This study is funded by the Longyan City Science and Technology Plan Project (Grant number 2020LYF17029).

The protocol described in this study has been reviewed and approved by the ethics committee of Longyan First Affiliated Hospital of Fujian Medical University in accordance with the ethical guidelines established by the Declaration of Helsinki. The safety and well-being of human participants are our topmost concern, and all procedures have been designed to minimize potential risks and discomfort. All data collected will be treated confidentially and used solely for the purposes of this research. Subjects will be free to withdraw from the study at any time, and their decision to participate or withdraw will not affect their relationship with the researchers or the institution.
1. General Information
<ol>
	<li>Select patients diagnosed with gastric LGIN. In this retrospective analysis, a total of 120 patients diagnosed with gastric LGIN by histopathological examination and treated with ESD at Longyan First Hospital, the Third Hospital of Xingtai, and Gansu Cancer Hospital from November 2020 to June 2022 were included.</li>
</ol>
2. Inclusion and exclusion criteria
<ol>
	<li>Inclusion criteria
	<ol>
		<li>Include patients diagnosed with gastric LGIN by histopathological examination and treated with ESD within 3 months of diagnosis.</li>
		<li>Ensure that all biopsy and surgical specimens are diagnosed according to the World Health Organization (WHO) pathological diagnostic criteria for tumors of the digestive system7, and all pathological sections are reviewed by two pathologists before and after surgery.</li>
		<li>Complete clinical data.</li>
	</ol>
	</li>
	<li>Exclusion criteria
	<ol>
		<li>Exclude patients who have recently received antibiotics, proton pump inhibitors (PPIs), bismuth preparations, and acid suppressants for the treatment of Helicobacter pylori (HP) infection.</li>
		<li>Exclude patients diagnosed with gastric high-grade intraepithelial neoplasia (HGIN), gastric cancer, or other metastatic tumors by histopathological examination.</li>
		<li>Exclude patients who underwent surgery.</li>
		<li>Exclude patients who underwent radiotherapy or chemotherapy.</li>
		<li>Exclude patients with incomplete clinical data.</li>
	</ol>
	</li>
</ol>
3. Research methods
<ol>
	<li>Endoscopic examination and surgery
	<ol>
		<li>Let all patients diagnosed with gastric LGIN by histopathological examination undergo routine white light endoscopy (WLE).
		<ol>
			<li>Prior to performing endoscopy, ensure that the patient undergoes some preparations, including fasting for more than 6 h. 
			NOTE: The examination usually involves local anesthesia of the throat to minimize discomfort.</li>
			<li>Insert the endoscope through the patient&#39;s mouth and gradually advance it through the esophagus into the stomach. The interior of the digestive tract is observed under WLE, including the color, shape, texture, and distribution of blood vessels in the mucosa, looking for possible lesions.</li>
			<li>Take tissue samples or perform other therapeutic operations depending on the observation results. After completing the examination, gradually withdraw the endoscope and end the examination.</li>
		</ol>
		</li>
		<li>Use ME-NBI technology to observe the microstructure of the gastric mucosal surface to determine the extent of the lesion further. 
		NOTE: ME-NBI is a formation mode that is switched by a button on the endoscope after observing the approximate mucosa in the WLE mode. It can observe microvascular (MV) and microstructure (MS) that cannot be observed with conventional endoscopy.
		<ol>
			<li>After finding the suspicious lesion in WLE, zoom in from far to near, from normal to the center of the lesion.</li>
			<li>Through ME-NBI examination, judge whether the demarcation line (DL) exists, the anisotropy of the MV and MS, and some special signs on the surface of the lesion to better observe the microstructure of the surface of the GI mucosa, and then make a judgment about the nature of the lesion, which may be lesions.</li>
		</ol>
		</li>
		<li>Next, make an electrocoagulation mark with a disposable mucosal incision knife 3-5 mm outside the lesion boundary in the patient, with a distance of approximately 2 mm between the mark points.</li>
		<li>Inject the prepared injection solution (250 mL of saline + 3 mg of epinephrine + 2 mL of indigo rouge) into the submucosa through a special endoscopic injection needle. 
		NOTE: Saline is mainly used to keep the submucosa moist and separate the mucosal layer from the muscular layer. Epinephrine constricts blood vessels and reduces bleeding. Indigo Carmine is a dye that helps doctors better visualize the distribution of blood vessels in the submucosa. The ingredients dispensed include 250 mL of saline + 3 mg of epinephrine + 2 mL of indigo rouge.</li>
		<li>Once the lesion is completely elevated, make a circumferential incision with a disposable mucosal incision knife at a point approximately 3 mm from the lesion marking point, followed by submucosal dissection until the lesion is completely removed.</li>
		<li>After dissection, carefully observe the wound and perform electrocoagulation using electrocoagulation forceps. If necessary, use metal hemostatic clips to clamp the wounds.</li>
	</ol>
	</li>
	<li>Pathological examination
	<ol>
		<li>Remove the tissue sample after complete excision using suction or snare.
		<ol>
			<li>Fix and preserve the removed tissue sample and mark the oral and anal sides for pathological examination and diagnosis. Fix the samples in 10% formalin for 24-48 h at 20-25 &#176;C. 
			NOTE: The pathological description included information on the general morphology of the lesion, volume and size, margins, histological classification, depth of infiltration, and gastric mucosal lesions.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>HP detection method
	<ol>
		<li>Use either the rapid urease test or the 13C breath test.
		<ol>
			<li>In the rapid urease test, take a sample of the patient&#39;s stomach lining with biopsy forceps under endoscopy and place it in a reagent containing urease.</li>
			<li>Check for the presence of H. pylori in the stomach lining. The urease enzyme breaks down the urea to produce ammonia, which makes the reagent alkaline and turns it red. Observe the change in color of the reagent and determine the presence of H. pylori infection. 
			NOTE: The 13C-breath test is a non-invasive, painless, and side-effect-free test performed by orally administering urea containing 13C-labeled urea and using an isotope ratio mass spectrometer to detect the amount of 13C-labeled carbon dioxide in the patient&#39;s exhaled breath. If Helicobacter pylori is present in the stomach, the bacteria will break down the 13C-labeled urea to produce 13C-labeled carbon dioxide, and the presence or absence of Helicobacter pylori infection can be determined by measuring the amount of 13C-labeled carbon dioxide in the exhaled gas. It is important to note that patients must discontinue proton pump inhibitors (PPIs) for at least 2 weeks and antibacterial drugs, bismuth, and certain herbal medicines with antibacterial properties for at least 4 weeks before performing the rapid urease test or 13C breath test. These medications can affect the accuracy of the test results. Patients must also fast or refrain from eating for at least 2 h during the 13C breath test to avoid the influence of food on the test results.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Data collection and grouping
	<ol>
		<li>Compile the general clinical data of the patients, including gender, age, Helicobacter pylori (HP) infection, lesion size, lesion location, morphology, gastric mucosal congestion, nodules status, surface ulceration and erosion, and ME observation.</li>
		<li>Divide all patients in the study into upgraded and non-upgraded groups according to whether the post-ESD pathological diagnosis was upgraded based on the evaluations performed. 
		NOTE: The upgraded group had a postoperative pathological diagnosis of gastric HGIN, early gastric cancer (EGC), or advanced gastric cancer, while the non-upgraded group had a postoperative pathological diagnosis of gastric LGIN or inflammation.</li>
	</ol>
	</li>
</ol>
4. Statistical methods
<ol>
	<li>Use statistical software to organize and analyze all data. Analyze categorical data statistically as frequencies (in percentages) and compare using chi-square tests or Fisher&#39;s exact tests. 
	NOTE: SPSS software was used in this study.</li>
	<li>Employ logistic regression analysis to identify risk factors associated with post-ESD pathological upgrading in patients diagnosed with gastric LGIN. A P-value less than 0.05 indicates a statistically significant difference.</li>
</ol>

