Gastric intestinal metaplasia subtype biomarker FOLH1 and use thereof

Abstract

Provided are a gastric intestinal metaplasia subtype biomarker FOLH1 and use thereof. The biomarker FOLH1 can be used to effectively distinguish between progressive intestinal metaplasia and maintained intestinal metaplasia. Risk stratification of intestinal metaplasia using the biomarker in combination with endoscopy and pathological biopsy can further move the early diagnosis window of gastric cancer back from intraepithelial neoplasia to an earlier stage, increasing the detection rate of progressive intestinal metaplasia.

Description

FOLH1, a biomarker of gastric mucosal intestinal metaplasia subtype, and its application

[0001] Cross-references to related applications

[0002] This application claims priority to the following application: Patent application No. 2024105051588, filed on April 25, 2024, entitled “FOLH1, a biomarker of gastric mucosal intestinal metaplasia subtype and its application”, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This invention belongs to the field of biomedical technology, specifically relating to FOLH1, a subtype biomarker of gastric mucosal intestinal metaplasia, and its applications. Background Technology

[0004] Gastric cancer has an insidious onset, with most cases diagnosed at an advanced stage. Therefore, early diagnosis is crucial for its prevention and treatment. Over 80% of gastric cancers are intestinal-type gastric cancers, following a developmental pattern: normal gastric mucosa → chronic inflammation → atrophic gastritis → intestinal metaplasia → intraepithelial neoplasia → gastric cancer. Gastric mucosal atrophy and intestinal metaplasia are classified as precancerous states, while intraepithelial neoplasia is classified as a precancerous lesion. The longer the diagnostic window for cancerous changes, the higher the probability of early detection of precancerous lesions / precancerous states that are likely to develop into gastric cancer. Current research on early diagnosis of gastric cancer mainly focuses on intraepithelial neoplasia. However, the diagnostic window for the transformation of intraepithelial neoplasia into gastric cancer is relatively short (approximately 2 years), while the diagnostic window for the transformation of gastric mucosal intestinal metaplasia into gastric cancer is longer (approximately 4.6 years). Therefore, risk stratification during the gastric mucosal intestinal metaplasia stage has significant clinical value for early screening and prevention of gastric cancer. Currently, there are no effective biomarkers for risk stratification of intestinal metaplasia-gastric cancer.

[0005] Intestinal metaplasia-gastric cancer risk stratification offers significant advantages for early screening and prevention of gastric cancer. The progression phase of intestinal metaplasia-gastric cancer is much longer than that of intraepithelial neoplasia-gastric cancer. This longer progression window makes the intestinal metaplasia-gastric cancer stage easier to detect clinically. Risk stratification and timely intervention during the intestinal metaplasia stage are more conducive to precise prevention and treatment of gastric cancer. my country has a high incidence of gastric cancer, with intestinal-type gastric cancer being the predominant type. As a precancerous lesion closely related to intestinal-type gastric cancer, the diagnosis, intervention, and treatment of gastric mucosal intestinal metaplasia are crucial aspects of secondary prevention of gastric cancer, playing a vital role in its prevention and treatment and effectively alleviating the economic and social burden of gastric cancer in my country. Therefore, there is an urgent clinical need for highly sensitive and specific intestinal metaplasia-gastric cancer risk stratification markers.

[0006] The existing methods for early screening of gastric cancer precancerous state / precancerous lesions mainly include the following aspects: (1) Gastric cancer risk scoring based on questionnaire scales. The existing gastric cancer risk scoring scales lack a unified consensus and are insufficient to provide a highly credible basis for clinical decision-making. Most of the current gastric cancer risk scoring scales are formed around some disease risk factors, such as alcohol consumption, diabetes, obesity, etc. However, the research on the correlation between risk factors and the development of gastric cancer generally comes from retrospective or observational studies, and the existing evidence is contradictory and the level of evidence is very low. (2) Classification of intestinal metaplasia pathological subtypes by special staining. Intestinal metaplasia exists in three histological types. Type I or "intestinal type" is called "complete intestinal metaplasia", and Type II and Type III or "colonic type" are called "incomplete intestinal metaplasia". Some studies have found that incomplete intestinal metaplasia has a higher risk of cancer, but the value of intestinal metaplasia subtype in predicting the risk of gastric cancer is still controversial, and the pathological subtype of intestinal metaplasia has limited value in predicting the risk of gastric cancer. The 2017 Chinese Consensus on Chronic Gastritis states that, based on actual pathological examination, intestinal metaplasia in chronic gastritis is mostly mixed-type, and the detection of incomplete / complete intestinal metaplasia is closely related to the number of biopsies, indicating a sampling error problem. The British Gastroenterological Society believes that only a small number of patients with invasive gastric cancer have incomplete intestinal metaplasia, and the traditional diagnosis of "complete intestinal metaplasia" or "incomplete intestinal metaplasia" is performed using enzyme histochemical staining, which is highly dependent on the diagnostic experience or professional level of the assessor. Due to insufficient evidence, intestinal metaplasia pathological subtype staining is not currently used clinically for risk prediction of intestinal metaplasia-gastric cancer. (3) Serum diagnostic markers related to gastric cancer. Gastric cancer is a disease with high incidence and mortality rates. Currently, common serum biomarkers include serum pepsinogen (PG), serum gastrin-17 (G-17), and serum gastric cancer-associated antigen MG7-Ag. However, these serum diagnostic biomarkers for gastric cancer have insufficient sensitivity and specificity and are not recommended for individual use in gastric cancer detection. Furthermore, the screening value of their combined use lacks high-level evidence. Moreover, guidelines issued by the Spanish Gastroenterological Association in 2021 and the British Gastroenterological Association in 2019 do not recommend the use of serum biomarkers for gastric cancer screening in the general population. Precise stratification of the risk of intestinal metaplasia-gastric cancer at the pathological and histological level necessitates the development of highly specific and sensitive biomarkers for this risk stratification. The application of intestinal metaplasia-gastric cancer risk stratification biomarkers can advance the early diagnosis of gastric cancer to the intestinal metaplasia stage, significantly extending the early diagnostic window for gastric cancer, improving the detection rate of intestinal metaplasia that is prone to progressing to gastric cancer, and reducing misdiagnosis and missed diagnosis. Summary of the Invention

[0007] In view of this, in order to address the technical problem of the continuously increasing incidence of intestinal metaplasia but the lack of an effective risk assessment method, this invention obtains the biomarker FOLH1 by performing spatial transcriptomics analysis on biopsy specimens from sequential cases of progressive and maintenance intestinal metaplasia. The application of this marker gene can effectively distinguish between progressive and maintenance intestinal metaplasia in the antrum of the stomach, predict the outcome of intestinal metaplasia, thereby advancing the diagnostic window for gastric cancer, reducing missed diagnoses caused by insufficient early diagnostic methods for gastric cancer, providing a basis for determining the follow-up time period for patients with intestinal metaplasia in the antrum of the stomach, and reducing the waste of endoscopic resources.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] The first aspect of the invention provides the use of a reagent for detecting the expression level of FOLH1 in a sample in the preparation of products for diagnosing and differentiating between maintenance and progressive intestinal metaplasia, predicting intestinal metaplasia outcomes, and / or assessing the efficacy of intestinal metaplasia treatment.

[0010] Furthermore, the reagent includes:

[0011] Reagents for detecting FOLH1 RNA levels in samples;

[0012] Reagents for detecting the DNA level of FOLH1 in a sample;

[0013] Reagents for detecting the protein expression level of FOLH1 in a sample; or

[0014] A reagent for detecting the number of FOLH1-positive cells in a sample.

[0015] Furthermore, the reagents for detecting the RNA level of FOLH1 in the sample include reagents for detecting the expression levels of FOLH1 mRNA, lncRNA, and / or miRNA in the sample;

[0016] The reagents used to detect the FOLH1 DNA level in the sample include reagents for detecting the FOLH1 DNA expression level, DNA methylation level, DNA acetylation level, DNA phosphorylation level, DNA hydroxymethylation level and / or SNP locus genotype in the sample.

[0017] Furthermore, the reagents for detecting the expression levels of FOLH1 mRNA, lncRNA, and / or miRNA in the sample include primers that specifically amplify FOLH1 and / or probes that specifically recognize FOLH1.

