Use of ANGPTL2 mRNA levels in formalin‑fixed paraffin‑embedded tissues as a biomarker to diagnose gastric cancer and to evaluate the extent of vascular invasion
- Authors:
- Published online on: October 23, 2018 https://doi.org/10.3892/ol.2018.9610
- Pages: 518-524
Abstract
Introduction
Gastric cancer is prevalent worldwide, with ~800,000 gastric cancer-associated mortalities occurring each year, making it the second most common cause of cancer-associated mortality (1). With the recent advances in medical technologies, gastric cancer can often be removed with minimally invasive surgical techniques if identified early (2). Endoscopic submucosal dissection is the least invasive and, therefore, the most widely used of these surgical procedures (2). It is important for all gastric cancer tissue to be removed by the endoscopic submucosal dissection, since residual cancerous tissue can lead to recurrence (3). Resected cancerous tissues are pathologically evaluated to determine whether all cancerous areas have been removed; however, pathological assessment is rarely straightforward. Interpretations of stained cancerous tissues are, however, not consistent between pathologists, meaning that different conclusions may be drawn by different pathologists (4).
Cancer markers could inform pathological evaluations of cancer. An ideal marker would be one that is identifiable in formalin-fixed paraffin-embedded (FFPE) tissue samples, which a high proportion of medical institutions prefer as these tissues store well and are easy to handle (5).
The first objective of the present study was to quantitatively determine the levels of angiopoietin-like protein 2 (ANGPTL2) in cancerous and noncancerous areas of FFPE tissues to evaluate whether ANGPTL2 is a marker relevant to pathological diagnoses of gastric cancer.
ANGPTL2, a member of the ANGPTL family, contributes to the onset and progression of chronic inflammation and to the associated diseases caused by this (6–10). ANGPTL2 also regulates angiogenesis in the body (11). Endo et al (9) considered ANGPTL2 to be a potential biomarker for diagnosing lung and breast cancer in humans.
Our previous studies showed ANGPTL2 to be widely expressed in gastric cancer cell lines and patients with gastric or colon cancer (12,13). These findings highlight the potential of ANGPTL2 as a biomarker for identifying gastric and colon cancer in clinical settings.
In the present study, mRNA levels of ANGPTL2 were determined in FFPE tumor tissues collected from patients with mucosal (M), submucosal (SM), tunica muscularis propria (MP), serosa-exposed (SE) and subserosal (SS) gastric cancer. A second objective was to evaluate whether ANGPTL2 mRNA is useful as a marker of the extent of vascular invasion of gastric cancer, which portends hematogenous metastasis (14).
Materials and methods
Patients and tissue samples
Serum samples were obtained from 15 patients who attended the clinic between May 2013 and November 2014 at the Nanpuh Hospital (Kagoshima, Japan). Patient characteristics, including sex, age, body mass index (BMI), serum carcinoembryonic antigen (CEA) levels and serum carbohydrate antigen 19-9 (CA19-9) levels, are summarized in Table I. Serum concentrations of CEA and CA19-9 were determined using an electro-chemiluminescence immunoassay using the LUMIPULSE G1200® (Fujirebio Diagnostics, Inc., Tokyo, Japan) according to the manufacturer's protocol.
Tissues removed from the patients were immersed in 10%-formaldehyde neutral buffer solution. FFPE tissues were prepared using the Tissue-Tek VIP6® (Sakura Finetek Japan Co., Ltd., Nagano, Japan).
Table II shows the sex, diagnosis, levels of ANGPTL2 mRNA, degree of differentiation, tumor invasion depth, lymph node metastasis, distant metastasis, tumor stage and degrees of lympho-vascular and vascular invasion for each patient. Lympho-vascular invasion was classified into four grades according to Japanese Classification of Gastric Carcinoma (15): ly0, no lymphatic invasion; ly1, minimal lymphatic invasion; ly2, moderate lymphatic invasion; and ly3, extensive lymphatic invasion. Vascular invasion was also classified into four grades according to Japanese Classification of Gastric Carcinoma (15): v0, no venous invasion; v1, minimal venous invasion; v2, moderate venous invasion; and v3, extensive venous invasion. Of the 15 patients with gastric cancer, 9 were diagnosed with adenocarcinoma, 5 with tubular adenocarcinoma and 1 with signet-ring cell carcinoma. The patient group included 3 patients diagnosed with M, 3 patients with SM, 4 patients with MP and 5 patients with SE or SS cancer.
Table II.Level of ANGPTL2 mRNA, degree of differentiation, tumor invasion depth, lymph node metastasis, distant metastasis, tumor stage, and degrees of lympho-vascular invasion and of vascular invasion. |
Among the patient group, 5 patients were diagnosed with clinical stage IA, 1 with stage IB, 4 with stage IIA, 2 with stage IIIA, 1 with stage IIIB, 1 with stage IIIC and 1 with stage IV. Cancer staging was based on routine histopathological analysis and clinical assessment, according to the Tumor Node Metastasis (TNM) classification (16). Tumors were classified according to the recommendations of the International Union Against Cancer/TNM system (17). The characteristics of the subjects are summarized in Table III.