Risk Prediction Model of Gastric Low-Grade Intraepithelial Neoplasia

<ol>
	<li>Bray, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+cancer+statistics+2018:+Globocan+estimates+of+incidence+and+mortality+worldwide+for+36+cancers+in+185+countries.">Global cancer statistics 2018: Globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries.</a> CA Cancer J Clin. 68 (6), 394-424 (2018).</li><li>Migita, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rnf126+as+a+marker+of+prognosis+and+proliferation+of+gastric+cancer.">Rnf126 as a marker of prognosis and proliferation of gastric cancer.</a> Anticancer Res. 40 (3), 1367-1374 (2020).</li><li>Zhang, H., Yang, X., Zhang, X., Huang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+significance+of+endoscopic+kyoto+classification+of+gastritis+in+the+gastric+cancer+risk+assessment:+A+systematic+review+and+meta-analysis.">The significance of endoscopic kyoto classification of gastritis in the gastric cancer risk assessment: A systematic review and meta-analysis.</a> Medicine. 102 (22), e33942 (2023).</li><li>Cheng, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+correa's+cascade+steps:+A+long-term+endoscopic+surveillance+among+non-ulcer+dyspepsia+and+gastric+ulcer+after+H.+pylori+eradication.">Evolution of the correa's cascade steps: A long-term endoscopic surveillance among non-ulcer dyspepsia and gastric ulcer after H. pylori eradication.</a> J Formos Med Assoc. 122 (5), 400-410 (2023).</li><li>Kim, B., Cho, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+screening+and+surveillance+for+gastric+cancer.">Endoscopic screening and surveillance for gastric cancer.</a> Gastrointest Endosc Clin N Am. 31 (3), 489-501 (2021).</li><li>Choi, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+risk+factors+for+discrepancy+after+endoscopic+submucosal+dissection+of+gastric+category+3+lesion+(low+grade+dysplasia).">The risk factors for discrepancy after endoscopic submucosal dissection of gastric category 3 lesion (low grade dysplasia).</a> Dig Dis Sci. 59 (2), 421-427 (2014).</li><li>Nagtegaal, I. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+2019+who+classification+of+tumours+of+the+digestive+system.">The 2019 who classification of tumours of the digestive system.</a> Histopathology. 76 (2), 182-188 (2020).</li><li>Zou, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+characteristics+in+predicting+prognosis+of+biopsy-diagnosed+gastric+low-grade+intraepithelial+neoplasia.">Endoscopic characteristics in predicting prognosis of biopsy-diagnosed gastric low-grade intraepithelial neoplasia.</a> Chin Med J (Engl). 135 (1), 26-35 (2022).</li><li>Ryu, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+outcomes+of+endoscopic+submucosa+dissection+for+high-grade+dysplasia+from+endoscopic+forceps+biopsy.">Clinical outcomes of endoscopic submucosa dissection for high-grade dysplasia from endoscopic forceps biopsy.</a> Gastric Cancer. 20 (4), 671-678 (2017).</li><li>Xu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors+for+under-diagnosis+of+gastric+intraepithelial+neoplasia+and+early+gastric+carcinoma+in+endoscopic+forceps+biopsy+in+comparison+with+endoscopic+submucosal+dissection+in+Chinese+patients.">Risk factors for under-diagnosis of gastric intraepithelial neoplasia and early gastric carcinoma in endoscopic forceps biopsy in comparison with endoscopic submucosal dissection in Chinese patients.</a> Surg Endosc. 30 (7), 2716-2722 (2016).</li><li>Kim, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+lesion+size+an+independent+indication+for+endoscopic+resection+of+biopsy-proven+low-grade+gastric+dysplasia.">Is lesion size an independent indication for endoscopic resection of biopsy-proven low-grade gastric dysplasia.</a> Dig Dis Sci. 59 (2), 428-435 (2014).</li><li>Zhao, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+commonly+is+the+diagnosis+of+gastric+low+grade+dysplasia+upgraded+following+endoscopic+resection?+A+meta-analysis.">How commonly is the diagnosis of gastric low grade dysplasia upgraded following endoscopic resection? A meta-analysis.</a> PLoS One. 10 (7), e0132699 (2015).</li></ol>

