Detection of digestive malignancies and post-gastrectomy complications via gastrointestinal fluid examination

Abstract

To date, gastric carcinoma (GC) is one of the common and fatal digestive malignancies worldwide. The prognosis of GC is not always satisfactory because of late diagnosis. Scholars are keen on discovering novel accurate and economical biomarkers in body liquids for GC screening to detect and evaluate the lesion before the results of imaging techniques are obtained. While traditional serum assays have limited sensitivity and specificity, gastrointestinal juice may provide relevant specific biomarkers because of its close contact with the tumor. Herein, the current progress in the relationship between gastrointestinal fluid analyses and GC is systematically and comprehensively reviewed. The detection of gastric juice pH, fluorescence spectrum, cytology, Helicobacter pylori-associated markers, nitrosamines, conventional tumor markers, amino acids, proteomics, microRNAs, long noncoding RNAs, protein-coding genes, vitamin C, etc., and combination tests of different category markers could provide important diagnostic and prognostic clues for gastrointestinal diseases. Particularly, early GC may be efficiently screened using gastric juice. Gastrointestinal fluid examination could also predict the adverse effects of postgastrectomy, such as pancreatic leakage, fistula, and abscess. Gastric fluid markers should be further studied to reveal the early predicators of malignancy and complications. The methods for obtaining the samples of gastrointestinal juice with minimum incision should also be comprehensively investigated.

Keywords

gastrointestinal fluid / gastric carcinoma / biomarker / diagnosis / prognosis / gastrectomy / adverse events

Introduction

To date, gastric carcinoma (GC) is one of the most common and fatal digestive malignancies worldwide [ 1], particularly in China [ 2], and usually has a poor prognosis because of late diagnosis. Early screening and treatment could significantly prolong the patients’ survival time. Early GC has a 5-year survival rate of 90% and even higher, whereas for advanced GC, surgery alone could not guarantee a long-term survival period [ 3]. Although timely treatment is associated with better clinical outcome, early screening is not always possible because of the asymptomatic and progressive nature of GC. Except for early detection, an accurate, economical, and noninvasive method, which could significantly improve prognosis, is not available [ 4, 5]. Specific and sensitive GC markers as diagnostic or prognostic surrogates have been a topic of interest and are comprehensively investigated with the hope of replacing invasive endoscopic and histological examinations. For screening early-stage gastric malignancies, the sensitivity and specificity of the existing GC serum biomarker detection modalities are significantly limited, and the positive rates of classical tumor biomarkers carcinoembryonic antigen (CEA) and carbohydrate antigen (CA) 19-9 in the sera of GC patients’ are only 4.2% and 6.3%, respectively. The CEA levels in almost all tumor node metastasis (TNM) stage I/II and 70% of stage III/IV in the sera of GC patients are normal [ 6]. Though a number of relatively sensitive imaging techniques are used in GC monitoring and diagnosis, scholars are still keen on discovering and revealing novel biomarkers in body fluids with the hope to screen and evaluate the malignancy and predict its prognosis when physical and imaging examination could not provide any answer.

Gastric juice is the most proximal fluid surrounding gastric tissue, and the tumor markers in it are ideally tissue specific and are excellent sources of GC biomarkers because they are directly released by the lesion without being eliminated by the liver. Moreover, these biomarkers are rarely destroyed in GC patients with weak or nonacidic gastric juices [ 7]. Unlike the serum and GC tissue that are heterogeneous and usually overloaded with abundant contents, gastric juice contains a concentrated molecular biopsy of the stomach and could accurately reflect gastric pathology [ 8]. However, gastric juice, a clinically important organic fluid, is routinely discarded during upper gastrointestinal endoscopy. Currently, research on gastric juice markers is scarce, and these markers need further investigation. The relationships between gastrointestinal juice detection and gastric diseases are reviewed. This study aimed to summarize the state of gastric marker discovery in the patients’ gastrointestinal juices, to outline key concerns in working with the fluid, and to discuss how the challenges of GC screening may be addressed with the new perspectives in GC diagnosis.

