to carcinogenesis, including GC SFRP2 was described to be one of tumor suppressor genes Our data showed that SFRP2
methylation rate in the case group was significantly higher than that in the control
group, and SFRP2 methylation caused a 6.06-fold higher risk of gastric carcinoma in relation to SFRP2 unmethylation (OR = 6.06, 95%CI: 3.28 - 11.21) This result was in
line with that derived from Zhang X’s study [6]
CONCLUSION
- Gastric carcinoma incidence among
male patients was greater than that of
female patients, with a ratio of 2.27/1 The
average age of cancer patients was 61.5
± 10.88, with a large proportion of them
was in the 60 - 69 years-old group
- According to Lauren’s system: The intestinal type was more common than the diffuse type (p > 0.05)
- In the WHO classification system (2000): Tubular adenocarcinoma was the major histologic patterns of gastric cancers, followed by undifferentiated carcinoma,
Trang 7signet ring cell carcinoma, and mucinous
adenocarcinoma Poorly differentiated
tumors occupied the highest percentage
- SFRP2 methylation associated with a
higher risk of GC (OR = 6.06, 95%CI:
3.28 - 11.21) This result suggests that
SFRP2 methylation has a potential to
become a diagnosis marker for GC
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Medicine Publisher 2007
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clinical, endoscopy and histopathology in
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Pharmacy, Hue University 2014
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treatment of 1/3 lower GC with radical surgery
combined with chemotherapy Doctor of
Medicine Thesis University of Medicine and
Pharmacy, Hue University 2017
4 Phan Van Cuong Incidence of stomach
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Detection of aberrant promoter methylation of RNF180, DAPK1 and SFRP2 in plasma DNA
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