The authors declare that they have no conflict of interest.

Gastric LGIN is a common gastrointestinal disease, and as the number of patients continues to increase, the prevention and treatment of the disease become increasingly crucial. Establishing a risk prediction model is essential in guiding clinical treatment and preventing disease occurrence8. Pathological upgrading of gastric LGIN refers to the deterioration of LGIN properties within a certain period, which can lead to the transformation of LGIN into HGIN or even more severe conditions. In clinical practice, the prediction of pathological upgrading has become a focus of attention for medical personnel. Currently, some prediction models based on clinical manifestations, imaging findings, and biomarkers have been studied, but these have the disadvantages of complex indicators and long-term consumption. Therefore, it is necessary to develop a simple and effective predictive model. According to the current domestic consensus recommendations, ESD should be actively adopted for the treatment of gastric LGIN patients with high-risk factors for pathological upgrading. However, an increasing number of studies have shown significant differences between biopsy pathological results and postoperative pathological results in gastric LGIN patients9. 
The study revealed that out of the 120 gastric LGIN patients observed, 49 underwent pathological upgrading post-surgery, resulting in a pathological upgrading rate of 40.83%. This rate is relatively elevated when compared to other relevant studies. Possible reasons for these findings include underestimation of the results obtained from pathological biopsy. Variations in endoscopists' qualifications may lead to variations in patients' condition and biopsy site assessment, resulting in superficial or inadequate sampling and site deviations. The accuracy of results may also be influenced by the cognitive ability and knowledge of pathologists. Additionally, biopsy accuracy may be affected by different endoscopic techniques. For instance, the application of high-definition gastroscopy to observe the microvascular and microstructure morphology of lesions and determine the most conspicuous site for targeted biopsy can considerably enhance the precision of biopsy.
This study demonstrated that the older the age of gastric LGIN patients, the higher the risk of pathological upgrading after surgery, which is similar to other domestic research results10. Consequently, for gastric LGIN patients over 60 years of age, close follow-up should be performed, and if the patient has more risk factors, timely surgery is recommended. Regarding the size of the lesion, this study showed that lesion size &#8805;2 cm is an independent risk factor for pathological upgrading, which is basically consistent with other research results11. The critical value of lesion size that may affect the occurrence of pathological upgrading after surgical treatment of gastric LGIN patients is still controversial, and clinical research is needed to find the most suitable critical value. Large sample size methods should be used for further research. The results showed that mucosal surface congestion, ulceration, and erosion are independent risk factors for pathological upgrading after surgery in patients with gastric LGIN, which is similar to relevant research12. Lesion surface ulceration and erosion are more likely to cause pathological upgrading after surgery, which may be because repeated damage to the gastric mucosa can promote dysplasia and intestinal metaplasia, thereby increasing the risk of gastric cancer. 
However, this study encountered certain limitations. Firstly, being a retrospective study made it impossible to fully control for bias and other confounding factors. In the future, prospective studies ought to be carried out to validate the conclusions and strengthen objectivity and inference ability. Secondly, due to the limited number of samples, the samples could not be segregated into a validation set for external validation of the model. Therefore, it is essential to gather additional patient data from several centers to augment the number of samples and expand the scope of observed variables. This will improve the model's predictive efficacy and the credibility of its outcomes. Furthermore, conducting external validation will reinforce the model's reliability. 
In conclusion, our research reveals that the risk prediction model, generated through analysis of independent risk factors for pathological upgrading in patients with gastric LGIN who underwent surgery, offers significant predictive value and can provide beneficial guidance for LGIN treatment in clinical contexts.