The screening of gastrointestinal diseases via gastric juice examination

The diagnosis of gastrointestinal diseases via gastric juice examination is summarized in Fig. 1.

pH

Multivariate analyses reveal that gastric juice bile stain, high acute corpus inflammatory score, and corpus atrophy are independent risk factors for gastric hypoacidity development with the odd ratios (ORs) of 3.1, 3.1, and 3.5, respectively. Patients with esophageal (1.9) and duodenal ulcer (2.1) have significantly lower gastric juice pH than healthy subjects (2.9). By contrast, patients with gastric ulcer (3.4) and GC (6.6) have significantly higher pH levels than healthy controls (2.9). Bile reflux, atrophy, and dense corpus neutrophil infiltrate are three independent determining factors of gastric juice acidity [ 9]. In Helicobacter pylori (Hp)-infected patients, the nitrite concentration in the gastric fluid increases, and the elevated degree is correlated with that of Hp density and inflammation. Gastric juice pH is higher in patients with protruded-type GCs than those with depressed-type tumors and in individuals with multiple malignant lesions than those with a single mass. Hypoacidity and Hp infection that is induced by high levels of gastric juice nitrite are associated with intestinal-type GC, particularly protruded malignancies [ 10]. Intragastric non-Hp bacteria growth often occurs during acid inhibition treatment. Non-Hp flora is present in the gastric fluid of about 40% of patients, and luminal non-Hp flora growth increases with the increase in the intragastric pH. Non-Hp flora significantly contaminates the gastric juice of patients treated with long-term acid suppression [ 11]. Our study further found that before gastrectomy, gastric pH has a low acidity and is significantly affected by age, tumor size, differentiation grade, and concentration of potassium and bicarbonate ions; advanced tumors are accompanied with significantly higher pH levels compared with early ones. Reflux symptom is significantly associated with the patients’ body mass index, levels of direct and indirect bilirubin, and total bile acid, whereas pH did not play a significant role, indicating that acidity is not an important factor in GC [ 7].

Fluorescence spectrum

Based on a study of 1870 patients, the maximum fluorescence intensity of the first peak (P1 FI) of the intrinsic fluorescence spectrum at 288 nm is significantly higher in the gastric juice of patients with GC than in patients with benign diseases. This intensity is also a clinical indicator of GC. Clinical verification in 1506 patients shows that a P1 FI of 76.5 is the optimal cut-off value in the receiver operating characteristic (ROC) curve for GC diagnosis with high sensitivity, specificity, and accuracy [ 12]. The mean P1 FI fluorescence intensity values of the gastric fluid in advanced GC (92.1) and early GC (90.8) groups are significantly higher than that in the normal mucosa/chronic nonatrophic gastritis group (55.7). The P1 FI of the gastric fluid is enhanced at the early stage of GC, suggesting that gastric juice fluorescence spectroscopy may be a potential novel screening tool for early GC detection [ 13].

Cytology

Endoscopic ultrasound (EUS)-guided fine needle aspiration (FNA) may reveal false-positive cytological results and cause needle-tract seeding. The presence of tumor cells within gastrointestinal luminal juices results from either tumor sloughing from luminal malignancies or being secondary to extraluminal site FNA. Luminal juices are commonly aspirated through the echoendoscope suction channel during EUS for cytological analysis among cancer patients and those with benign diseases. Among prospectively enrolled patients, a luminal fluid cytology examination shows positive results for malignancy in luminal (e.g., esophageal, 48%) and extraluminal (e.g., pancreatic, 10%) cancer patients. The positive luminal fluid cytology rate in patients with luminal cancers is affected by FNA. The cytological examination of luminal juice aspirates does not reveal tumor cells in patients with benign diseases. Thus, malignant cells are commonly found in the gastrointestinal luminal juice of patients with luminal cancers and in pancreatic cancer patients after EUS-guided FNA [ 14].

Hp

Hp is a bacterium that is associated with digestive inflammation and peptic ulcers and is a GC risk factor. Among the DNAs extracted from endoscopically collected gastric juice, the Hp cag pathogenicity island genes (i.e., cag1/5/T/E/A), which is useful for the accurate assessment of cagA-positive strain infection, could be detected by PCR, which is a simple procedure with a high sensitivity. Among patients with cagA amplification, cagE is amplified in patients with early GC and chronic active gastritis. However, most of the five genes are unamplified. cag PAI gene deletion is found in patients with early GC, stomach ulcer, and active chronic gastritis. Thus, Hp pathogenicity might not be determined by cag PAI genes [ 15]. In a multicenter study including 17 907 patients in 10 endoscopy units, Hp infection and oxyntic mucosa (AGOM) are greatly underdiagnosed in routine endoscopic practices because of the intrinsic limitations of the conventional tests and inappropriateness/lack of biopsy planning. By contrast, gastric juice analysis is a simple, affordable, and effective approach that prevents underdiagnosis and allows the detection of atrophic gastritis and Hp in 96% and 98% of cases, respectively. Thus, gastric fluids provide a valuable clinicopathologic information source, and if properly analyzed, these fluids can detect major risk factors of GC (Hp and atrophic gastritis), overcoming the screening limitations and also saving time and cost [ 16].