[0018] The reagents used to detect the DNA expression level, DNA methylation level, DNA acetylation level, DNA phosphorylation level, DNA hydroxymethylation level, and / or SNP genotype of FOLH1 in the sample include reagents that detect the levels using sequencing technology.

[0019] Furthermore, the reagent used to detect the protein expression level of FOLH1 in the sample includes an affinity protein that specifically binds to the protein encoded by FOLH1;

[0020] The reagents for detecting the number of FOLH1-positive cells in the sample include reagents for detecting the number of FOLH1-positive cells by immunohistochemistry.

[0021] Furthermore, the affinity protein includes antibodies that specifically bind to proteins encoded by FOLH1, antibody functional fragments, agglutinants, receptors, and / or conjugated antibodies.

[0022] Furthermore, the samples include tissue samples, blood samples, serum samples, plasma samples, exosome samples, cell samples, fecal samples, and / or urine samples from the subject.

[0023] Furthermore, the product is a diagnostic kit, diagnostic chip, and / or test strip for diagnosing and differentiating between maintenance intestinal metaplasia and progressive intestinal metaplasia, predicting intestinal metaplasia outcomes, and / or assessing the efficacy of intestinal metaplasia treatment.

[0024] Furthermore, the subjects are either human or non-human animals.

[0025] In this invention, FOLH1 comprises the FOLH1 gene and the FOLH1 protein. The FOLH1 gene is transcribed and translated into the FOLH1 protein product in the research subject (subject). The FOLH1 gene has a Gene ID of 2346, and its full name is folate hydrolase 1 [Homo sapiens (human)]. Detailed information about the gene can be obtained from the NCBI database (https: / / www.ncbi.nlm.nih.gov / gene / ) based on the aforementioned Gene ID.

[0026] In some embodiments, the FOLH1 has a sequence known in the art or a derivative thereof. In some embodiments, the FOLH1 is a molecule comprising the following sequences: (a) FOLH1 molecules having sequences such as Gene ID:2346 (human), Gene ID:53320 (house mouse), Gene ID:85309 (Norwegian mouse), Gene ID:505865 (cattle), Gene ID:100060132 (horse), Gene ID:133763336 (rabbit); (b) molecules that hybridize with the sequence defined in (a) under stringent conditions; (c) molecules having sequence homology of 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5% or more, or any value or range thereof) to the sequence of the molecules shown in (a) or (b), such as FOLH1 molecules obtained through codon optimization.

[0027] In some implementations, the sequencing technology refers to any reagent capable of detecting the DNA expression level, DNA methylation level, DNA acetylation level, DNA phosphorylation level, DNA hydroxymethylation level, and / or SNP locus genotype of FOLH1 in a sample. Exemplarily, the sequencing technology includes, but is not limited to: Sanger sequencing technology, Illumina sequencing technology, Ion Torrent sequencing technology, PacBio sequencing technology, and Oxford Nanopore long-read sequencing technology.

[0028] In this invention, the sample refers to a composition obtained from or derived from the target subject, which contains cellular entities and / or other molecular entities to be characterized and / or identified, for example, based on physical, biochemical, chemical and / or physiological characteristics.

[0029] In some embodiments, the sample may be a tissue sample, blood, or other fluid sample of biological origin, such as a biopsy tissue sample or tissue culture or cells derived therefrom, obtained from a subject (the subject may be a human or a non-human mammal, preferably a human). The source of the tissue sample may be solid tissue, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy tissue or aspirates; blood or any blood component; body fluids; cells from any stage of an individual's pregnancy or development; or plasma. The term "sample" includes biological samples that have been processed in any way after their acquisition, such as by reagent treatment, stabilization, or enrichment for certain components (such as proteins or polynucleotides), or embedding in a semi-solid or solid matrix for sectioning purposes. In this invention, there are no particular limitations on the sample; detection of FOLH1 in any sample for the purpose of diagnosing maintenance intestinal metaplasia and progressive intestinal metaplasia, predicting intestinal metaplasia outcomes, and / or assessing the efficacy of intestinal metaplasia treatment falls within the scope of this invention.

[0030] In specific embodiments, the samples described in this invention include, but are not limited to: tissues, blood, tissue-derived cells, blood-derived cells, serum, plasma, lymph, synovial fluid, exosomes, cell extracts, feces, urine, saliva, sputum, synovial fluid, pleural effusion, peritoneal effusion, serous cavity effusion, lymph, cerebrospinal fluid, uterine fluid, digestive juices, bile, bronchial lavage fluid, organs, and any combination thereof. In a preferred embodiment, the sample is selected from the subject's tissues (e.g., intestinal metaplasia tissue in the gastric antrum).

[0031] In some implementations, the reagent is used to detect the expression level of FOLH1 in test samples from subjects using spatial transcriptomics analysis (DSP), sequencing technology, nucleic acid hybridization technology, nucleic acid amplification technology, and protein immunoassay technology.

[0032] In some embodiments, the subject refers to any animal, including both human and non-human animals. The term non-human animals includes all vertebrates, such as mammals, including non-human primates (especially higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any livestock or pets; as well as non-mammals, such as chickens, amphibians, reptiles, etc. In specific embodiments of the present invention, the subject is preferably a human.

[0033] In this invention, the inventors' team initially conducted a real-world clinical sequential case study (gastric mucosal intestinal metaplasia tissue samples taken at different time points within the same medical institution). All enrolled cases were patients pathologically diagnosed with intestinal metaplasia. Based on their disease outcomes, intestinal metaplasia was categorized into two types: progressive intestinal metaplasia and maintenance intestinal metaplasia. For progressive intestinal metaplasia, the starting point was the time when the first endoscopic biopsy pathological result in the sequential case diagnosed antral intestinal metaplasia. If, within 5 years, the endoscopic biopsy pathological result diagnosed high-grade intraepithelial neoplasia / intramucosal carcinoma / adenocarcinoma of the antrum, it was considered progressive intestinal metaplasia. For maintenance intestinal metaplasia, the starting point was the time when the first endoscopic biopsy pathological result diagnosed antral intestinal metaplasia, and the last endoscopic biopsy pathological result diagnosed antral intestinal metaplasia.

[0034] A second aspect of the invention provides a product for diagnosing and differentiating between maintenance intestinal metaplasia and progressive intestinal metaplasia, predicting intestinal metaplasia outcomes, and / or assessing the efficacy of intestinal metaplasia treatment.

[0035] Furthermore, the product includes the reagent described in the first aspect of the present invention for detecting the expression level of FOLH1 in a sample.

[0036] Furthermore, the product includes diagnostic kits, diagnostic chips, and / or test strips for diagnosing and differentiating between maintenance and progressive intestinal metaplasia, predicting intestinal metaplasia outcomes, and / or assessing the efficacy of intestinal metaplasia treatment.

[0037] Furthermore, the samples include tissue samples, blood samples, serum samples, plasma samples, exosome samples, cell samples, fecal samples, and / or urine samples from the subject.

[0038] Furthermore, the samples are derived from subjects, who may be human or non-human animals.

[0039] In some embodiments, the detection kit includes primers, probes, or chips that specifically bind to FOLH1. In some embodiments, the detection chip includes a solid support and probes that specifically recognize FOLH1 attached to the solid support.

[0040] In some embodiments, the test kit further includes one or more substances selected from the group consisting of: a container, instructions for use, a positive control, a negative control, a buffer, an adjuvant, or a solvent. The test kit includes RT-PCR kits, DNA microarray kits, ELISA kits, protein microarray kits, rapid test kits, or MRM (multiple reaction monitoring) kits.

[0041] In a specific embodiment, the RT-PCR kit may further include elements necessary for a reverse transcription polymerase chain reaction. The RT-PCR kit contains a pair of primers specifically targeting a gene encoding a marker protein. Each primer is a nucleotide having a nucleic acid sequence specifically targeting the gene. The RT-PCR kit may also include test tubes or suitable dishes, reaction buffers (at different pH values ​​and magnesium concentrations), deoxynucleotides (dNTPs), enzymes (e.g., Taq polymerase and reverse transcriptase), deoxyribonuclease inhibitors, ribonuclease inhibitors, DEPC-water, and sterile water.

