Non-invasive Detection Of Gastric Cancer Research Articles

Urinary Biomarkers for the Noninvasive Detection of Gastric Cancer.

Gastric cancer (GC) is associated with high morbidity and mortality rates. Thus, early diagnosis is important to improve disease prognosis. Endoscopic assessment represents the most reliable imaging method for GC diagnosis; however, it is semi-invasive and costly and heavily depends on the skills of the endoscopist, which limit its clinical applicability. Therefore, the search for new sensitive biomarkers for the early detection of GC using noninvasive sampling collection methods has attracted much attention among scientists. Urine is considered an ideal biofluid, as it is readily accessible, less complex, and relatively stable than plasma and serum. Over the years, substantial progress has been made in screening for potential urinary biomarkers for GC. This review explores the possible applications and limitations of urinary biomarkers in GC detection and diagnosis.

The Reprimo-Like Gene Is an Epigenetic-Mediated Tumor Suppressor and a Candidate Biomarker for the Non-Invasive Detection of Gastric Cancer.

Reprimo-like (RPRML) is an uncharacterized member of the Reprimo gene family. Here, we evaluated the role of RPRML and whether its regulation by DNA methylation is a potential non-invasive biomarker of gastric cancer. RPRML expression was evaluated by immunohistochemistry in 90 patients with gastric cancer and associated with clinicopathologic characteristics and outcomes. The role of RPRML in cancer biology was investigated in vitro, through RPRML ectopic overexpression. Functional experiments included colony formation, soft agar, MTS, and Ki67 immunofluorescence assays. DNA methylation-mediated silencing was evaluated by the 5-azacytidine assay and direct bisulfite sequencing. Non-invasive detection of circulating methylated RPRML DNA was assessed in 25 gastric cancer cases and 25 age- and sex-balanced cancer-free controls by the MethyLight assay. Downregulation of RPRML protein expression was associated with poor overall survival in advanced gastric cancer. RPRML overexpression significantly inhibited clonogenic capacity, anchorage-independent growth, and proliferation in vitro. Circulating methylated RPRML DNA distinguished patients with gastric cancer from controls with an area under the curve of 0.726. The in vitro overexpression results and the poor patient survival associated with lower RPRML levels suggest that RPRML plays a tumor-suppressive role in the stomach. Circulating methylated RPRML DNA may serve as a biomarker for the non-invasive detection of gastric cancer.

Abstract 2437: Clinical, biological and translational significance in gastric cancer of reprimo-like gene, an uncharacterized member of the reprimo gene family