Gastric cancer is one of the most common malignant tumors, especially in East Asia, with high incidence and mortality rates. It is one of the most common cancers in China, with new diagnoses and deaths accounting for about half of the global total1. It is a major cause of morbidity and mortality in the Chinese population2. Gastric cancer ranks third in the world in terms of the highest mortality rate among tumors, and its prognosis is highly dependent on the stage of the lesion1. The 5-year survival rate for advanced-stage patients is less than 30%, while the 5-year survival rate for early-stage patients is usually more than 90%. Therefore, early diagnosis and treatment of gastric cancer are essential to prevent and control the disease3. 
The Correa cascade reaction is widely recognized as one of the major patterns of gastric cancer development, suggesting that the carcinogenic process of gastric cancer gradually progresses from atrophic gastritis to intestinal metaplasia, intraepithelial neoplasia, and finally to adenocarcinoma4. With the application of modern technology and the increasing popularity of gastroscopy, an increasing number of gastric mucosal precancerous lesions have been detected, including magnifying endoscopy (ME), staining endoscopy, and narrow-band imaging (NBI)5. 
Low-grade intraepithelial neoplasia (LGIN) is one of the precancerous lesions of gastric cancer and is closely related to gastric cancer. However, some patients with low-grade intraepithelial neoplasia showed pathological upgrading after endoscopic submucosal dissection (ESD) compared with biopsy findings6. Therefore, there is some controversy in clinical practice regarding the choice of follow-up or treatment for patients with biopsy-proven LGIN. This article explores the risk factors for pathological upgrading after ESD treatment in patients with gastric LGIN establishes and evaluates a risk prediction model for LGIN occurrence, and provides more scientific and valuable reference opinions for the clinical management of gastric LGIN patients.

<table><tbody><tr><td>Disposable mucosal incision knife</td><td>Olympus (Japan)</td><td>KD-650Q</td><td></td></tr><tr><td>Endoscopic image processing device</td><td>Olympus (Japan)</td><td>CV-290</td><td></td></tr><tr><td>Hemostasis Clips</td><td>MICRO-TECH(Nanjing)</td><td>ROCC-D-26-195</td><td></td></tr><tr><td>High-frequency hemostatic forceps</td><td>Olympus (Japan)</td><td>FD-410LR</td><td></td></tr><tr><td>Indicarminum</td><td>MICRO-TECH(Nanjing)</td><td>MTN-DYZ-15</td><td></td></tr><tr><td>Injection Needles</td><td>MICRO-TECH(Nanjing)&amp;nbsp;</td><td>IN02-25423230</td><td></td></tr><tr><td>Magnifying gastroscope</td><td>Olympus (Japan)</td><td>GIF-H290Z</td><td></td></tr><tr><td>Orthodontic rubber band</td><td>3M Unitek Corporation</td><td></td><td>6.4 mm 3.5 oz</td></tr><tr><td>Therapeutic gastroscopy</td><td>Olympus (Japan)</td><td>GIF-2TQ260M</td><td></td></tr><tr><td>Transparent cap</td><td>Olympus (Japan)</td><td>D-201-11804</td><td></td></tr></tbody></table>

establishment and evaluation of a risk prediction model for pathological escalation of gastric low-grade intraepithelial neoplasia

The study aims to explore the risk factors for pathological escalation after endoscopic surgery for gastric low-grade intraepithelial neoplasia (LGIN) and to establish and evaluate a risk prediction model for LGIN. A total of 120 patients diagnosed with gastric LGIN by biopsy and endoscopic submucosal dissection (ESD) between November 2020 and June 2022 were retrospectively analyzed. Gender, age, Helicobacter pylori (HP) infection, lesion size, lesion location, morphology, gastric mucosal congestion, nodules status, surface ulceration and erosion, and ME-observation of all patients were collected and divided into upgraded and non-upgraded groups according to the biopsy and ESD postoperative pathological diagnosis results. Independent risk factors for pathological escalation after ESD surgical treatment were screened by logistic regression analysis, and a risk prediction model was established. Among the 120 patients with gastric LGIN, 49 patients developed postoperative pathological upgrading; the rate of pathological upgrading was 40.83%. Among them, 42 patients were upgraded to high-grade intraepithelial neoplasia (HGIN), 1 case was upgraded to advanced gastric cancer, and 6 cases were upgraded to early gastric carcinoma (EGC). Univariate analysis showed that age, lesion size, gastric mucosal congestion, surface ulcers, and erosion were significantly different between the groups (p &lt; 0.05). Multivariate Logistic regression analysis revealed that age &#8805;60 years, focal length &#8805;2 cm, gastric mucosal congestion, and surface ulceration and erosion were independent risk factors for postoperative pathological escalation in patients with gastric LGIN. Final joint probability forecasting model for P = 1/[1 + e(26.515-0.161 x &#946;1-0.357 x &#946;2+0.039 x &#946;3-0.269 x &#946;4)]. Age, lesion size &#8805;2 cm, gastric mucosal congestion, and lesion surface ulceration and erosion are risk factors for postoperative pathological upgrading in patients with gastric LGIN. The risk prediction model established in this study based on risk factors has predictive value and can provide a scientific reference for the clinical treatment of patients with gastric LGIN.