[0042] In a specific embodiment, the ELISA kit may further include elements necessary for performing the ELISA. The ELISA kit may contain an antibody specifically targeting a protein (the FOLH1 protein described in this invention). The antibody has high selectivity and affinity for the labeled protein, no cross-reactivity with other proteins, and may be a monoclonal antibody, polyclonal antibody, or recombinant antibody. Furthermore, the ELISA kit may contain an antibody specifically targeting a control protein. Additionally, the ELISA kit may further include reagents capable of detecting the bound antibody, such as a labeled secondary antibody, a chromophore, an enzyme (e.g., conjugated to an antibody), a substrate thereof, or a substance capable of binding the antibody.

[0043] In some implementations, the detection chip (microarray chip) refers to a solid support containing linked nucleic acid or peptide probes. The array typically contains a variety of different nucleic acid or peptide probes attached to a substrate surface at different known locations. These arrays, also known as microarrays, can typically be produced using mechanosynthesis or photoguided synthesis methods, which combine photolithography and solid-phase synthesis methods. The array can contain a flat surface or can be nucleic acids or peptides on beads, gels, polymer surfaces, fibers such as optical fibers, glass, or any other suitable substrate. The array can be packaged in a way that allows for diagnostic or other manipulation of a fully functional device.

[0044] In a specific implementation, the detection chip includes a gene chip and a protein chip. The gene chip includes a solid support and oligonucleotide probes ordered immobilized on the solid support, wherein the oligonucleotide probes specifically correspond to part or all of the sequence represented by FOLH1. The protein chip includes a solid support and specific antibodies or ligands for the FOLH1-encoded protein immobilized on the solid support. The solid support can be made of various commonly used materials in the chip field, including but not limited to: plastic products, microparticles, membrane carriers, etc.

[0045] A third aspect of the invention provides the use of reagents for detecting FOLH1 expression levels in samples in the preparation of systems and / or devices for diagnosing and differentiating between maintenance and progressive intestinal metaplasia, predicting intestinal metaplasia outcomes, and / or assessing the efficacy of intestinal metaplasia treatment.

[0046] Furthermore, the reagent is the reagent for detecting the expression level of FOLH1 in the sample as described in the first aspect of the present invention.

[0047] Furthermore, the samples include tissue samples, blood samples, serum samples, plasma samples, exosome samples, cell samples, fecal samples, and / or urine samples from the subject.

[0048] Furthermore, the samples are derived from subjects, who may be human or non-human animals.

[0049] A fourth aspect of the present invention provides a system and / or apparatus for diagnosing and differentiating between maintenance intestinal metaplasia and progressive intestinal metaplasia, predicting intestinal metaplasia outcomes, and / or assessing the efficacy of intestinal metaplasia treatment.

[0050] Furthermore, the system and / or device includes a processor, an input module, and an output module;

[0051] The processor is used to perform logical operations on the input information using bioinformatics methods; the input module is used to input the FOLH1 expression level in the subject sample, and a computer-readable medium containing instructions, which, when executed by the processor, executes an algorithm at the FOLH1 input expression level; the output module is used to output the subject's intestinal metaplasia type, intestinal metaplasia outcome, and / or the therapeutic efficacy of intestinal metaplasia.

[0052] Furthermore, the samples include tissue samples, blood samples, serum samples, plasma samples, exosome samples, cell samples, fecal samples, and / or urine samples from the subject.

[0053] Furthermore, the subjects are either human or non-human animals.

[0054] In this invention, the system and / or apparatus is a method for distinguishing different components, elements, parts, sections, or assemblies at different levels. However, if other terms can achieve the same purpose, they can be replaced by other expressions. Those skilled in the art will know that this invention can be implemented as a device, method, or computer program product. Therefore, the disclosure of this invention can be specifically implemented in the following forms: entirely hardware, entirely software (including firmware, resident software, microcode, etc.), or a combination of hardware and software. Furthermore, in some specific embodiments, this invention can also be implemented as a computer program product in one or more computer-readable media containing computer-readable program code.

[0055] The fifth aspect of the invention provides the use of the reagent described in the first aspect of the invention for detecting the expression level of FOLH1 in a sample in the diagnosis of maintenance intestinal metaplasia and progressive intestinal metaplasia, predicting intestinal metaplasia outcomes and / or evaluating the efficacy of intestinal metaplasia treatment.

[0056] Furthermore, the samples include tissue samples, blood samples, serum samples, plasma samples, exosome samples, cell samples, fecal samples, and / or urine samples from the subject.

[0057] Furthermore, the samples are derived from subjects, who may be human or non-human animals.

[0058] The sixth aspect of the present invention provides a method for diagnosing and differentiating between maintenance intestinal metaplasia and progressive intestinal metaplasia, predicting intestinal metaplasia outcomes, and / or assessing the efficacy of intestinal metaplasia treatment.

[0059] Furthermore, the method includes: detecting the FOLH1 expression level in samples from subjects to diagnose and differentiate between maintenance intestinal metaplasia and progressive intestinal metaplasia, predicting intestinal metaplasia outcomes, and / or assessing the efficacy of intestinal metaplasia treatment.

[0060] Furthermore, the detection of FOLH1 expression level can be achieved by detecting the FOLH1 RNA level, the FOLH1 DNA level, the FOLH1 protein level, or the number of FOLH1-positive expression cells.

[0061] Furthermore, the subject-derived samples include subject-derived tissue samples, blood samples, serum samples, plasma samples, exosome samples, cell samples, fecal samples, and / or urine samples.

[0062] Furthermore, the subjects are either human or non-human animals.

[0063] The present invention also provides a computer-readable storage medium. The computer-readable storage medium stores a computer program that, when executed by a processor, implements the system and / or apparatus described in the fourth aspect of the present invention.

[0064] The present invention also provides a method for inhibiting the development of maintenance intestinal metaplasia into progressive intestinal metaplasia, the method comprising the following steps: administering a therapeutically effective amount of a FOLH1 promoter to a subject in need, the FOLH1 promoter comprising substances that increase FOLH1 expression levels, substances that enhance FOLH1 activity, substances that delay FOLH1 metabolism, and / or any combination thereof.

[0065] In some embodiments, the FOLH1 promoter includes naturally purified substances, modified naturally purified substances, semi-synthetic substances, and / or chemically synthesized substances. In specific embodiments, the FOLH1 promoter includes a vector expressing FOLH1, nanoparticles carrying the FOLH1 gene, a viral vector carrying the FOLH1 gene, a PEG-modified protein encapsulating the FOLH1 gene or protein, protein microspheres encapsulating the FOLH1 gene or protein, liposomes encapsulating the FOLH1 gene or protein, extracellular vesicles encapsulating the FOLH1 gene or protein, and / or any combination thereof.

[0066] The method provided above for diagnosing and differentiating between maintenance intestinal metaplasia and progressive intestinal metaplasia, predicting intestinal metaplasia outcomes, and / or assessing the efficacy of intestinal metaplasia treatment specifically includes the following steps:

[0067] (1) Collect samples from the subjects;

[0068] (2) Detect the expression level of FOLH1 in samples from subjects;

[0069] (3) Based on the detected FOLH1 expression level, diagnose and differentiate between maintenance intestinal metaplasia and progressive intestinal metaplasia, predict intestinal metaplasia outcomes and / or assess the efficacy of intestinal metaplasia treatment.

[0070] The present invention also provides a biomarker for diagnosing and differentiating between maintenance intestinal metaplasia and progressive intestinal metaplasia, predicting intestinal metaplasia outcomes and / or assessing the efficacy of intestinal metaplasia treatment, wherein the biomarker is FOLH1.

[0071] This invention also provides the application of the biomarker FOLH1 in diagnosing and differentiating between maintenance and progressive intestinal metaplasia, predicting intestinal metaplasia outcomes, and / or assessing the efficacy of intestinal metaplasia treatment.

[0072] In this invention, any method that uses the biomarker FOLH1 described in this invention to diagnose and differentiate between maintenance intestinal metaplasia and progressive intestinal metaplasia, predict intestinal metaplasia outcomes, and / or assess the efficacy of intestinal metaplasia treatment falls within the scope of protection of this invention. It is not limited to the specific method of using the biomarker FOLH1. As long as it can achieve or substantially achieve the purpose of diagnosing and differentiating between maintenance intestinal metaplasia and progressive intestinal metaplasia, predicting intestinal metaplasia outcomes, and / or assessing the efficacy of intestinal metaplasia treatment, it is within the scope of protection of this invention.