Abstract Background: Gastric cancer (GC) is one of the leading causes of cancer death. Elucidating the molecular bases of GC might contribute to develop opportune diagnostic strategies. Reprimo-like (RPRML) is a poorly characterized member of the Reprimo gene family. The founding member of this family, Reprimo, is reported to be a putative tumor suppresser gene and candidate biomarker for GC diagnosis. We have recently reported the downregulation of RPRML expression in GC tissues compared to paired normal adjacent tissues, and that in vitro expression is regulated by DNA methylation. We hypothesize that RPRML promoter methylation may act as a non-invasive biomarker for GC. Methods: RPRML expression was evaluated by immunohistochemistry (IHC) in 91 GC cases from The Gastric Cancer Task Force (NCT03158571). Two pathologists scored staining by multiplying the proportion and the intensity of stained cells (range 0-3). Cox proportional hazard ratio was applied to determine an association between RPRML IHC score and global survival, after adjusting by stage. In addition, we investigated the effect of RPRML overexpression in GC cell behavior in vitro through the stable transfection and sorting of RPRML coupled to GFP or GFP alone in the AGS cell line. Functional assays included Ki67 immunofluorescence, colony formation, soft agar, transwell migration and wound healing assays. All experiments were performed in biological triplicate and Kruskal-Wallis statistical analysis applied. The Methylight assay was used to quantify methylated RPRML DNA copies in plasma samples from 25 GC cases (tumor bank repository BTHCUCH) and 25 healthy donors under an endoscopic surveillance program (PREVECAN). ROC curve analysis was performed to determine the cut-off value for the highest sensitivity and specificity. Results: Among 91 GC cases, RPRML expression showed a median IHC score of 0.024 (range 0-2.45). By using the median as a cut-off point, low RPRML IHC score was associated with poor overall survival of stage III and IV GC patients (HR=2.13, 95%CI:1.1-4.1, p=0.02). In vitro overexpression of RPRML inhibited cell proliferation (p=0.04) and resulted in reduced clonogenic capacity (p=0.001) and anchorage independent growth (p≤0.0001). Furthermore, RPRML overexpression retarded migration and wound healing in AGS cells. Methylation of RPRML DNA in plasma samples discriminated between GC patients and controls with an AUC of 0.66. Utilizing a cut-off value of 0.15 copies/ul, sensitivity was 56% (95%CI: 34.9 - 75.6) and specificity 72% (95%CI: 50.6 - 87.9). Conclusion: RPRML downregulation associated with poor survival of advanced stage GC patients. In accordance, ectopic overexpression of RPRML in vitro inhibited biological characteristics associated with tumor progression, suggesting that RPRML may have a tumor suppressor role in GC. Future analysis will determine if the methylation of RPRML is a candidate marker for the non-invasive detection of GC. Grants: CONICYT-FONDAP 15130011 and Fondecyt 1191928 Citation Format: Maria Alejandra Alarcon, Wilda Olivares, Andres Rodriguez, Maria Jose Maturana, Rocío Bustos, Iva Polakovicova, Miguel Cordova-Delgado, Matías Muñoz-Medel, Marcelo Garrido, Gareth I. Owen, Tomas De Mayo, Gonzalo Carrasco-Avino, Julio Amigo, Arnoldo Riquelme, Robinson Gonzalez, Carlos Barrientos, Edmundo Aravena, Franz Villarroel-Espíndola, Alejandro H. Corvalan. Clinical, biological and translational significance in gastric cancer of reprimo-like gene, an uncharacterized member of the reprimo gene family [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2437.

Gold@Silica Nanoparticles Functionalized with Oligonucleotides: A Prominent Tool for the Detection of the Methylated Reprimo Gene in Gastric Cancer by Dynamic Light Scattering.

Reprimo (RPRM) is a tumor suppressor gene involved in the development of gastric cancer. Hypermethylation of the RPRM promoter region has been found in tumor tissue and plasma samples from patients with gastric cancer. These findings suggest that circulating methylated DNA of RPRM could be a candidate for a noninvasive detection of gastric cancer. We designed a nanosystem based on the functionalization of silica coated gold nanoparticles with oligonucleotides that recognize a specific DNA fragment of the RPRM promoter region. The functionality of the oligonucleotide on the surface of the nanoparticle was confirmed by polymerase chain reaction (PCR). The nanoparticles were incubated with a synthetic DNA fragment of methylated DNA of RPRM and changes in the size distribution after hybridization were evaluated by dynamic light scattering (DLS). A difference in the size distribution of nanoparticles hybridized with genomic DNA from the KATO III gastric cancer cell line was observed when was compared with DNA from the GES-1 normal cell line. These results showed that this nanosystem may be a useful tool for the specific and sensitive detection of methylated DNA of RPRM in patients at risk of developing gastric cancer.

A novel urinary microRNA biomarker panel for detecting gastric cancer.