Incidence of pathological upgrade in gastric LGIN patients after ESD 
A total of 120 gastric LGIN patients were included in this study, of which 49 (40.83%) experienced pathological upgrades after ESD. Among them, 42 cases were upgraded to HGIN, 1 case was upgraded to advanced gastric cancer, and 6 cases were upgraded to EGC. A total of 71 cases did not experience pathological upgrade, among which 2 cases were downgraded to inflammation, and 69 cases remained as LGIN, with a rate of pathological downgrade of 1.67%.
Univariate analysis of pathological upgrade after ESD in gastric LGIN patients 
As shown in Table 1, there were no statistically significant differences (p &#62; 0.05) in gender, HP infection, lesion location, surface nodules, presence of a demarcation line (DL) in the ME layer, lesion morphology, microstructure (MS), and microvascular (MV) morphology between the postoperative pathological upgrade group and the non-upgrade group of gastric LGIN patients. However, there were statistically significant differences (p &#60; 0.05) in age, lesion size, gastric mucosal congestion, and surface ulceration and erosion.
Multivariate logistic regression analysis of pathological upgrade after ESD in gastric LGIN patients 
Factors with p &#60; 0.05 screened out by univariate analysis, including age, lesion size, gastric mucosal congestion, and surface ulceration and erosion, which were used as independent variables, and pathological upgrade after ESD in gastric LGIN patients was used as the dependent variable for logistic regression analysis (Table 2). Table 3 showed that age &#8805;60 years, lesion diameter &#8805;2 cm, gastric mucosal congestion, and surface ulceration and erosion were independent risk factors for pathological upgrade after ESD in patients with gastric LGIN. The final joint probability prediction model was P = 1 / [1 + e(26.515-0.161x&#946;1-0.357x&#946;2+0.039x&#946;3-0.269x&#946;4)] based on the prediction formula for logistic regression: P(y = 1/x) = 1/(1+e-(&#946;0+&#946;1x1+&#946;2x2+&#946;3x3+&#946;4x4)), where y represents the dichotomous dependent variable, x represents the vector of independent variables, &#946; represents the regression coefficient, and e represents the base of the natural logarithm. Additionally, technical term abbreviations will be defined when first used. This formula indicates that given the independent variable x, the probability of event y = 1 occurring is P(y = 1/x). When the sum of &#946;0 + &#946;1 x 1 + &#946;2 x 2 + &#946;3 x 3 + &#946;4 x 4 increases, so too does the value of P(y = 1/x), indicating a higher probability of the event y = 1 occurring; and conversely, a lower probability of the event y = 1 occurring. Here, &#946;1 is the regression coefficient of age, &#946;2 is the regression coefficient of lesion size, &#946;3 is the regression coefficient of gastric mucosal congestion, and &#946;4 is the regression coefficient of surface ulceration and erosion.
Case presentation 
As a representative case, a 50-year-old female patient presented with rough mucosa and erosion at the gastric angle during a physical examination. The endoscopic biopsy confirmed low-grade intraepithelial neoplasia (LGIN). Further investigations using magnification endoscopy, narrow band imaging (NBI), and pigment endoscopy suggested the patient was at risk of developing high-grade intraepithelial neoplasia (HGIN). The patient was advised to undergo endoscopic submucosal dissection (ESD) treatment, and the final pathological diagnosis was HGIN (as indicated in Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65868/65868fig01.jpg" /> 
Figure 1: Case presentation. (A,B) Pre-operation, WLE showed rough gastric mucosa with erosion. (C,D) Irregular MV and MS were observed in the lesions observed by ME + NBI. (E) The contours of the lesions could be seen by indigo blush staining. (F) The lesions were labeled under the endoscope. (G) Stripped after submucosal water injection. (H) Wound hemostatic treatment. (I) Fixed specimens were sent for pathological examination. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65868/65868fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Univariate analysis of post-ESD pathological upgrade (%). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65868/Table1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Multivariate logistic regression analysis variable assignment table for post-ESD pathological upgrade. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65868/Table 2.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: Multivariate logistic regression analysis of post-ESD pathological upgrade. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65868/Table 3.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Establishment and Evaluation of a Risk Prediction Model for Pathological Escalation of Gastric Low-Grade Intraepithelial Neoplasia at JoVE.com

Author Spotlight: Advancing Early Detection and Treatment of Gastrointestinal Tumors

Here, we performed a systematic evaluation of patients diagnosed with gastric low-grade intraepithelial neoplasia by previous endoscopic biopsy and obtained a pathological diagnosis by complete resection of the lesion by endoscopic submucosal dissection (ESD), analyzing factors that potentially increase the risk of pathological escalation.