[0073] Compared with the prior art, the present invention has the following advantages:

[0074] (1) Based on spatial transcriptomics analysis and clinical sequential cases, this invention first discovered that the biomarker FOLH1 can effectively distinguish between progressive intestinal metaplasia and maintenance intestinal metaplasia. Combined with endoscopy and pathological biopsy, risk stratification of intestinal metaplasia can be performed, which can further advance the early diagnosis window of gastric cancer from intraepithelial neoplasia, improve the detection rate of progressive intestinal metaplasia, reduce the waste of endoscopic resources, and solve the technical problem of the current continuous increase in the incidence of intestinal metaplasia but lack of effective risk assessment methods. It has important scientific significance and clinical application value.

[0075] (2) Compared with gastric cancer risk scores based on questionnaires, this invention uses real-world sequential samples for DSP detection and verification, ensuring the authenticity and objectivity of the results. Furthermore, this invention is based on the gold standard for diagnosing intestinal metaplasia—endoscopic pathological biopsy—and performs immunohistochemical staining on biopsy specimens, a mature technique with stable and reliable results.

[0076] (3) Compared with the classification by intestinal metaplasia pathological subtype, the detection of this invention is not significantly correlated with the number of biopsies, is not affected by sampling errors, and is applied by immunohistochemistry technology routinely carried out in clinical practice. The technology is mature and stable, has low dependence on pathology technicians, and is highly reproducible.

[0077] (4) Compared with diagnostic markers related to gastric cancer, the biomarker FOLH1 provided by this invention can be accurately applied to intestinal metaplasia, with high accuracy, high sensitivity and high specificity. Furthermore, it is based on immunohistochemistry of endoscopic pathological biopsy samples, avoiding the instability of serological tests.

[0078] In summary, this invention solves the predicament of existing technologies that lack direct relevant research to distinguish between progressive and maintenance intestinal metaplasia, as well as technical problems such as low sensitivity and specificity, and poor patient compliance due to various reasons, thus bringing convenience to the early screening and prevention of intestinal metaplasia. Attached Figure Description

[0079] Figure 1 is a simulation diagram of the Correa pattern of gastric mucosal precancerous state progressing to gastric cancer;

[0080] Figure 2 shows a panoramic view of all ROIs obtained based on DSP;

[0081] Figure 3 shows representative images of intestinal metaplasia-gastric cancer based on pathological histology;

[0082] Figure 4 is a heatmap visualizing the correlation results of ROI gene expression. The horizontal and vertical axes, from left to right, represent epithelial cells_1_001, immune cells_1_001, epithelial cells_1_002, immune cells_1_002, epithelial cells_1_003, immune cells_1_003…epithelial cells_1_0026, immune cells_1_0026, epithelial cells_2_001, immune cells_2_001, epithelial cells_2_002, immune cells_2_002…epithelial cells_2_03 3. Immune cells_2_033, the vertical axis from top to bottom represents epithelial cells_1_001, immune cells_1_001, epithelial cells_1_002, immune cells_1_002, epithelial cells_1_003, immune cells_1_003… epithelial cells_1_0026, immune cells_1_0026, epithelial cells_2_001, immune cells_2_001, epithelial cells_2_002, immune cells_2_002… epithelial cells_2_033, immune cells_2_033;

[0083] Figure 5 shows the statistical results of FOLH1 differential expression in different groups in the training set, where *P<0.05; **P<0.01, N1: initial group of maintenance intestinal metaplasia; N2: final group of maintenance intestinal metaplasia; P1: initial group of progressive intestinal metaplasia; P2: final group of progressive intestinal metaplasia.

[0084] Figure 6 shows the ROC curve results of FOLH1 in different groups in the training set. In the figure, A: N1 / N2, P1, P2; B: N1 / N2; C: N1 / P1; D: N1 / P2; N1: initial group of maintenance intestinal metaplasia; N2: final group of maintenance intestinal metaplasia; P1: initial group of progressive intestinal metaplasia; P2: final group of progressive intestinal metaplasia.

[0085] Figure 7 shows an immunohistochemical staining image of FOLH1;

[0086] Figure 8 shows the statistical results of FOLH1 differential expression in different groups in the validation set, where *P<0.05; **P<0.01; ***P<0.001. N1: Initial group of maintenance intestinal metaplasia; N2: Outcome group of maintenance intestinal metaplasia; P1: Initial group of progressive intestinal metaplasia; P2: Outcome group of progressive intestinal metaplasia;

[0087] Figure 9 shows the ROC curve results of FOLH1 in different groups in the validation set. In the figure, A: N1 / N2, P1, P2; B: N1 / N2; C: N1 / P1; D: N1 / P2; N1: initial group of maintenance intestinal metaplasia; N2: final group of maintenance intestinal metaplasia; P1: initial group of progressive intestinal metaplasia; P2: final group of progressive intestinal metaplasia. Detailed Implementation

[0088] The present invention will be further illustrated below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. To facilitate understanding of the invention, the following terms used herein are explained:

[0089] As used herein, the terms “comprising” or “including” mean that any one or more of the stated elements or components are included, without excluding other elements or other components.

[0090] As used herein, the term "biomarker" is synonymous with "marker," referring to a molecular indicator possessing specific biological, biochemical, or other characteristics that can be used to determine the presence or absence of a particular disease or condition and / or the severity of a particular disease or condition. In a specific embodiment of the invention, the biomarker is FOLH1.

[0091] As used herein, the term "expression level" is synonymous with "level," referring to the absolute or relative amount of the biomarker FOLH1 described in this invention. The expression level of the biomarker FOLH1 described in this invention can be determined by various techniques. In particular, the absolute or relative amount of the biomarker FOLH1 described in this invention can be detected by methods well known to those skilled in the art.

[0092] In some implementations, methods for detecting FOLH1 RNA levels in samples include, but are not limited to: reverse transcription polymerase chain reaction (RT-PCR), RNA sequencing (RNA-seq), and in situ hybridization (ISH).

[0093] RT-PCR is a commonly used method for detecting RNA levels. First, total RNA is extracted from the sample. Then, reverse transcriptase is used to reverse transcribe the RNA into cDNA. The cDNA is then used as a template for PCR amplification. The amount of amplified product is used to determine the RNA expression level of FOLH1. Real-time quantitative PCR (qRT-PCR), developed from RT-PCR, can perform relative or absolute quantification of RNA and has advantages such as high sensitivity and specificity.

[0094] RNA sequencing technology can perform high-throughput sequencing of all RNA in a sample, thereby comprehensively and accurately detecting the RNA expression level of FOLH1, and can also discover new transcripts and alternative splicing events. Its advantage is that it can provide richer transcriptome information.

[0095] In situ hybridization can detect the expression and localization of FOLH1 RNA in tissues or cells. Labeled nucleic acid probes are used to hybridize with FOLH1 RNA in tissues or cells, and the hybridization signal is then observed through color development or fluorescent labeling to determine the distribution and expression level of FOLH1 RNA in the tissues or cells.

[0096] In some implementations, methods for detecting the DNA level of FOLH1 in a sample include, but are not limited to: polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH), and gene sequencing.

[0097] In some implementations, methods for detecting the protein expression level of FOLH1 in a sample include, but are not limited to: Western blot, immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA).

[0098] In some implementations, methods for detecting the number of FOLH1-positive cells in a sample include, but are not limited to, flow cytometry (FCM) and immunofluorescence staining (IF).

[0099] The present invention does not impose any particular limitations on the methods for detecting the RNA level of FOLH1 in a sample, the DNA level of FOLH1 in a sample, the protein expression level of FOLH1 in a sample, or the number of FOLH1-positive cells in a sample. Those skilled in the art can make conventional choices according to actual needs.

[0100] In this paper, the term "primer" refers to a 7-50 nucleic acid sequence that forms complementary base pairs to the template strand and serves as a starting point for template replication. Primers are usually synthesized, but naturally occurring nucleic acids can also be used. The primer sequence does not necessarily need to be identical to the template sequence, as long as it is sufficiently complementary to hybridize with the template.

[0101] As used herein, the term "probe" refers to a nucleic acid fragment, such as RNA or DNA, ranging from a few to hundreds of bases long, that can specifically bind to mRNA and can determine the presence of a specific mRNA through labeling. Probes can be prepared in the form of oligonucleotide probes, single-stranded DNA probes, double-stranded DNA probes, and RNA probes.