Table III.Level of ANGPTL2 mRNA, degree of differentiation, lymph node metastasis, distant metastasis, tumor stage, and degrees of lympho-vascular invasion and of vascular invasion by degree of tumor invasion depth. |
Six consecutive slices, each 3-µm thick, were prepared from the FFPE tissues. Hematoxylin and eosin (H&E) staining was performed on one slice to identify the cancerous areas. Tissue slices were stained with hematoxylin (for 10 min) and eosin (1 min) at room temperature. A pathologist observing the H&E-stained slices classified the cancerous and noncancerous areas. For patients with M cancer, the cancerous and noncancerous areas in the M layer of the remaining five slices were obtained using microdissection. For patients with SM, MP or SE/SS cancer, the cancerous and noncancerous areas in the SM layer were obtained using microdissection. Tissues were also stained with Victoria blue (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) for 12 h at room temperature to determine venous invasion.
Written informed consent was obtained from all patients. The study design was approved by the Ethics Committee of Nanpuh Hospital (Kagoshima Kyosaikai, Public Interest Inc. Association, Japan). Clinical examinations were performed according to the principles of the Declaration of Helsinki.
RNA extraction from FFPE tissue
Total RNA was extracted from FFPE tissue slices using the PureLink FFPE Total RNA Isolation kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
RT-qPCR was performed with equipment of the Division of Gene Research, Kagoshima University. The RT reaction was performed using random primers (Toyobo Co., Ltd., Osaka, Japan) and ReverTra Ace® (Toyobo Co., Ltd.), according to the manufacturer's protocol, using 100 ng RNA. Cycle conditions were 95°C for 1 min, followed by 45 cycles of denaturation for 15 sec at 95°C, annealing and extension steps for 30 sec at 60°C each.
The amplification was performed using the StepOnePlus™ Real-Time PCR System (Applied Biosciences; Thermo Fisher Scientific, Inc.) using a SYBR Green Realtime PCR Master Mix kit (Toyobo Co., Ltd.) according to the manufacturer's protocol. The specific primers for human ANGPTL2 (purchased from Thermo Fisher Scientific, Inc.) were 5′-GCCACCAAGTGTCAGCCTCA-3′ (forward) and 5′-TGGACAGTACCAAACATCCAACATC-3′ (reverse). Human β-actin, used as a control, was amplified using the following specific primers: Forward, 5′-AAGCCACCCCACTTCTCTCTAA-3′; and reverse, 5′-AATGCTATCACCTCCCCTGTGT-3′ (Thermo Fisher Scientific, Inc.). With the ANGPTL2 mRNA level in the noncancerous areas taken to be 1.0, ANGPTL2 mRNA levels in the cancerous areas were calculated with the 2−ΔΔCq method (18).
Statistical analysis
Data are presented as the mean ± standard deviation. Data were analyzed using SPSS version 23 (IBM SPSS, Armonk, NY, USA). The correlations of the ANGPTL2 mRNA concentration with the patient age, BMI and serum CEA and CA19-9 levels were analyzed using Pearson's correlation analysis. The correlations of the ANGPTL2 mRNA concentration with the degree of differentiation, tumor invasion depth, lymph node metastasis, distant metastasis, tumor stage, degree of lympho-vascular invasion and degree of vascular invasion were analyzed using Spearman's rank correlation analysis. A receiver operating characteristic (ROC) curve was plotted to evaluate the ability of ANGPTL2 mRNA level to predict vascular invasion. Youden's index method (19) was used to determine the optimal cutoff for the ANGPTL2 mRNA level for assessing the presence of vascular invasion in gastric cancer. P<0.05 was considered to indicate a statistically significant difference.
Results and Discussion
To the best of our knowledge, this is the first study to investigate whether ANGPTL2 in FFPE tissues is a useful biomarker for diagnosing gastric cancer. To prepare FFPE tissues, a tissue specimen removed from a patient was fixed in a formalin solution to cross-link biological molecules, and then embedded in paraffin. This procedure may denature mRNA and other biological components. von Ahlfen et al (20), however, successfully extracted the mRNA for telomere-binding protein from FFPE tissues (21), which indicated that ANGPTL2 mRNA may be extractable from FFPE tissues. In the present study, the cancerous and noncancerous areas were distinguished from one another by pathological diagnosis using H&E-stained cross-sections.