Hp in the gastric mucus layer is considered a main causative agent of gastritis, peptic ulcers, and GC. During inflammation, the association between the gastric epithelial cell pathogen and mucins is considered crucial. Hp affects the soluble MUC 1 mucin expression and Lewis a and b structures existing in the gastric fluid. The carbohydrate structures of MUC 1 mucin are also considered important in the infection mechanism, and the Lewis b structure of the secretory MUC 5AC mucin could be a bacterium receptor. Epithelial MUC 1 mucin may be implicated in the infection mechanism. In 90% of patients, higher amounts of MUC 1 mucin are observed at the end of Hp eradication treatment, and results for Lewis a and b structures are similar. In the cases of MUC 1 and Lewis b, the discrepancies are statistically significant [ 17]. Among Hp-infected patients who are treated with omeprazole or omeprazole and antibiotics, the level of MUC 1 is low in nearly all cases before therapy, whereas at the end of treatment, MUC 1 mucin increases in both groups of patients. Thus, Hp suppresses the MUC 1 mucin expression on cellular membranes [ 18].

Antral somatostatin interacts with gastric acid secretion. Among patients with Hp-positive gastric ulcer, before dual therapy treatment, the somatostatin output and acid are inversely correlated to the neutrophil infiltration severity in the corpus and antrum, respectively. After a successful Hp eradication, basal acid output (BAO) and basal and gastrin-stimulated somatostatin output increase. Eradication did not affect the maximal acid output (MAO). A positive correlation was also observed between somatostatin output and gastric acid secretion in the basal and stimulated conditions, regardless of Hp infection. Thus, gastric BAO recovery may be caused by an improvement in the neutrophil infiltration of the corpus. However, the increase in parietal cell volume or atrophy changes did not affect the gastric BAO recovery [ 19]. The increased cell proliferation, severe inflammation, and marked acid suppression observed in subjects with Hp-associated enlarged-fold gastritis indicate that enlarged-fold gastritis may be a GC risk factor. The mutagenicity of gastric juice from enlarged-fold gastritis patients is significantly higher than in the Hp-negative controls and Hp-positive patients without enlarged folds, and Hp eradication significantly decreases the gastric juice mutagenicity in enlarged-fold gastritis patients [ 20].

Nitrosamines

A chemometrically optimized isolation procedure combined with gas chromatography–mass spectrometry (GC–MS) has been proposed for the quantitative detection of nitrosamines in gastric juice samples of patients with gastrointestinal tract problems. By using the regression model based on the central composite design, Akyuz et al. [ 21] modified and optimized the extraction conditions of nitrosamine (10.7 min for the extraction time, 4.2 for the pH, and 23% for the 2-propanol percentage in the extraction solution), and nitrosamine recoveries ranging from 94.0% (NDMA) to 99.3% (NDPheA) with a precision as indicated by the relative standard deviations (SDs) within the range of 0.7% (NDPheA) and 2.6% (NDMA) were obtained. The detection limits were obtained using GC–MS based on S/N = 3 ranging from 0.3 pg/ml to 1.1 pg/ml. The classification results of the gastric juice samples in different patient groups are very satisfactory, allowing all patients to be correctly grouped. A new mathematical model has been developed, allowing the classification of gastric juices with a 93.1% success rate based on the MNPIZ-to-DNPIZ ratio, which might be considered as a biomarker for the classification of gastric juices of patients and might act as an indicator of an increased risk for GC.

Conventional tumor markers

In the gastric fluid, the high levels of CEA and CA 19-9 have been found to be correlated with precancerous lesions and GC [ 22, 23]. Among patients with GC, benign gastroduodenal diseases, and inguinal hernia and patients with no other pathologies, the mean gastric juice CA 19-9 and CEA levels are significantly higher than the serum levels. Gastric fluid CEA levels in GC patients are significantly higher than in patients with benign gastroduodenal diseases compared with the controls, whereas no significant differences are observed between patients with benign gastroduodenal diseases and controls. No significant correlation is observed between histopathological characteristics and gastric fluid CEA positivity, which does not show any prognostic value [ 22]. Muretto et al. [ 24] studied healthy controls and patients with suspected GC or epigastric distress/dyspepsia, asking participants to swallow a tiny gelatin capsule (length, 14 mm; diameter, 5 mm) that contains a pierced plastic cover and surrounds a piece of absorbent paper, leaving the capsule in the gastric cavity for 1 h to allow absorbent paper saturation with gastric juice, and a 45–50 cm long nylon thread that is connected to the inner capsule to remove the device from the gastric cavity was used. After processing the absorbent paper for radioimmunoassay, CA 19-9 and CEA levels are correlated to the upper endoscopy findings and biopsies of the gastric mucosa or suspected lesions. The endogastric capsule seldom causes adverse effects, and the mean levels of both markers increase according to the gastric lesion severity in cancer patients. Patients with precancerous lesions and cancers show significantly higher levels of CA 19-9 and CEA than those with normal findings or gastritis. The values may distinguish normal or minor pathological changes and precancerous lesions or cancers. Thus, the endogastric capsule is a simple and noninvasive tool for measuring gastric juice CA 19-9 and CEA levels and may represent a novel method for GC screening.