[0102] As used herein, the term "antibody" refers to a specific immunoglobulin targeting an antigenic site. In this invention, an antibody refers to an antibody that specifically binds to the FOLH1 protein described herein, and can be manufactured according to conventional methods in the art. Antibodies can take the form of polyclonal or monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) antibodies, multispecific antibodies (such as bispecific antibodies), monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antigen-binding site, and any other modified immunoglobulin molecule containing an antigen-binding site, provided that the antibody exhibits the desired biological binding activity.

[0103] As used herein, the term "peptide" refers to a class of substances that have a high binding capacity to a target substance (the biomarker protein described in this invention) and do not undergo denaturation during heat or chemical treatment. Furthermore, due to their small size, they can be used as fusion proteins by attaching to other proteins. Specifically, because they can specifically attach to high molecular weight protein chains, they can be used as diagnostic kits and drug delivery substances.

[0104] In this article, the term "Digital Spatial Profiler (DSP)" is used. DSP is one of the most advanced genomics technologies currently available, capable of highly precise analysis of gene and cellular changes during life development, both temporally and spatially. It can accurately select multiple lesion regions on a single paraffin-embedded tissue section, enabling in-situ co-analysis of hundreds of proteins and thousands of mRNAs, truly achieving spatial localization of cells within tissues while simultaneously detecting their gene expression. DSP technology has significant advantages in tumor microenvironment research, tumor heterogeneity, and tumor immunity studies; however, no researchers have yet used DSP technology to conduct research on risk stratification of intestinal metaplasia-gastric cancer.

[0105] In some implementations, this invention performs spatial transcriptomics analysis on biopsy specimens from sequential cases of progressive and maintenance intestinal metaplasia. The analysis focuses on differentially expressed genes in the epithelial cells of the intestinal metaplasia glands in biopsy samples from progressive and maintenance intestinal metaplasia in the gastric antrum, yielding the biomarker FOLH1. The application of this marker gene can effectively differentiate between progressive and maintenance intestinal metaplasia in the gastric antrum and effectively predict the outcome of intestinal metaplasia, thereby advancing the diagnostic window for gastric cancer, reducing missed diagnoses due to insufficient early detection methods, providing a basis for determining the follow-up timeframe for patients with gastric antrum intestinal metaplasia, reducing the waste of endoscopic resources, and demonstrating broad clinical application prospects.

[0106] As used herein, the term "diagnosis" refers to the discovery, judgment, or recognition of an individual's health status or condition based on one or more symptoms, data, or other information relevant to that individual. An individual's health status may be diagnosed as healthy / normal (i.e., without disease or ailment), or as unhealthy / abnormal (i.e., with disease or ailment), or as a specific degree of disease severity or progression (e.g., maintenance intestinal metaplasia, progressive intestinal metaplasia). The term "diagnosis" includes the early detection of a disease / symptom associated with a specific disease or ailment; the characteristics or classification of the disease; the discovery of disease progression, cure, or recurrence; and the discovery of an individual's response to the disease after treatment or intervention. In a specific embodiment of the invention, the diagnosis includes distinguishing between progressive intestinal metaplasia and maintenance intestinal metaplasia.

[0107] As used herein, the term "effective amount" refers to the amount that has a therapeutic effect or the amount required to produce a therapeutic effect in a subject. For example, a therapeutically or pharmaceutically effective amount is the amount of drug required to produce the desired therapeutic effect, which can be reflected by the results of clinical trials, animal model studies, and / or in vitro studies. A pharmaceutically effective amount depends on several factors, including but not limited to: the subject's characteristics (such as height, weight, sex, age, and medication history), the severity of the disease, etc.

[0108] The reagents and raw materials used in this invention are readily available to those skilled in the art and can be obtained commercially unless otherwise specified. Experimental methods not specified in this invention are generally performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

[0109] Example 1: Clinical Sample Collection and Screening

[0110] 1. Experimental materials

[0111] Sample source: Wangjing Hospital as the central unit, in conjunction with various branch units.

[0112] Sample characteristics: Intestinal metaplastic paraffin-embedded sample.

[0113] Reagents and instruments: wax blocks, formaldehyde, xylene, disodium hydrogen phosphate, potassium dihydrogen phosphate, glycerol protein adhesive tablets, paraffin, hematoxylin, eosin, sodium iodate, glacial acetic acid, glycerol, anhydrous ethanol, xylene, hydrochloric acid, neutral resin, scalpel (for trimming), paintbrush (for spreading slides), etc. Digital Spatial Multi-Target Analysis System (DSP) (GeoMx DSP, Nanostring).

[0114] 2. Experimental Methods

[0115] Clinical sample collection

[0116] (1) Sample source

[0117] The clinical cases were derived from Wangjing Hospital as the central unit, in conjunction with various branch centers in Beijing. Sequential samples from the same patient who underwent multiple endoscopic biopsies at this hospital were selected from the pathological tissue sample bank; all samples were paraffin-embedded tissue.

[0118] (2) Diagnostic criteria

[0119] 1) Intestinal metaplasia

[0120] ① Gastroscopy diagnostic criteria: Intestinal metaplasia is visible under endoscopy as pale yellow nodules measuring 2-3 mm in size, which can be single or multiple, with a villous or fine granular surface, slightly flattened and protruding from the gastric mucosa; the reflectivity of intestinal metaplasia is stronger than that of normal tissue under endoscopy, and small nodules can be seen as isolated or multiple, with a soft and smooth surface, and a porcelain white translucent appearance; gastric areas are generally distributed in strips or diffusely, with strip-like enlargement, feather-like or fish-scale-like arrangement; grayish-white irregular granules are diffuse.

[0121] ② Pathological diagnosis and grading

[0122] Intestinal metaplasia: Intestinal metaplasia refers to the replacement of gastric mucosal epithelial cells by intestinal-type epithelial cells. The degree of intestinal metaplasia is calculated when the area of ​​the intestinal metaplasia zone accounts for 1 / 3 of the total area of ​​the glands and surface epithelium.

[0123] Mild: The area of ​​intestinal metaplasia is ≤ 1 / 3 of the total area of ​​glands and surface epithelium;

[0124] Moderate: 1 / 3 of the total area of ​​glands and surface epithelium ≤ intestinal metaplasia area ≤ 2 / 3 of the total area of ​​glands and surface epithelium;

[0125] Severe: The area of ​​intestinal metaplasia is ≥ 2 / 3 of the total area of ​​glands and surface epithelium.

[0126] AB-HID / PAS staining can differentiate IM subtypes and confirm the diagnosis of IM. The diagnostic grading criteria for intestinal metaplasia include four levels: 0 indicates none, + indicates mild, ++ indicates moderate, and +++ indicates severe.

[0127] 2) Intraepithelial neoplasia

[0128] Mild: The basic structure is preserved, with only slight disorder. There may be a small number of irregular branches and budding. The nuclei are slightly larger and thinner, deeply stained, and round, oval, or columnar. They are arranged relatively densely and located at the base of the cell. There is a slight increase in nuclear division.

[0129] Moderate: Cellular structure is significantly disordered. Nuclei are enlarged, oval or rod-shaped, varying in size, densely stained, and arranged haphazardly and irregularly, exhibiting a pseudostratified structure, with frequent nuclear division. Epithelial cells are columnar, mucus secretion is significantly reduced, goblet cells are rare, and Paneth cells are absent. Glandular ducts are densely arranged, with tortuous branches, irregular luminal structure, and varying shapes and sizes.

[0130] Severe: Cellular atypia is very pronounced, sometimes making it difficult to distinguish from well-differentiated mucosal endothelial carcinoma. Epithelial cells are tall columnar (intestinal type), without goblet cells and Paneth cells; or they are cuboidal or amorphous (gastric type), with lost mucus secretion. Nuclei are significantly enlarged, varying in size and shape, disordered and irregularly arranged, with an increased nucleus-to-cell ratio, dense staining or loose reticular formation, prominent nucleoli, and frequent mitotic figures. Glandular structure is significantly disordered, with extremely irregular shape, size, and arrangement, commonly exhibiting budding, branching, papillary formation, shared cell walls, and back-to-back structures.