Establishment and Evaluation of a Risk Prediction Model for Pathological Escalation of Gastric Low-Grade Intraepithelial Neoplasia

The study aims to explore the risk factors for pathological escalation after endoscopic surgery for gastric low-grade intraepithelial neoplasia (LGIN) and ...

establishment-evaluation-risk-prediction-model-for-pathological

Nanyang Technological University

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Cancer Research

965 Views.  Longyan First Affiliated Hospital of Fujian Medical University. De scope van ons onderzoek is het opsporen, diagnosticeren en behandelen van eerdere gastro-intestinale tumoren. De vraag die ik zou willen beantwoorden is hoe het risico op pathologische escalatie van maagkernen tot epitheliale kern kan worden voorspeld om de diagnose en eerste behandeling van patiënten te krijgen. Recente ontwikkelingen op het gebied van spijsverteringsendoscopie omvatten minimaal invasieve technieken, verbeterde beeldvormingsmodaliteiten, revisiebiopsieën en robotgeassis...

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Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell proliferation and increased nutrient absorption, are critical in recovery, yet poorly understood. Understanding the molecular mechanism behind the adaptive responses is crucial to facilitate the identification of nutrients or drugs to enhance adaptation. Different approaches and models have been described throughout the literature, but a detailed descriptive way to essentially perform the procedures is needed to obtain reproducible data. Here, we describe a method to estimate important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis in mice. We demonstrate the detection of proliferating cells using a cell cycle specific marker, as well as using small intestinal weight, crypt depth, and villus height as endpoints. Some of the critical steps within the described method are the removal and weighing of the small intestine and the rather complex software system suggested for the measurement of this technique. These methods have the advantages that they are not time-consuming, and that they are cost-effective and easy to carry out and measure.

Here, we present a protocol to establish important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis. We demonstrate the detection of proliferating cells using a cell cycle specific marker and using small intestinal weight, crypt depth, and villus height as endpoints.

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell ...

Diagnosis of Neoplasia in Barrett&#8217;s Esophagus using Vital-dye Enhanced Fluorescence Imaging

The ability to differentiate benign metaplasia in Barrett&#8217;s Esophagus (BE) from neoplasia in vivo remains difficult as both tissue types can be flat and indistinguishable with white light imaging alone. As a result, a modality that highlights glandular architecture would be useful to discriminate neoplasia from benign epithelium in the distal esophagus. VFI is a novel technique that uses an exogenous topical fluorescent contrast agent to delineate high grade dysplasia and cancer from benign epithelium. Specifically, the fluorescent images provide spatial resolution of 50 to 100 &#956;m&#160;and a field of view up to 2.5 cm,&#160;allowing endoscopists to visualize glandular morphology. Upon excitation, classic Barrett&#8217;s metaplasia appears as continuous, evenly-spaced glands and an overall homogenous morphology; in contrast, neoplastic tissue appears crowded with complete obliteration of the glandular framework. Here we provide an overview of the instrumentation and enumerate the protocol of this new technique. While VFI affords a gastroenterologist with the glandular architecture of suspicious tissue, cellular dysplasia cannot be resolved with this modality. As such, one cannot morphologically distinguish Barrett&#8217;s metaplasia from BE with Low-Grade Dysplasia via this imaging modality. By trading off a decrease in resolution with a greater field of view, this imaging system can be used at the very least as a red-flag imaging device to target and biopsy suspicious lesions; yet, if the accuracy measures are promising, VFI may become the standard imaging technique for the diagnosis of neoplasia (defined as either high grade dysplasia or cancer) in the distal esophagus.

Vital-dye enhanced fluorescence imaging (VFI) is a novel in vivo technique that combines high-resolution epithelial imaging with exogenous topical fluorescent contrast to highlight glandular morphology and delineate neoplasia (high grade dysplasia and cancer) in the distal esophagus.

The ability to differentiate benign metaplasia in Barrett&#8217;s Esophagus (BE) from neoplasia in vivo remains difficult as both tissue types can be flat ...

Bioengineering

Establishing a Competing Risk Regression Nomogram Model for Survival Data

The Kaplan&#8211;Meier method and Cox proportional hazards regression model are the most common analyses in the survival framework. These are relatively easy to apply and interpret and can be depicted visually. However, when competing events (e.g., cardiovascular and cerebrovascular accidents, treatment-related deaths, traffic accidents) are present, the standard survival methods should be applied with caution, and real-world data cannot be correctly interpreted. It may be desirable to distinguish different kinds of events that may lead to the failure and treat them differently in the analysis. Here, the methods focus on using the competing regression model to identify significant prognostic factors or risk factors when competing events are present. Additionally, nomograms based on a proportional hazard regression model and a competing regression model are established to help clinicians make individual assessments and risk stratifications in order to explain the impact of controversial factors on prognosis.

Presented here is a protocol to build nomograms based on the Cox proportional hazards regression model and competing risk regression model. The competing method is a more rational method to apply when competing events are present in the survival analysis.

The Kaplan&#8211;Meier method and Cox proportional hazards regression model are the most common analyses in the survival framework. These are relatively ...

Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is a useful measure to provide an accurate analysis of the methylation status of gastrointestinal (GI) malignancies. Given the multiple potential translational uses of DNA methylation analysis, practicing clinicians and others new to DNA methylation studies need to be able to understand step by step how these genome-wide analyses are performed. The goal of this protocol is to provide a detailed description of how this method is used for the biomarker identification in GI malignancies. Importantly, we describe three critical steps that are needed to obtain accurate results during genome-wide analysis. Clearly and concisely written, these three methods are often lacking and not noticeable to those new to epigenetic studies. We used 48 samples of a GI malignancy (gastric cancer) to highlight practically how genome-wide DNA methylation analysis can be performed for GI malignancies.

Herein, we describe a procedure for genome-wide analysis of DNA methylation in gastrointestinal cancers. The procedure is of relevance to studies that investigate relationships between methylation patterns of genes and factors contributing to carcinogenesis in gastrointestinal cancers.

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is ...

Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all human squamous cell carcinomas. Despite recent progress in defining the molecular changes accompanying ESCC initiation and development, patient prognosis remains poor. The functional annotation of these molecular changes is the necessary next step and requires models that both capture the molecular features of ESCC and can be readily and inexpensively manipulated for functional annotation. Mice treated with the tobacco smoke mimetic 4-nitroquinoline 1-oxide (4NQO) predictably form ESCC and esophageal preneoplasia. Of note, 4NQO lesions also arise in the oral cavity, most commonly in the tongue, as well as the forestomach, which all share the stratified squamous epithelium. However, these mice cannot be simply manipulated for functional hypothesis testing, as generating isogenic mouse models is time- and resource-intensive. Herein, we overcome this limitation by generating single cell-derived three-dimensional (3D) organoids from mice treated with 4NQO to characterize murine ESCC or preneoplastic cells ex vivo. These organoids capture the salient features of ESCC and esophageal preneoplasia, can be cheaply and quickly leveraged to form isogenic models, and can be utilized for syngeneic transplantation experiments. We demonstrate how to generate 3D organoids from normal, preneoplastic, and SCC murine esophageal tissue and maintain and cryopreserve these organoids. The applications of these versatile organoids are broad and include the utilization of genetically engineered mice and further characterization by flow cytometry or immunohistochemistry, the generation of isogeneic organoid lines using CRISPR technologies, and drug screening or syngeneic transplantation. We believe that the widespread adoption of the techniques demonstrated in this protocol will accelerate progress in this field to combat the severe burden of ESCC.

This protocol describes the key steps to generate and characterize murine oral-esophageal 3D organoids that represent normal, preneoplastic, and squamous cell carcinoma lesions induced via chemical carcinogenesis.

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all ...

Nomogram Development for Survival Prediction in MPCC Patients

Competing-Risk Nomogram for Predicting Cancer-Specific Survival in Multiple Primary Colorectal Cancer Patients after Surgery

Cancer-specific survival (CSS) in multiple primary colorectal cancer (MPCC) patients is competitively affected by death from other causes. This study aimed to investigate CSS and associated risk factors by competing risk analysis in MPCC patients. The data of this study is from the SEER database. Using univariable and multivariable analysis of the competitive risk model to weaken the impact of competitive events, it explores the risk factors of CSS and develops a nomogram model. Then, the performance of the model is verified by the ROC curve, calibration curve, and DCA. The study encompasses a total of 8931 patients, with 6255 in the training cohort and 2676 in the validation cohort. Univariable and multivariable analyses showed that sex, Tumor Node Metastasis (TNM) stage, and tumor grade are independent risk factors for cancer-specific survival in MPCC patients. Based on the risk factors, we developed a diagram model to predict CSS. ROC curve, calibration curve, and DCA also show good results. In conclusion, a nomogram is developed that serves as a valuable tool for predicting CSS in MPCC patients, providing clinicians with crucial insights for personalized treatment planning.

The study found that male gender, poor tumor grade, and advanced Tumor Node Metastasis stage were associated with poorer cancer-specific survival (CSS) in multiple primary colorectal cancer (MPCC) patients after surgery. We developed a nomogram to predict the CSS of MPCC patients and contribute to clinical treatment decision-making.

Cancer-specific survival (CSS) in multiple primary colorectal cancer (MPCC) patients is competitively affected by death from other causes. This study ...

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett&#8217;s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett&#8217;s esophagus and esophageal adenocarcinoma.