Gastric cancer (GC) is one of the most common causes of cancer deaths worldwide; however, reliable and non-invasive screening methods for GC are not established. Therefore, we conducted this study to develop a biomarker for GC detection, consisting of urinary microRNAs (miRNAs). We matched 306 participants by age and sex [153 pairs consisting of patients with GC and healthy controls (HCs)], then randomly divided them across three groups: (1) the discovery cohort (4 pairs); (2) the training cohort (95 pairs); and (3) the validation cohort (54 pairs). There were 22 urinary miRNAs with significantly aberrant expressions between the two groups in the discovery cohort. Upon multivariate analysis of the training cohort, urinary expression levels of miR-6807-5p and miR-6856-5p were significantly independent biomarkers for diagnosis of GC, in addition to Helicobacter pylori (H. pylori) status. A diagnostic panel that combined these 2 miRNAs and H. pylori status distinguished between HC and GC samples with an area under the curve (AUC) = 0.736. In the validation cohort, urinary miR-6807-5p and miR-6856-5p showed significantly higher expression levels in the GC group, and the combination biomarker panel of miR-6807-5p, miR-6856-5p, and H. pylori status also showed excellent performance (AUC = 0.885). In addition, this biomarker panel could distinguish between HC and stage I GC patients with an AUC = 0.748. Urinary expression levels of miR-6807-5p and miR-6856-5p significantly decreased to undetectable level after curative resection of GC. This novel biomarker panel enables early and non-invasive detection of GC.

Discovery and Validation of Salivary Extracellular RNA Biomarkers for Noninvasive Detection of Gastric Cancer.

Biomarkers are needed for noninvasive early detection of gastric cancer (GC). We investigated salivary extracellular RNA (exRNA) biomarkers as potential clinical evaluation tools for GC. Unstimulated whole saliva samples were prospectively collected from 294 individuals (163 GC and 131 non-GC patients) who underwent endoscopic evaluation at the Samsung Medical Center in Korea. Salivary transcriptomes of 63 GC and 31 non-GC patients were profiled, and mRNA biomarker candidates were verified with reverse transcription quantitative real-time PCR (RT-qPCR). In parallel, microRNA (miRNA) biomarkers were profiled and verified with saliva samples from 10 GC and 10 non-GC patients. Candidate biomarkers were validated with RT-qPCR in an independent cohort of 100/100 saliva samples from GC and non-GC patients. Validated individual markers were configured into a best performance panel. We identified 30 mRNA and 15 miRNA candidates whose expression pattern associated with the presence of GC. Among them, 12 mRNA and 6 miRNA candidates were verified with the discovery cohort by RT-qPCR and further validated with the independent cohort (n = 200). The configured biomarker panel consisted of 3 mRNAs (SPINK7, PPL, and SEMA4B) and 2 miRNAs (MIR140-5p and MIR301a), which were all significantly down-regulated in the GC group, and yielded an area under the ROC curve (AUC) of 0.81 (95% CI, 0.72-0.89). When combined with demographic factors, the AUC of the biomarker panel reached 0.87 (95% CI, 0.80-0.93). We have discovered and validated a panel of salivary exRNA biomarkers with credible clinical performance for the detection of GC. Our study demonstrates the potential utility of salivary exRNA biomarkers in screening and risk assessment for GC.

1177TiP - Early non-invasive detection of gastric cancer with plasma pepsinogens in Croatian patients

1177TiP - Early non-invasive detection of gastric cancer with plasma pepsinogens in Croatian patients

Noninvasive detection of gastric cancer.

Gastric cancer (GC) is the fifth most common cancer and the third common cause of cancer death worldwide. Endoscopy is the most effective method for GC screening, but its application is limited by the invasion. Therefore, continuous efforts have been made to develop noninvasive methods for GC detection and promising results have been reported. Here, we review the advances in GC detection by protein and nucleic acid tumor markers, circulating tumor cells, and tumor-associated autoantibodies in peripheral blood. Some potential new noninvasive methods for GC detection are also reviewed, including exhaled breath analysis, blood spectroscopy analysis and molecular imaging.

Differential Proteomic Analysis of Human Saliva using Tandem Mass Tags Quantification for Gastric Cancer Detection.