[0131] 3) Gastric cancer: The pathological manifestations of gastric cancer that progresses from intestinal metaplasia are mainly malignant lesions of gastric mucosal epithelial cells. The cells change from columnar to square, with large nuclei, increased nucleocytoplasmic ratio, prominent nucleoli, obvious glandular atypia, increased mitotic figures, pathological nuclear division, loss of cell polarity, and the nucleus extending to the lateral side of the glandular lumen; it is an invasive carcinoma confined to the mucosa or submucosa.

[0132] 4) H. pylori infection: Observe H. pylori on the surface of the gastric mucus layer, surface epithelium, pitted epithelium and glandular epithelium.

[0133] 0: No HP was observed on the special staining slide;

[0134] +: Occasionally, or less than 1 / 3 of the total length of the specimen, a few HPs are seen;

[0135] ++: HP distribution reaches or exceeds 1 / 3 but less than 2 / 3 of the total specimen length or is continuous, thin and sparse on the epithelial surface;

[0136] +++: *Helicobacter pylori* (HP) is present in clusters, basically distributed along the entire length of the specimen. *Helicobacter pylori* (IM) mucosal surfaces usually do not have HP colonization; it is advisable to search in non-IM areas. For specimens with significant inflammatory reactions but no HP found in HE-stained sections, special staining should be performed for careful searching.

[0137] (3) Inclusion criteria

[0138] ① Patients who meet the diagnostic criteria for intestinal metaplasia during their first visit;

[0139] ②No gender restriction, age 18 and above;

[0140] ③ Patients who have undergone two or more consecutive gastroscopy examinations and have received pathological diagnosis;

[0141] ④ Those with complete medical records.

[0142] (4) Exclusion criteria

[0143] ①Those with a history of stomach surgery;

[0144] ② The patient was diagnosed with other complications besides the aforementioned intestinal metaplasia in the antrum of the stomach, such as reparative hyperplasia, dysplasia, intraepithelial neoplasia, gastric cancer, etc.

[0145] ③ Patients with gastrointestinal ulcers, suspected malignant transformation, existing malignant transformation, or metastatic tumors;

[0146] ④ Patients with severely missing clinical medical records.

[0147] (5) Grouping criteria

[0148] A sequential case refers to the same patient who has undergone multiple gastroscopic biopsies at this hospital.

[0149] Progressive intestinal metaplasia: The starting point was the time when the first gastroscopy biopsy pathology result in the sequential cases diagnosed intestinal metaplasia in the antrum, and the end point was the time when the biopsy pathology result in the diagnosis of high-grade intraepithelial neoplasia / intramucosal carcinoma / adenocarcinoma in the antrum within 5 years.

[0150] Maintenance intestinal metaplasia: Sequential cases were selected based on an interval of ≥5 years between two endoscopic biopsies. The starting point was the time when the pathological result of the first endoscopic biopsy diagnosed intestinal metaplasia in the antrum, and the ending point was the time when the pathological result of the last endoscopic biopsy diagnosed intestinal metaplasia in the antrum.

[0151] 3. Experimental results

[0152] Patients selected based on the above diagnostic and inclusion / exclusion criteria were numbered and included in the database. A total of 31 patients were enrolled in the maintenance intestinal metaplasia group, and 31 patients were enrolled in the progressive intestinal metaplasia group. Sequential samples from 4 patients in each group were randomly selected. Sixteen paraffin-embedded tissue blocks were uniformly sectioned, stained with hematoxylin and eosin (HE), and diagnosed by two senior pathologists, awaiting subsequent DSP testing. Detailed information on 8 patients with intestinal metaplasia is shown in Table 1.

[0153] Table 1 DSP Sample Information

[0154] All other patients were enrolled in the validation group. All paraffin-embedded sections were stained with hematoxylin and eosin (HE) and then diagnosed by two senior pathologists. Specific information is shown in Tables 2-3.

[0155] Table 2. Sample information from the maintenance intestinal metaplasia validation group.

[0156] Table 3. Sample information from the validation group for progressive intestinal metaplasia.

[0157] Example 2 Spatial Transcriptomics Analysis (DSP) Sequencing

[0158] 1. Experimental subjects

[0159] DSP samples: 16 paraffin-embedded samples, 4 pairs of sequential intestinal metaplasia samples from the maintenance group and 4 pairs of sequential intestinal metaplasia samples from the progression group.

[0160] 2. Experimental methods

[0161] 2.1 Preparation of tissue sections

[0162] ① Section thickness: 5μm; ② Sectioning time: Fresh sections are recommended. If preservation is required, the preservation time should not exceed 14 days; ③ Sectioning location: If the sample surface is exposed to air, the first 2-3 sections should be discarded; ④ Preservation period: 3-year FFPE block; ⑤ Tissue sections must be placed within the effective position on the slide (35.3mm long × 14.1mm wide). If there are multiple sections on each slide, ensure that the tissue spacing is at least 2-3mm. The specific placement can be determined according to the size of the tissue; ⑥ It is recommended to use cationic anti-detachment slides (SuperFrost™ Plus slides) to prepare sections to prevent tissue detachment.

[0163] 2.2 Sample quality control

[0164] DV200: Percentage of RNA fragments >200nt; DV200 cannot be correlated with DSP RNA quality; ① DV200 <30% indicates poor sample quality and is not recommended; ② DV200 between 40% and 60% shows no real difference and may or may not have any effect; ③ DV200 >60% indicates very good sample quality and is recommended.

[0165] 2.3 Fixation and processing of tissue sections

[0166] 2.3.1 Paraffin-embedded tissue sections

[0167] 1) Unlock before use. 2) Adjust the cutting angle. The recommended setting angle for the Leica biosystem blade and blade holder is 1 to 5°. 3) When sectioning, press down the wax block (as the wax block surface is relatively flat, do not easily adjust the blade head) and adjust the section thickness. 4) Always lock the blade head when loading and unloading the wax block to prevent hand injury. 5) Ensure the blade holder handle is aligned with the bevel of the blade holder. Over-adjusting will damage the blade holder; remember this. 6) For roughing, use the manual wheel or press the roughing button to expose the tissue to a certain level, at which a targeted section can be obtained. 7) After cutting, rotate the large wheel continuously to cut out the wax strip. Hold the brush in your left hand to support the lower end of the wax strip, and use tweezers in your right hand to pick up the wax strip. Place the wax strip face up in the slide spreader to flatten it (you can also put the wax strip in a 30% ethanol aqueous solution, relying on the surface tension of the ethanol to open up the wrinkles, and then transfer the wax strip to warm water). Use tweezers to separate the excess wax. 8) Take a labeled glass slide, vertically insert it into the water to retrieve the section, generally so that the tissue is located about 2 / 3 of the way down the slide. 9) Stand the glass slide upright on the edge of the baking table, drain the excess water, and then place the sections on the slide spreader in the order of retrieval (baking temperature 65-70℃) for baking, waiting for staining. 10) After finishing work, please lock the machine and clean and wipe it.

[0168] 2.3.2 HE staining

[0169] HE staining procedure: 1) Dewax sections to water. 2) Stain with hematoxylin for 10 minutes. 3) Rinse thoroughly with tap water. 4) Stain with differentiation solution for 5 seconds. 5) Rinse thoroughly with tap water. 6) Stain with blue solution for 5 seconds. 7) Rinse thoroughly with tap water. 8) Stain with 0.1% eosin for 1 minute. 9) Stain with 95% ethanol for 2 minutes. 10) Stain with 95% ethanol for 2 minutes. 11) Stain with 100% ethanol I for 3 minutes. 12) Stain with 100% ethanol II for 3 minutes. 13) Stain with xylene I for 7 minutes. 14) Stain with xylene II for 7 minutes. 15) Mount with neutral resin for fixation.

[0170] 2.3.3 Morphological markers

[0171] 1) In situ hybridization incubation with RNA probes. 2) Selection of DSP morphological markers. 3) Staining with morphological markers.

[0172] 4) Load the instrument and set it up.

[0173] 2.3.4 Selecting Regions of Interest (ROIs)

[0174] 1) Clean the bottom of the slide with 70% ethanol. 2) Add 6 mL of buffer to each tissue section. 3) Ensure the slide is clamped in the release position. 4) Load the slide into the DSP instrument and have it recognized. 5) Define the scanning area; select ROIs if desired. 6) Click “Approve ROIs” to enter UV cutting mode. 7) Collect the cut oligos into the well plate using a capillary needle.