Of the 15 patients studied, 12 (80%) had a higher ANGPTL2 mRNA level in cancerous areas compared with the reference level (set as 1.0) in noncancerous areas (Fig. 1). In total, 2 of the 3 patients with M cancer, 2 of the 3 patients with SM cancer, 3 of the 4 patients with MP cancer and all 5 patients with SE/SS cancer had an ANGPTL2 mRNA level >1.0 (Fig. 1). This finding indicated that ANGPTL2 mRNA is useful as a biomarker for identifying cancerous areas in FFPE tissues, at least for male patients (owing to the small female sample size). Furthermore, the results indicated that the ANGPTL2 mRNA level may have higher diagnostic precision in advanced cancer.
The association between ANGPTL2 mRNA expression and BMI, as well as other factors, was also evaluated. As shown in Table IV, ANGPTL2 mRNA levels in FFPE tissues were not correlated with age (correlation coefficient, r=−0.31; P=0.26), BMI (r=0.09; P=0.75), CEA level (r=0.04; P=0.90), CA19-9 level (r=0.03; P=0.91), degree of tumor differentiation (r=0.21; P=0.46), depth of tumor invasion (r=0.35; P=0.21), degree of lymph node metastasis (r=0.31; P=0.26), degree of distant metastasis (r=0.00; P=1.00), tumor stage (r=0.33; P=0.23) or degree of lympho-vascular invasion (r=0.07; P=0.80). ANGPTL2 mRNA levels were, however, correlated with the degree of vascular invasion (r=0.66; P=0.01).
Table IV.Correlations between angiopoietin-like protein 2 mRNA concentration and various parameters. |
Micrographs of cancerous areas stained with Victoria blue and H&E in patients with a high ANGPTL2 mRNA level and a high degree of vascular invasion [patient no. 4 (mRNA level 2.52) and patient no. 14 (mRNA level 2.81)] are shown in Fig. 2. The arrows in Fig. 2A and B show the tumor cells in the blood vessels. Fig. 2C shows the micrograph of cancerous areas, stained with H&E, of patients with a low ANGPTL2 mRNA level [patient no. 5 (mRNA level 0.42)]. The arrow in Fig. 2C indicates no tumor cells in the blood vessels.
Since primary cancer with a high degree of vascular invasion has often already metastasized (14,22,23), accurate pathological diagnoses of vascular invasion could inform assessments of metastatic status. Pathological diagnoses of vascular invasion, however, are elusive. A biomarker of vascular invasion would aid the detection of metastatic cancer. As shown in Table IV, the ANGPTL2 mRNA level was correlated with vascular invasion.
An ROC analysis was conducted to explore this possibility. Fig. 3 contains the ROC curve (n=15) for the analysis of ANGPTL2 mRNA levels and the degree of vascular invasion. An area under the curve of 0.92 (95% confidence interval, 0.78–1.00; P=0.01) indicated a high diagnostic potential. The results of ROC analysis also indicated that ANGPTL2 mRNA may be useful for assessing gastric cancer metastasis.
Next, the optimal cutoff using by Youden's index method for the ANGPTL2 mRNA level for assessing the presence of vascular invasion in gastric cancer. The optimal cutoff was determined to be a relative expression level of 1.50. All patients with vascular invasion had a level ≥1.50, while only 20% lacking vascular invasion had a level at or above this cutoff. Thus, the cutoff of 1.50 produces a high true-positive rate and low false-positive rate.
The present study demonstrated that ANGPTL2 mRNA in FFPE tissues is a potential biomarker for informing the pathological diagnosis of gastric cancer. Since numerous medical institutions retain FFPE tissues, this discovery may lead to a widely usable diagnostic procedure for more accurately assessing gastric cancer compared with the conventional pathological diagnosis. The present study also showed that ANGPTL2 mRNA may be predictive of vascular invasion, which is an indicator of metastasis in gastric cancer. An increase in the number of samples of cancer metastasis is necessary to clarify whether the ANGPTL2 mRNA level is actually correlated with metastasis.
The present study focused on ANGPTL2, and comparisons with other tumor markers were not performed. Previous studies have reported microRNAs, human epidermal growth factor receptor 2 and proteomic profiling in FFPE as gastric cancer biomarkers (24–26). Our future studies will measure and compare other biomarkers.
Acknowledgements
The present study was supported in part by the Division of Gastrointestinal Surgery, the Division of Diagnostic Pathology, and the Division of Clinical Laboratory of Nanpuh Hospital.
Funding
No funding was received.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
MY, TT, HN and TY conceived and designed the experiments. Data collection and experiments were performed by ST, EH, AT and ET. TY analyzed the data and all authors contributed to the writing of the manuscript.
Ethics approval and consent to participate
The study design was approved by the Ethics Committee of Nanpuh Hospital (Kagoshima Kyosaikai, Public Interest Inc. Association, Japan). Written informed consent was obtained from all patients.
Patient consent for publication
Written informed consent was obtained from all patients.
Competing interests
The authors declare that they have no competing interests.
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