To compare peridistal gastrectomy (DG) gastric juice CA 19-9 [ 25] and CA 72-4 [ 26] levels and to reveal their potential significances, we selected pathologically diagnosed GC patients who underwent DG and collected their perioperative gastric juice samples whose CA 19-9 and CA 72-4 levels were detected. We found that both markers in the gastric juice before surgery were significantly higher among patients with advanced-stage diseases and significantly correlated with tumor TNM stage, regarding tumor size, invasion level of gastric wall, differentiated grade, and metastatic lymph node number as the significant influential factors. The levels in the gastric juice after operation were significantly correlated with the radical degree, regarded number of resected lymph nodes, and cutting edge classification as the influencing factors; the difference of gastric juice markers between pre- and post-resection was significantly correlated with tumor TNM stage, radical resection degree, regarded tumor size, numbers of metastatic and harvested lymph nodes, sum of distances from tumor to cutting edges, and cutting edge classification as the significant influential factors. Thus, peri-DG gastric juice CA 19-9 and CA 72-4 reveal much information about malignancies and radical gastrectomy and may suggest prognosis.

Amino acids

Early GC is largely asymptomatic and may easily be overlooked by conventional gastroscopy. Currently, no useful marker is observed for the early detection of GC, and biomarkers for identification are needed urgently. The concentrations of L-proline, L-glutamate, L-alanine, L-serine, and D-alanine in the gastric juice of early GC patients with Hp infection are significantly higher than the others. Except for D-alanine, no correlation was observed between the concentrations of D-amino acids and presence of cancer or Hp [ 27]. The levels of tyrosine (median, 19.4 vs. 3.8 mg/ml), phenylalanine (24.6 vs. 5.3 mg/ml), and tryptophan (8.3 vs. 1.0 mg/ml) in the gastric fluid are significantly higher in early GC patients than in patients with non-neoplastic gastric diseases. The high levels of aromatic amino acids in gastric juice are significantly associated with GC. Gastric juice tyrosine, phenylalanine, and tryptophan levels increase in the early phase of gastric carcinogenesis. Thus, tyrosine, phenylalanine, and tryptophan in the gastric juice could be used as biomarkers for early GC detection, which is an economical, efficient, and convenient method [ 28].

To develop a rapid high-performance liquid chromatography (HPLC) approach for the determination of tryptophan in the gastric juice and to help differentiate GC and benign gastric diseases, Lian et al. [ 29] performed HPLC on a restricted access material, choosing phosphate buffered solution (PBS, 90 mmol/L, pH 3.5)-acetonitrile (V/V, 80/20) as the mobile phase, conducted separation using a constant flow rate of 1.0 ml/min, and detected and measured fluorescence emission signal intensity of gastric juice samples from patients with GC and benign gastric diseases at 330 nm excited by ultraviolet light at 288 nm. A linear relationship in the range of 0.20–100 mg/L between the tryptophan concentration and fluorescence emission signal intensity was obtained, and the detection limit was 0.05 mg/L. The developed HPLC method based on strong cation-exchange restricted access columns for determining the gastric juice tryptophan concentration has excellent stability and precision and is compatible with the gastric juice analysis with the potential to screen GC.