[0175] 2.3.5 NGS Library Preparation and Quality Control

[0176] 1) Counting - PCR setup - Establishing the ROIs pool - Purifying PCR materials in the ROIs pool - Quality control - Results. 2) PCR setup. 3) Establishing the ROIs pool. 4) Purification and quality control of PCR materials in the ROIs pool: ① Dilute the library 1:8 (2μL library + 14μL Elution Buffer); ② Quantify library yield using QuBit; ③ Detect the expected fragment size (approximately 130-150bp) using Agilent 4200 Tapestation electrophoresis; ④ The purified library can be stored at -20℃ for a long period until sequencing. 5) Obtain a qualified NGS library.

[0177] 2.3.6 Illumina Sequencing

[0178] After library construction and quality control, sequencing was performed using the Illumina NovaSeq sequencing platform. Illumina sequencing works by binding PCR products to oligos on the sequencer's chip, followed by sequencing-by-synthesis. Therefore, adapters and barcodes are added to the PCR products to ensure proper sequencing. After obtaining the sequencing data, subsequent data analysis was performed. The Illumina sequencing workflow is as follows: 1) Reads1, Index1, Index2, and Reads2 sequences were obtained through sequencing. 2) The initial analysis results were clustered using a temperature and enzyme gradient clustering algorithm to obtain a feature vector matrix of specific paired-end sequence fragments. The temperature and enzyme levels can be adjusted according to the designed sequencing space size. Finally, several corrected sequence fragments based on this matrix were obtained. 3) The obtained sequence fragments are clustered together with other fragments containing paired-end sequence markers and read on the machine. The measurement results are fed back to the chip to determine the location. Using the location information of the sequence fragments, they are reassembled into a complete sequence and signal-to-noise ratio analysis is performed to obtain high-accuracy genome sequence information.

[0179] 3. Data Analysis

[0180] 3.1 Gene probe quantification

[0181] After sequencing is completed, the raw sequencing data (fastq format) is imported into the NanoString DSP probe quantification pipeline (geomxngspipeline software). By combining the fixed sequence of each probe RNA, the quantification results of each probe can be obtained.

[0182] 3.2 ROIs Quality Assessment

[0183] The quality of ROIs is mainly evaluated from the following three aspects:

[0184] 3.3 Technical Signal Quality Control

[0185] (1) The total number of reads for each ROI shall not be less than 1000. (2) The alignment rate shall not be less than 80%. (3) The sequencing saturation shall not be less than 50%.

[0186] 3.4 Technical Background and Quality Control

[0187] (1) The geometric mean of negative probes is greater than 4. (2) The total number of reads in PCR control samples without template is not higher than 1000.

[0188] 3.5 DSP Parameters

[0189] (1) The number of cell nuclei in each ROI is not less than 100. (2) The area of ​​each ROI is not less than 8000 μm. 2 (3) Probe quality control analysis: Before gene normalization, probe RNA will be subjected to quality control analysis, mainly from the following two aspects: ① According to the ratio of the geometric mean of a probe's value in all ROIs to the geometric mean of the other 4 probes corresponding to the gene in all ROIs, if the ratio of the geometric mean of a probe's value in all ROIs to the geometric mean of the other 4 probes corresponding to the gene in all ROIs (GEOMEAN probes in all segments / GEOMEAN probes within the target) is less than 0.1, then this probe will be excluded and will not participate in subsequent analysis. ② According to the GRUBBS OUTLIER test, this step is to filter out the outliers of the 5 probes of the same gene. If a probe is higher or lower than other probes of the same gene, and if the GRUBBS OUTLIER test fails in a certain proportion (usually set to 20%) of ROIs, then the probe will be filtered out.

[0190] 4. Quantitative analysis of genes

[0191] After quality control analysis of the probes, unqualified probes were removed, and the geometric mean of the remaining probes for each gene was calculated to obtain the gene expression value (CTA) in each ROI. ROI and gene filtering: In addition to the gene probes, 80 negative probes were added to each ROI. A LOQ (Limit of Quantitation) value was calculated for each ROI; the LOQ value = the geometric mean of the negative probes.

[0192] 4.1 The filtering criteria for ROIs are: if the expression levels of 10% of the genes in the ROI are above the LOQ value, then the ROI will be retained;

[0193] 4.2 The filtering criterion for genes is: if a gene is higher than the corresponding LOQ value in 10% of ROIs, the gene will be retained.

[0194] 5. Gene standardization methods

[0195] Considering the impact of area, cell nucleus count, sequencing data volume, and other technical noise on gene quantification for each ROI, gene standardization is necessary to eliminate these influences before analyzing gene differences between ROIs. NanoString recommends using the Q3 correction method, specifically: calculate a Q3 value (upper quartile) for all gene expression values ​​in each ROI. Assuming the Q3 value for a given ROI is M, and the geometric mean of the Q3 values ​​for all ROIs is N, then multiply the expression value of each gene in that ROI by a coefficient N / M. This operation is performed for each ROI. After standardization, the Q3 values ​​for each ROI are identical, facilitating subsequent comparisons of differences.

[0196] 6. Experimental Results

[0197] (1) Gene Quantitative Results Display ROIs

[0198] Figure 1 illustrates the pattern of gastric cancer development. Figure 2 shows one of the panoramic views of ROIs marked by DSP morphology on pathological tissue sections of all enrolled cases. First, ROIs were selected and numbered on the pathological tissue sections of each case according to their grouping to achieve the research objective. A total of 97 ROIs were obtained in this study (i.e., in the training set, the ratio of maintenance intestinal metaplasia ROIs to progressive intestinal metaplasia ROIs was 51:46) for subsequent differentially expressed gene analysis. Figure 3 shows images of intestinal metaplasia and gastric cancer lesions from pathological tissue samples taken from lesions before and after the same sequential case. HE imaging revealed the lesions of intestinal metaplasia and gastric adenocarcinoma. DSP morphological marking of the same lesion is the basis for subsequent DSP analysis.

[0199] (2) Correlation analysis among ROIs

[0200] Correlation analysis can assist in screening for abnormal samples among biologically replicated ROIs. The x and y axes represent the ROIs between two groups of samples, respectively. Pearson correlation indicates the correlation between the two groups, with the Pearson correlation coefficient r ranging from |r|≤1. The correlation coefficient among biologically replicated ROIs is r≥0.96, indicating a very strong correlation in gene expression among ROIs. However, due to individual differences, regional spatial differences, and other factors, the r value may vary. Specific results are shown in Figure 4.

[0201] (3) Bioinformatics analysis results

[0202] Gene expression results for each ROI in progressive and maintenance intestinal metaplasia were obtained using the DSP analysis platform, and differentially expressed genes were screened. The screening criteria were: adjusted p-value < 0.05; Log2(fold change) > 0.6.

[0203] Figure 5 shows the expression of FOLH1 in each group of the training set. The results show that FOLH1 exhibits significant differential expression among the groups in the training set.

[0204] ROC curves were plotted based on the expression levels of FOLH1 in each ROI of the training set. We found that the AUC value of FOLH1 was 0.8750 compared to the initial intestinal metaplasia group (N1) and the initial intestinal metaplasia group (P1) in the maintenance group, with a sensitivity of 100.00% and a specificity of 75.00%; the AUC value was 0.9063 compared to the intestinal metaplasia outcome group (P2) in the progression group, with a sensitivity of 100.00% and a specificity of 75.00%; and the AUC value was 0.8438 compared to the intestinal metaplasia outcome group (N2) in the maintenance group, with a sensitivity of 87.50% and a specificity of 75.00%. The AUC value of FOLH1 in the initial intestinal metaplasia group (N1) in the maintenance group compared to all other groups was 0.8750, with a sensitivity of 96.00% and a specificity of 75.00% (see Table 4 and Figure 6). The results showed that FOLH1 can serve as an effective biomarker for risk stratification of intestinal metaplasia and gastric cancer, and has extremely high application value in diagnosing and differentiating between maintenance intestinal metaplasia and progressive intestinal metaplasia, with high accuracy, high specificity and high sensitivity.

[0205] Table 4. ROC curve parameters of FOLH1 in each group of the training set.

[0206] Note: N1: Initial group of maintenance intestinal metaplasia; N2: Outcome group of maintenance intestinal metaplasia; P1: Initial group of progressive intestinal metaplasia; P2: Outcome group of progressive intestinal metaplasia.