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Medicine

Intraoperative Gastroscopy for Tumor Localization in Laparoscopic Surgery for Gastric Adenocarcinoma

Determining resection margins for gastric cancer, which are not exposed to the serosal surface of the stomach, is the most important procedure during totally laparoscopic gastrectomy (TLG). The aim of this protocol is to introduce a procedure for intraoperative gastroscopy, in order to directly mark tumors during TLG for gastric cancer in the middle third of the stomach. Patients who were diagnosed with adenocarcinoma in the middle third of the stomach were enrolled in this case series. Before surgery, additional gastroscopy for tumor localization is not performed. Under general anesthesia, laparoscopic mobilization of the stomach is performed first. After the first portion of the duodenum is mobilized from the pancreas and clamped, the surgeon moves to the other side for the gastroscopic procedure. On the insertion of a gastroscope through the oral cavity into the stomach, 2 - 3 cc of indigo carmine is administered via an endoscopic injector into the gastric muscle layer at the proximal margin of the stomach. The location of stained serosa in the laparoscopic view is used to guide distal subtotal gastrectomy, however, total gastrectomy is performed if the tumor is too close to the esophagogastric junction. A specimen is sampled after distal gastrectomy to confirm sufficient length from resection margin to tumor before reconstruction. In our case series, all patients had tumor-free margins and required no additional resection. There was no morbidity related to the gastroscopic procedure, and the time required for the procedure has gradually decreased to about five minutes. Intraoperative gastroscopy for tumor localization is an accurate and tolerated method for gastric cancer patients undergoing totally laparoscopic distal gastrectomy.

In early gastric cancer, the aim of surgery is to precisely remove the distal stomach including the primary tumor. To do this, accurate localization of the tumor is crucial, especially in totally laparoscopic surgery. This protocol describes a procedure for intraoperative gastroscopy in totally laparoscopic subtotal gastrectomy.

Determining resection margins for gastric cancer, which are not exposed to the serosal surface of the stomach, is the most important procedure during ...

Gene Regulation and Targeted Therapy in Gastric Cancer Peritoneal Metastasis: Radiological Findings from Dual Energy CT and PET/CT

Gastric cancer remains fourth in cancer incidence worldwide with a five-year survival of only 20%-30%. Peritoneal metastasis is the most frequent type of metastasis that accompanies unresectable gastric cancer and is a definitive determinant of prognosis. Preventing and controlling the development of peritoneal metastasis could play a role in helping to prolong the survival of gastric cancer patients. A non-invasive and efficient imaging technique will help us to identify the invasion and metastasis process of peritoneal metastasis and to monitor the changes in tumor nodules in response to treatments. This will enable us to obtain an accurate description of the development process and molecular mechanisms of gastric cancer. We have recently described experiment using dual energy CT (DECT) and positron emission tomography/computed tomography (PET/CT) platforms for the detection and monitoring of gastric tumor metastasis in nude mice models. We have shown that weekly continuous monitoring with DECT and PET/CT can identify dynamic changes in peritoneal metastasis. The sFRP1-overexpression in gastric cancer mice models showed positive radiological performance, a higher FDG uptake and increasing enhancement, and the SUVmax (standardized uptake value) of nodules demonstrated an obvious alteration trend in response to targeted therapy of TGF-&#946;1 inhibitor. In this article, we described the detailed non-invasive imaging procedures to conduct more complex research on gastric cancer peritoneal metastasis using animal models and provided representative imaging results. The use of non-invasive imaging techniques should enable us to better understand the mechanisms of tumorigenesis, monitor tumor growth, and evaluate the effect of therapeutic interventions for gastric cancer.

This protocol describes the value of dual energy CT and PET/CT imaging methods in tumor imaging and efficacy evaluation. This article demonstrates the research methods and results acquired by dual energy CT and PET/CT to evaluate the gene regulation and targeted treatment of gastric cancer peritoneal metastasis.

Gastric cancer remains fourth in cancer incidence worldwide with a five-year survival of only 20%-30%. Peritoneal metastasis is the most frequent type of ...

A Preclinical Human-Derived Autologous Gastric Cancer Organoid/Immune Cell Co-Culture Model to Predict the Efficacy of Targeted Therapies

Tumors expressing programmed cell death-ligand 1 (PD-L1) interact with programmed cell death protein 1 (PD-1) on CD8+ cytotoxic T lymphocytes (CTLs) to evade immune surveillance leading to the inhibition of CTL proliferation, survival, and effector function, and subsequently cancer persistence. Approximately 40% of gastric cancers express PD-L1, yet the response rate to immunotherapy is only 30%. We present the use of human-derived autologous gastric cancer organoid/immune cell co-culture as a preclinical model that may predict the efficacy of targeted therapies to improve the outcome of cancer patients. Although cancer organoid co-cultures with immune cells have been reported, this co-culture approach uses tumor antigen to pulse the antigen-presenting dendritic cells. Dendritic cells (DCs) are then cultured with the patient's CD8+ T cells to expand the cytolytic activity and proliferation of these T lymphocytes before co-culture. In addition, the differentiation and immunosuppressive function of myeloid-derived suppressor cells (MDSCs) in culture are investigated within this co-culture system. This organoid approach may be of broad interest and appropriate to predict the efficacy of therapy and patient outcome in other cancers, including pancreatic cancer.

The goals of the protocol are to use this approach to 1) understand the role of the immunosuppressive gastric tumor microenvironment and 2) predict the efficacy of patient response, thus increasing the survival rate of patients.

Tumors expressing programmed cell death-ligand 1 (PD-L1) interact with programmed cell death protein 1 (PD-1) on CD8+ cytotoxic T lymphocytes (CTLs) to ...

Establishment and Evaluation of a Risk Prediction Model for Pathological Escalation of Gastric Low-Grade Intraepithelial Neoplasia

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