Proteomics

The overall GC survival remains poor owing to the absence of a reliable approach that identifies highly curable early-stage diseases. Gastric fluid is a GC protein marker source. The multiprotein profiling of gastric juices obtained from the pathologic anatomic site could reveal diagnostic proteomic fingerprints. The proteomic analyses of gastroduodenal juices offer an alternative strategy in studying diseases, such as peptic ulcers and GC. The protein contents of gastric juice in patients with gastric ulcer (1.06 mg/ml) and GC (2.61 mg/ml) are significantly higher than in healthy subjects (0.48 mg/ml), and patients with duodenal ulcer (0.26 mg/ml) have significantly lower protein levels of gastric juice than in healthy subjects (0.48 mg/ml). Old age and gastric hypoacidity are independent factors that affect the protein contents of gastric juice with ORs of 32.9 and 3.2, respectively. The electrophoresis images of gastric juice could be classified into three patterns (basic, specific, and nonspecific bands). A thorough proteomic analysis reveals that a1-antitrypsin precursor is the principal peptide in the specific band pattern and is highly expressed in advanced and early GCs, significantly correlating the a1-antrypsin precursor with gastric hypoacidity and indicating that gastric juice a1-antitrypsin precursor is a novel valid protein biomarker of GC and ulcers [ 30]. A noninvasive method that obtains gastric juice, followed by proteomic analysis, may be a novel approach to screen GC. However, this method does not contribute to the research advancement that aims to discover the possible protein origin.

In healthy individuals, the digestive enzymes pepsin A/B and gastric lipase are the main proteins detected by 2D gel electrophoresis and found in 40% of GC cases. Interestingly, a significant amount of a1-antitrypsin is observed in the cases. By contrast to GC cases (60%), a1-antitrypsin is detected in only 5% of patients with chronic atrophic gastritis (CAG), and the detection frequency increases as the disease develops. The a1-antitrypsin concentration of gastric juice is significantly higher in GC patients than in healthy controls and patients with digestive ulcer. The AUC for identifying GC patients is 0.96 (95% confidence interval, 0.93–0.99). The sensitivity and specificity of a1-antitrypsin concentration in gastric juice are 96% and 92%, respectively. The a1-antitrypsin assay of gastric juice through string test is validated in patients for GC screening [ 31]. Thus, a noninvasive a1-antitrypsin string test may serve as a novel screening tool for identifying GC cases. Zymography shows that a 60 kDa protease is significantly associated with a1-antitrypsin, and MS analysis reveals that the gastric a1-antitrypsin has a protease-cleaved form. Thus, a1-antitrypsin and 60 kDa protease in gastric juice may be satisfactory diagnostic and prognostic markers of GC-associated conditions [ 32].

A 2D analysis of gastric juices reveals discrepant protein profiles for neutral and acidic samples, highlighting pH effects on protein compositions. Via the 2D LC–MS/MS analysis of pooled samples, a total of 284 and 347 proteins are identified in acidic and neutral samples, respectively (FDR≤1%), of which 265 proteins (72.4%) are overlapped. However, most of the proteins in acidic samples are identified from peptides in the filtrate (<3 kDa). Consistent with this result, in acidic samples, the immunoblotting analysis of six potential GC biomarkers seldom detects full-length proteins [ 33]. The majority of GC patients have neutral gastric fluid compared with noncancer controls. Therefore, sample stratification, proteomic approach, and validation strategy can significantly affect the biomarker finding interpretations. Three precipitation techniques (ethanol, acetone, and trichloroacetic acid) could all successfully extract proteins from gastroduodenal juices with acetone, resulting in excellent resolution and minimal protein degradation compared with other approaches. A total of 134 different proteins are detected in the in-gel tryptic digestion, followed by the LC–MS/MS analysis of endoscopic pancreatic function test (ePFT)-collected gastroduodenal fluid samples, and 67 proteins are identified in at least two of the three samples. Gene ontology analysis classifies these proteins into peptidases and extracellularly localized proteins [ 34]. Thus, ePFT, followed by acetone precipitation and coupled with LC–MS/MS, could be used to safely collect gastroduodenal juices from the upper gastrointestinal tract for MS-based proteomic analyses.

Kon et al. [ 35] generated protein profiles from the gastric juice samples of GC and gastritis patients using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (TOF-MS) on multiple protein chip arrays, comparing proteomic characteristics by the significant analyses of the microarray algorithm and two-way hierarchical clustering. By the significance analysis of microarrays, a total of 60 proteomic features were significantly upregulated, and 46 proteomic features were downregulated in GC samples. The multimarker clustering showed two discrepant proteomic profiles that are independent of ethnicity and age, and about 95% of tumor samples were clustered together. Selected peptide sequencing has revealed that pepsinogen C and pepsin A activation peptide were significantly downregulated, and a-defensin was significantly upregulated. Thus, the simple and reproducible multimarker proteomic assays could supplement the clinical gastroscopic evaluation of symptomatic patients that improve the diagnostic accuracy of GC and premalignant lesions. Chang et al. [ 36] investigated peptide biomarkers from gastric juice samples using MALDI-TOF-MS. This experiment revealed the clear pattern discrepancies of mass spectra among samples from healthy controls and patients with different gastric diseases, and five peptides with high sensitivity and specificity were found in GC patients. The sequences of these five peptides, including two pepsinogen fragments, albumin fragment, leucine zipper protein fragment, and a1-antitrypsin fragment, were identified by MS analyses and immunodepletion assays.