Novel biomarkers and non-invasive diagnostic methods are urgently needed for the screening of gastric cancer to reduce its high mortality. We employed quantitative proteomics approach to develop discriminatory biomarker signatures from human saliva for the detection of gastric cancer. Salivary proteins were analyzed and compared between gastric cancer patients and matched control subjects by using tandem mass tags (TMT) technology. More than 500 proteins were identified with quantification, and 48 of them showed significant difference expression (p < 0.05) between normal controls and gastric cancer patients, including 7 up-regulated proteins and 41 down-regulated proteins. Five proteins were selected for initial verification by ELISA and three were successfully verified, namely cystatin B (CSTB), triosephosphate isomerase (TPI1), and deleted in malignant brain tumors 1 protein (DMBT1). All three proteins could differentiate gastric cancer patients from normal control subjects, dramatically (p < 0.05). The combination of these three biomarkers could reach 85% sensitivity and 80% specificity for the detection of gastric cancer with accuracy of 0.93. This study provides the proof of concept of salivary biomarkers for the non-invasive detection of gastric cancer. It is highly encouraging to turn these biomarkers into an applicable clinical test after large scale validation.

Detection of OSR2, VAV3, and PPFIA3 Methylation in the Serum of Patients with Gastric Cancer.

Aim. This study was to evaluate the diagnostic value of OSR2, VAV3, and PPFIA3 hypermethylation in gastric cancer (GC) patients. Patients and Methods. By using methylation-specific polymerase chain reaction (MSP), we detected the methylation status in tissue and serum samples from 48 gastric cancer (GC) patients and 25 normal individuals. Results. We found that OSR2, VAV3, and PPFIA3 were methylated in 70.8% (34/48), 54.2% (26/48), and 60.4% (29/48) of GC tissue, respectively. On the contrary, those genes were barely methylated in their paired paracancerous histological normal tissues (PCHNTs) (all P values < 0.01). We next analyzed the methylated OSR2, VAV3, and PPFIA3 in serum DNA. Compared with 25 normal individuals, those three genes were significantly hypermethylated in GC patients serum samples (all P values < 0.01). Regarding their diagnostic value in serum samples, the combined sensitivity of at least one positive among the three markers in serum was 83.3%, with a specificity of 88%. Conclusion. Our test suggested that methylation of OSR2, VAV3, and PPFIA3 genes in serum sample may offer a good alternative in a simple, promising, and noninvasive detection of GC.

Loss of Expression of Reprimo, a p53-induced Cell Cycle Arrest Gene, Correlates with Invasive Stage of Tumor Progression and p73 Expression in Gastric Cancer.

Reprimo (RPRM), a downstream effector of p53-induced cell cycle arrest at G2/M, has been proposed as a putative tumor suppressor gene (TSG) and as a potential biomarker for non-invasive detection of gastric cancer (GC). The aim of this study was to evaluate the epigenetic silencing of RPRM gene by promoter methylation and its tumor suppressor function in GC cell lines. Furthermore, clinical significance of RPRM protein product and its association with p53/p73 tumor suppressor protein family was explored. Epigenetic silencing of RPRM gene by promoter methylation was evaluated in four GC cell lines. Protein expression of RPRM was evaluated in 20 tumor and non-tumor matched cases. The clinical significance of RPRM association with p53/p73 tumor suppressor protein family was assessed in 114 GC cases. Tumor suppressor function was examined through functional assays. RPRM gene expression was negatively correlated with promoter methylation (Spearman rank r = -1; p = 0.042). RPRM overexpression inhibited colony formation and anchorage-independent growth. In clinical samples, RPRM gene protein expression was detected in 75% (15/20) of non-tumor adjacent mucosa, but only in 25% (5/20) of gastric tumor tissues (p = 0.001). Clinicopathological correlations of loss of RPRM expression were significantly associated with invasive stage of GC (stage I to II-IV, p = 0.02) and a positive association between RPRM and p73 gene protein product expression was found (p<0.0001 and kappa value = 0.363). In conclusion, epigenetic silencing of RPRM gene by promoter methylation is associated with loss of RPRM expression. Functional assays suggest that RPRM behaves as a TSG. Loss of expression of RPRM gene protein product is associated with the invasive stage of GC. Positive association between RPRM and p73 expression suggest that other members of the p53 gene family may participate in the regulation of RPRM expression.