microRNAs

microRNAs (miRNAs and miRs) play important roles in GC genesis and progression, and conventional serological tests for GC screening have limited sensitivity and specificity. Several serum miRNAs have been used as biomarkers for GC; however, most of them are also found in several cancer types. GC patients have significantly different levels of gastric juice miR-106a and miR-21 than in patients with benign gastric diseases, and significant correlations were found between miR-106a/miR-21 levels and Borrmann tumor types. miR-21 levels in intestinal-type GC are significantly higher than in the diffuse- or mixed-type GC. The AUC is up to 0.969 for miR-21 [ 37]. Thus, both miRNAs in the gastric fluid are potential biomarkers of GC screening. miR-129-1/2 is aberrantly expressed in GC. GC patients have significantly lower levels of miR-129-1/2-3p in gastric juices than in patients with benign gastric diseases, suggesting that miR-129-1/2-3p in gastric juices may be potential biomarkers of GC screening. Moreover, the miRNA detection of gastric juice may be a convenient noninvasive approach for GC diagnosis [ 38]. miR-421 plays important roles during gastric tumorigenesis and is a potential tumor marker in cancer diagnosis. The miR-421 levels in gastric juice are significantly different in GC patients than in patients with benign gastric diseases. However, the miR-421 levels in gastric juice are not significantly associated with the main clinicopathological features, such as tumor size and Lauren’s and Borrmann’s tumor classifications. For early GC detection, the use of gastric juice miR-421 shows a marked improvement compared with the use of serum CEA alone [ 39]. Thus, miR-421 in gastric juice may also be a useful biomarker for GC screening.

Long noncoding RNAs

Long noncoding RNAs (lncRNAs) are implicated in GC. The levels of AA174084 in gastric juice from GC patients are significantly higher than in patients with normal mucosa, minimal gastritis, gastric ulcers, and atrophic gastritis, suggesting that AA174084 may be a potential marker for early GC diagnosis [ 40]. The levels of ABHD11-AS1 in gastric juice from GC patients are significantly higher than in patients with normal mucosa or minimal gastritis, atrophic gastritis, and gastric ulcers. Moreover, higher ABHD11-AS1 levels are also associated with Lauren classification, tumor size, tumor stage, and serum CEA levels, indicating that the levels of ABHD11-AS1 in gastric juice may be a potential biomarker for GC screening [ 41].

Vitamin C

Hp infection could increase nitrite and decrease vitamin C levels in gastric fluid. In the presence of Hp infection, the low levels of total vitamin C in gastric juice, which plays a role in the neutralization of various pathogenic factors associated with Hp infection, including the destruction of free radicals that damage tissue and cell DNA, and the inhibition of the formation of the strongly carcinogenic N-nitroso compounds, which are carcinogens formed from nitrite, may play a role in gastric carcinogenesis. Therefore, the susceptibility of Hp-infected patients to GC may be caused by the increased levels of N-nitroso compounds in gastric juice. However, most Hp-infected patients do not develop GC. Dabrowska-Ufniarz et al. [ 42] classified Hp-infected patients into four groups based on the results of histological examinations: group I (control), patients with normal gastric mucosa; group II, patients with chronic gastritis; group III, patients with metaplasia; and group IV, GC patients. In addition, the mean vitamin C concentration in gastric juice was 18.2 mg/ml. In group II, the mean concentration was 6.3 mg/ml. In group III, the mean concentration was 3.9 mg/ml, and in group IV 3.2 mg/ml. Statistically significant differences of vitamin C concentration were found among group I and groups II, III, and IV and among groups II and III and IV. No differences of vitamin C concentrations in gastric juice were found between patients with metaplasia and GC. These results indicate that the low levels of vitamin C in gastric juice might play a role in the earlier stages of tumorigenesis. pH and nitrite levels increase, and vitamin C levels decrease in the gastric juices of patients with atrophic gastritis and GC compared with other patients. However, in patients with a similar gastric acidity (pH 5–8), the nitrite concentrations in gastric juices are significantly higher, and vitamin C levels are lower in patients with GC than in patients with atrophic gastritis [ 43]. Thus, although hypochlorhydria increases intraluminal nitrite and decreases intraluminal vitamin C levels, which increases the intraluminal N-nitroso compound formation, GC patients may have additional factors that enhance the alterations.

Others

Telomerase is a ribonucleoprotein polymerase that is essential for cell immortality. A high percentage of GC tissue expresses telomerase. In gastric lavage fluid, telomerase is detected in 80% of GC patients and 28% of patients with peptic ulcer. However, it is not found in normal controls with significant differences [ 44]. Thus, the presence of telomerase activity in gastric lavage fluid of patients with GC compared with those without might represent a new method for GC diagnosis. A pilot study showed the feasibility of an endogastric capsule as a valuable tool for DNA collection from exfoliated cells in gastric juice from the gastric mucosa and a significantly different E-cadherin (CDH1) promoter hypermethylation status between diffused GC patients and controls [ 45]. The capsule may represent a novel noninvasive tool for the analysis of a specific epigenetic change in diffused GC patients and may be a cost-effective approach for the early detection of sporadic and hereditary diffused GC in CDH1 germ-line mutation carriers.

Combination tests of different category markers

The pepsinogens I and I/II ratios of gastric juice are more accurate than that in the serum. The combination test using the gastric pepsinogen I/II ratio and melanoma-associated gene (MAGE, A1 to A6) is the most accurate. The sensitivity for stage I GC is not affected by tumor location or pathologic type [ 46]. Thus, the combination test is another potential tool for GC detection. Type IV collagen and hyaluronic acid (HA) are the main components of the basement membrane and extracellular matrix (ECM), respectively. The contents of gastric juice type IV collagen and HA are significantly higher in patients with GC than those with precancerous lesions and controls. GC patients with lymph node metastasis have a significantly higher level of gastric juice type IV collagen and HA than those without metastasis [ 47]. Thus, the analysis of gastric juice type IV collagen and HA may be a simple method for diagnosing GC and evaluating metastasis.

Gastric secretion may provide valuable information, particularly when Hp infection results in CAG/intestinal metaplasia (IM) preceding carcinoma. Prostaglandin E2 (PGE2) and peptide growth factors (epidermal growth factor [EGF] and transforming growth factor a [TGF-a]) are harbingers of injury and repair. In adenocarcinoma patients, gastric juice PGE2 increases fivefold than the controls, and PGE2 increases almost threefold than the CAG patients. The EGF levels in adenocarcinoma patients are fourfold higher than the controls, and EGF level in adenocarcinoma patients is almost threefold higher than the CAG patients. In patients with CAG/IM, the EGF levels are also nearly three times higher than the controls. The TGF-a levels in adenocarcinoma patients are half the value of the controls and CAG patients. In patients with CAG/IM, the levels are as low as 1/5 of the controls or CAG patients [ 48]. Thus, testing the gastric juice levels of PGE2, EGF, and TGF-a in patients with endoscopy- and biopsy-proven CAG may be helpful in the follow-up of patients whose conditions may potentially progress to IM and ultimately adenocarcinoma, which could be considered as an adjunct to histological assessment particularly given that the best surveillance regimens of biopsy specimens are inherited with sampling errors.

Nitrite and nitrate levels in gastric fluids of Hp-positive patients with gastritis and ulcers are higher than Hp-negative patients, and the concentrations of total nitrite and nitrate and proinflammatory cytokines (tumor necrosis factor [TNF]-a and interleukin [IL]-2R, IL-6, and IL-8) in the gastric fluids of Hp-positive GC patients are higher than Hp-negative controls. IL-1b level is significantly increased in the gastric fluid of infected GC patients. However, IL-1b level is not increased in the serum [ 49]. Thus, an increase in cytokine-induced NO combination in the gastric mucosa is not restricted to the locally infected gastric tissue and could also be detected in the gastric fluid of Hp-positive subjects. Moreover, the increase in cytokine-induced NO combination might play an important role in the pathogenesis and development of common gastric diseases.

The detection methods that use gastrointestinal juice in predicting the side effects of postgastrectomy

The detection methods that use gastrointestinal juice in predicting the side effects of postgastrectomy are illustrated in Fig. 2.