Sa1897 Novel Evidence for Interleukin-8 (IL-8) As a Predictive Biomarker of Response to 5fluorouracil Based Chemotherapy in Colorectal Cancer

BACKGROUND. Early and non-invasive detection of gastric cancer (GC) is remaining a challenge in many high-risk areas. OBJECTIVES. To investigate the feasibility of gastric cancer and peptic ulcer disease detection by measuring volatile organic compounds (VOCs) in the exhaled breath by a nanomaterial-based sensor technology. METHODS. Alveolar exhaled breath samples were collected from 99 GC patients, 53 peptic ulcer disease (PUD) patients and 342 controls in Latvia (Caucasian population) having been investigated by upper endoscopy. Nanomaterial-based sensors Gold nano particles (GNP) and single-wall carbon nanotube (SWCNT) were used to discriminate the GC and the PUD groups from the healthy controls by loading discriminant factor analysis (DFA) pattern recognition. Classification success was calculated by (i) building an algorithm for 70% of the samples as a training set and (ii) randomly blinding 30% of the samples as a validation set. RESULTS. The blind DFA models showed: (i) An excellent discrimination between the GC patients and controls (91% accuracy); (ii) An excellent discrimination between the GC patients and PUD patients (86% accuracy); (iii) Good discrimination between the PUD patients and controls (80% accuracy). CONCLUSIONS: The obtained results are demonstrating the good potential of VOC detection in exhaled breath by nanomaterial-based sensor technology in diagnosing GC and PUD. Performance characteristics of nanomaterial-based sensor technology to detect gastric cancer and peptic ulcer disease

Sa1896 Nanomaterial-Based Sensor Technology Can Detect Gastric Cancer and Peptic Ulcer Disease With a High Accuracy From an Exhaled Air Sample

BACKGROUND. Early and non-invasive detection of gastric cancer (GC) is remaining a challenge in many high-risk areas. OBJECTIVES. To investigate the feasibility of gastric cancer and peptic ulcer disease detection by measuring volatile organic compounds (VOCs) in the exhaled breath by a nanomaterial-based sensor technology. METHODS. Alveolar exhaled breath samples were collected from 99 GC patients, 53 peptic ulcer disease (PUD) patients and 342 controls in Latvia (Caucasian population) having been investigated by upper endoscopy. Nanomaterial-based sensors Gold nano particles (GNP) and single-wall carbon nanotube (SWCNT) were used to discriminate the GC and the PUD groups from the healthy controls by loading discriminant factor analysis (DFA) pattern recognition. Classification success was calculated by (i) building an algorithm for 70% of the samples as a training set and (ii) randomly blinding 30% of the samples as a validation set. RESULTS. The blind DFA models showed: (i) An excellent discrimination between the GC patients and controls (91% accuracy); (ii) An excellent discrimination between the GC patients and PUD patients (86% accuracy); (iii) Good discrimination between the PUD patients and controls (80% accuracy). CONCLUSIONS: The obtained results are demonstrating the good potential of VOC detection in exhaled breath by nanomaterial-based sensor technology in diagnosing GC and PUD. Performance characteristics of nanomaterial-based sensor technology to detect gastric cancer and peptic ulcer disease

Screening and Triage Test for Early Detection of Gastric Cancer (STEAD-GC): A noninvasive trial for early detection of gastric cancer.