Pancreatic leakage

Pancreatic leakage is a serious postgastrectomy adverse effect. Based on the data from 24 072 patients, the mean amylase and lipase values of the group with complications of pancreatic leakage are significantly higher than in patients with no pancreatic leakage-associated complications in the drainage fluid. No statistically significant difference was observed in the mean volume of the drained fluid through the tube between the two groups on postoperative day (POD) 1. However, a significant correlation was observed between the lipase and amylase values in the drainage fluid (r = 0.812) [ 50]. Among postoperative amylase and lipase values in the drainage fluid and the volume of drainage fluid, the amylase level is remarkably differentiated between patients with and without pancreatic leakage than other biomarkers.

Pancreatic fistula

Pancreatic fistula is one of the most serious and important potential adverse effects after D2/3 total and distal gastrectomy (4% to 6%), although its incidence has decreased. The management of postgastrectomy drainage tube is critical, not only for anastomotic leaks but also for the diagnosis and management of pancreatic fistula. High amylase drainage content and the presence of pancreatic fistula may be caused by several reasons: the operation itself, including splenectomy or the resection of both the pancreatic tail and spleen, and the extended lymphadenectomy. However, even the “gentle and soft” pancreatic manipulation may be a risk factor. The amylase concentration in the drainage fluid after GC operation can be considered a predicator of pancreatic fistula. The measurement of drainage fluid amylase level is often performed for the detection of pancreatic fistula formation after total gastrectomy. Pancreatic fistula formation can be detected by changes in drainage fluid properties. Amylase drainage content>3 times the serum amylase is an important risk factor for the prediction of pancreatic fistula [ 51]. The incidence of pancreatic fistula in patients with dark-red drainage fluid is significantly higher than those with light-red fluid, and the diagnosis sensitivity and specificity by fluid properties are 100.0% and 77.7%, respectively. Pancreatic fistula formation also correlates with drainage amylase levels with diagnosis sensitivity and specificity by a drainage amylase level of 5000 U of 100.0% and 82.2%, respectively. No differences in the above-mentioned parameters are observed between the two diagnostic methods [ 52]. Thus, drainage fluid inspection can provide an accurate diagnosis of pancreatic fistula formation, similar to drainage amylase measurement, indicating that routine drainage amylase measurement is probably not necessary in every patient.

Abscess

The insertion of drainage tubes at GC operation can be useful for the prediction and management of postoperative adverse effects. However, drainages should be removed no sooner than they are deemed unnecessary. The amylase concentration in the drainage fluid following total gastrectomy for GC is an important risk factor for surgical adverse events. Amylase concentration is significantly higher in patients who underwent splenectomy, pancreatosplenectomy, proximal/total gastrectomy, and extended lymphadenectomy and who eventually developed intra-abdominal abscess. An amylase concentration of≥1000 IU/L on POD 1 is an independent risk factor for intra-abdominal abscess in the pancreas. With a negative predictive value of 97.7%, complications in the pancreas are highly unlikely when the amylase concentration is less than 1000 IU/L, and the early removal of the drainage tube could be recommended for these patients [ 53]. The rate of positive bacterial cultures in the drainage fluid (D-cultures) is 6.4% on POD 1. According to a univariate analysis, the drain amylase level and D-culture status on POD 1 are significantly associated with the incidence of intra-abdominal abscesses. In addition, a multivariate analysis demonstrates that D-culture positivity on POD 1 is the only significantly independent predictor of intra-abdominal abscess [ 54]. Thus, the bacterial culture positivity of drainage fluid during the early postoperative period has a significant effect on the development of intra-abdominal abscesses after gastrectomy.

Conclusions

Gastric juice is a digestive fluid that is in direct contact with stomach tissue, and the stomach receives fluids from the lower intestinal tract. The contents inside are mostly relieved by the lesions without going through metabolism and are rarely destroyed in the neutral gastric luminal environment exiting in most of the GC patients, making them ideal biomarkers with satisfactory sensitivity and specificity (Fig. 3 and Table 1). However, notably, previous studies revealed varying sample sizes and study quality, which might affect the robustness of the results. Furthermore, the current investigations on the gastric juice mainly focus on disease diagnosis with prognostic significance that is seldom reported. The gastric fluid markers should be further investigated for the early detection of malignancies. However, the methods used in obtaining gastric juice samples with minimum invasion should be comprehensively studied. Active and multicenter prospective trials with strict inclusion and exclusion criteria and a sufficient follow-up period and confounding factors that are appropriately controlled should be the main focus to guarantee the reality and reliability of the results and to obtain significant clinical significances. Meanwhile, novel markers and detection methods should be further explored on the basis of international collaboration and communication to achieve a consensus on none or minimally invasive, accurate, convenient, and economical diagnostic methods for digestive diseases via gastric fluid examination.

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