17 Background: Gastric cancer (GC) is the second leading cause of cancer-related deaths worldwide. Previously, we identified a potential biomarker for non-invasive detection of GC, the DNA methylation of the promoter region of Reprimo, a p53-dependent G2 arrest mediator candidate (Clin Cancer Res 2008;14:6264-9). Furthermore, we developed a quantitative assay (MethyLight) for a mass screening of GC (DDW2011-1029128). Here we reported the preliminary findings of our ongoing prospective trial STEAD-GC (Screening and Triage test for Early Detection of Gastric Cancer) which is being conducted in Chile, a country with a high mortality rate for GC. Methods: Twenty GC cases (tumor, non-tumor tissues and plasma samples) and 41 symptomatic chronic gastritis cases (29 tissues and 12 pairs of tissue and plasma samples) were evaluated for Reprimo levels by MethyLight after DNA extraction and bisulfite conversion. Results: Concentrations of DNA were similar in both groups (Avg 32.2 ng/ml [range: 8.9-70.8 ng/ml]). The average DNA levels of Reprimo were higher in GC [964,215 copies/ml, 539,593 copies/ml and 80,113 copies/ml in tumor, non-tumor and plasma, respectively] but lower in symptomatic chronic gastritis [137,721 copies/ml and 8,387 copies/ml, tissue and plasma, respectively]. Methods: Twenty GC cases (tumor, non-tumor tissues, and plasma samples) and 41 symptomatic chronic gastritis cases (29 tissues and 12 pairs of tissue and plasma samples) were evaluated for Reprimo levels by MethyLight after DNA extraction and bisulfite conversion. Conclusions: By using our previous cut-off of 15,125 copies/ml, our method correctly identified 10 out of 12 gastritis cases and 16 out of 20 GC cases (p value &lt;0.001). Our data confirms our non-invasive method for early detection of GC may be suitable for a mass screening of GC.

S1928 Reprimo: A Novel Non-Invasive Biomarker for Gastric Cancer Detection

Although gastric cancer is one of the highest mortality cancers in the world, no methods for non-invasive diagnosis have been described. Aberrant hypermethylation, an epigenetic mechanism of inactivation of tumor suppressor genes, is an emerging approach to noninvasive diagnosis of gastric cancer. We have previously shown high prevalence of aberrant hypermethylation of Reprimo (RPRM) a novel tumor suppressor gene, in plasma from gastric cancer but not in plasma from healthy donors (Clin Cancer Res. 2008;14:6264-9). This finding suggested that RPRM promoter methylation could be a sensitive and specific biomarker for the non-invasive detection of gastric cancer. Here we explore in a prospective and blind fashion specificity and sensitivity of RPRM for non-invasive diagnoses of gastric cancer. Methods. We prospectively collect 83 gastric cancer patients from two hospitals and 223 healthy donors from corresponding blood banks. Detection of RPRM was perform by methylation specific PCR (MSP). We perform endoscopy in healthy donors that were RPRM(+) in plasma. Results. Aberrant hypermethylation of RPRM was identified in 67/74 (90.5%) and 75/79 (94.9%) of tumor and plasma from gastric cancer patients, respectively. Among healthy donors, methylation of RPRM was identified in 29.1% (65 of 223) of tested cases. These differences were statistically significant (p< 0.001 by Fisher's exact test). The sensibility and specificity of RPRM detection in plasma were 89% (95% CI: 81%-98%) and 67% (95% CI: 58%-75%) respectively. We perform endoscopy in 5 RPRM positive healthy donors, and no cancer was found. Only one healthy donor was diagnosed with chronic gastritis. Discussion. Our data indicate that detection of RPRM promoter methylation in cell free plasma DNA is a promissory method for non-invasive detection of gastric cancer. RPRM could be included in the serologic biopsy along with pepsinogen and gastrin 17 not only for the identification of gastric atrophy, the precursor lesion of gastric cancer but also for direct identification of gastric cancer. Supported by FONDECYT grant 1080563 from Government of Chile