[0207] Example 3 Immunohistochemical verification

[0208] 1. Experimental materials

[0209] Paraffin-embedded tissue, PM2235 microtome, HI1210 slide spreader, HI1210 slide oven, high-pressure steam sterilizer (YX280), three-in-one constant temperature water bath (SH.W21.600), BX51 microscope, FOLH1 antibody (BM4078), etc.

[0210] 2. Experimental Methods

[0211] Patients selected based on the above diagnostic criteria and inclusion / exclusion criteria were numbered and included in the database. The validation group consisted of 31 cases of maintenance intestinal metaplasia and 31 cases of progressive intestinal metaplasia. All tissue sections were uniformly stained with hematoxylin and eosin (HE), and two senior pathologists were asked to repeat the diagnosis to confirm compliance with immunohistochemistry requirements (paraffin-embedded samples that did not meet the requirements for immunohistochemistry were also included in the study if the HE staining results for progressive intestinal metaplasia were clear).

[0212] (1) Immunohistochemical steps

[0213] 1) Paraffin-embedded tissue sections were sliced ​​at 2.5 micrometers, developed in a 48°C slide dryer, dried at 63°C, and baked for 1 hour; 2) Tissue dewaxing and hydration (soaking in dewaxing solution I for 15 minutes → dewaxing solution II for 15 minutes → anhydrous ethanol I for 5 minutes → anhydrous ethanol II for 5 minutes → 95% ethanol for 2 minutes → 80% ethanol for 1 minute → washing 3 times with distilled water); 3) Soaking in 0.01M phosphate-buffered saline (PBS) for 5 minutes 3 times; 4) Autoclave heat antigen retrieval: retrieval was performed using citrate buffer (pH 6.0); preheated to boiling; sections were placed in the corresponding autoclave, timed for 3 minutes after EDTA was ejected, and timed for 2.5 minutes after citrate was ejected; cooled to room temperature for 20 minutes; soaked in 0.01M PBS for 5 minutes, repeated 3 times; soaked in 3% H2O2 for 10 minutes; soaked again in 0.01M PBS. Soak in PBS for 5 minutes, repeat 3 times; after adding primary antibody, incubate the slides overnight at 37°C in a humidity chamber; soak in 0.01M PBS for 5 minutes, repeat 3 times; after adding secondary antibody, incubate the slides at 37°C for 30 minutes; soak in 0.01M PBS for 5 minutes, repeat 3 times; DAB staining (microscopic examination); hematoxylin counterstain for 5 minutes, then rinse 3 times with water in a glass jar; after separation with color-separating solution, rinse 3 times with water in a glass jar; after inversion with blue solution, rinse 3 times with water in a glass jar; microscopic examination to observe the staining of cell nuclei; ascending dehydration and clearing (soak in 80% ethanol for 1 minute → soak in 95% ethanol I for 2 minutes → soak in 95% ethanol II for 2 minutes → soak in anhydrous ethanol I for 5 minutes → soak in anhydrous ethanol II for 5 minutes → soak in clearing solution I for 1 minute → soak in clearing solution II for 5 minutes → soak in clearing solution III for 5 minutes); mount with neutral resin and microscopic examination.

[0214] (2) Analysis of immunohistochemical results

[0215] All immunohistochemistry slides were placed under an optical microscope, and two senior pathologists randomly selected 3-5 high-power fields to acquire images. The integrated optical density value of FOLH1 was then calculated using Image Pro Plus 6.0 software. ROC curves were plotted based on the integrated optical density values ​​to analyze the diagnostic value of FOLH1 as a marker for progressive and maintenance intestinal metaplasia.

[0216] (3) Statistical methods

[0217] SPSS 26.0 software system was used for statistical analysis of the obtained data. For normally distributed measurement data, independent samples t-tests were used for analysis, and the results were expressed as mean ± standard deviation (Mean ± SD). For data that did not conform to a normal distribution, nonparametric tests were used, and p < 0.05 was considered statistically significant.

[0218] 3. Experimental Results

[0219] By examining the immunohistochemical pathological slides, we found that in the validation set, FOLH1 showed significant differences in intestinal metaplasia between the maintenance group and the progressive group, as shown in Figures 7 and 8.

[0220] ROC curves were plotted based on the expression levels of FOLH1 in each group of the validation set. We found that the AUC value of FOLH1 was 0.9584 compared to the initial intestinal metaplasia group (N1) in the maintenance group and the initial intestinal metaplasia group (P1) in the progression group, with a sensitivity of 100.00% and a specificity of 87.1%. The AUC value was 0.9392 compared to the intestinal metaplasia outcome group (P2) in the progression group, with a sensitivity of 100.00% and a specificity of 83.87%. The AUC value was 0.7076 compared to the intestinal metaplasia outcome group (N2) in the maintenance group, with a sensitivity of 48.39% and a specificity of 93.55%. The AUC value of FOLH1 in the initial intestinal metaplasia group (N1) in the maintenance group compared to all other groups was 0.8644, with a sensitivity of 85.23% and a specificity of 83.87%. These results indicate that FOLH1 can effectively distinguish between intestinal metaplasia in the maintenance group and intestinal metaplasia in the progression group (see Table 5 and Figure 9). The above results further demonstrate that FOLH1 can serve as an effective biomarker for risk stratification of intestinal metaplasia and gastric cancer, and has extremely high application value in diagnosing and differentiating between maintenance intestinal metaplasia and progressive intestinal metaplasia, with high accuracy, high specificity and high sensitivity.

[0221] Table 5. ROC curve parameters of FOLH1 in each group of the validation set.

[0222] Note: N1: Initial group of maintenance intestinal metaplasia; N2: Outcome group of maintenance intestinal metaplasia; P1: Initial group of progressive intestinal metaplasia; P2: Outcome group of progressive intestinal metaplasia.

Claims

1. Application of reagents for detecting FOLH1 expression levels in samples in the preparation of products for diagnosing and differentiating between maintenance and progressive intestinal metaplasia, predicting intestinal metaplasia outcomes, and / or assessing the efficacy of intestinal metaplasia treatment.

2. The application according to claim 1, characterized in that, The reagents include: Reagents for detecting FOLH1 RNA levels in samples; Reagents for detecting the DNA level of FOLH1 in a sample; Reagents for detecting the protein expression level of FOLH1 in a sample; or A reagent for detecting the number of FOLH1-positive cells in a sample.

3. The application according to claim 2, characterized in that, The reagents used to detect the RNA level of FOLH1 in the sample include reagents for detecting the expression levels of FOLH1 mRNA, lncRNA, and / or miRNA in the sample. The reagents used to detect the FOLH1 DNA level in the sample include reagents for detecting the FOLH1 DNA expression level, DNA methylation level, DNA acetylation level, DNA phosphorylation level, DNA hydroxymethylation level and / or SNP locus genotype in the sample.

4. The application according to claim 3, characterized in that, The reagents used to detect the expression levels of FOLH1 mRNA, lncRNA, and / or miRNA in the sample include primers that specifically amplify FOLH1 and / or probes that specifically recognize FOLH1. The reagents used to detect the DNA expression level, DNA methylation level, DNA acetylation level, DNA phosphorylation level, DNA hydroxymethylation level, and / or SNP genotype of FOLH1 in the sample include reagents that detect the levels using sequencing technology.

5. The application according to claim 2, characterized in that, The reagents used to detect the protein expression level of FOLH1 in the sample include affinity proteins that specifically bind to the protein encoded by FOLH1. The reagents for detecting the number of FOLH1-positive cells in the sample include reagents for detecting the number of FOLH1-positive cells by immunohistochemistry.

6. The application according to claim 5, characterized in that, The affinity proteins include antibodies that specifically bind to proteins encoded by FOLH1, antibody functional fragments, agglutinants, receptors, and / or conjugate antibodies.

7. The application according to any one of claims 1-6, characterized in that, The samples include tissue samples, blood samples, serum samples, plasma samples, exosome samples, cell samples, fecal samples, and / or urine samples from the subject.

8. The application according to claim 1, characterized in that, The product is a diagnostic kit, diagnostic chip, and / or test strip used to diagnose and differentiate between maintenance intestinal metaplasia and progressive intestinal metaplasia, predict intestinal metaplasia outcomes, and / or assess the efficacy of intestinal metaplasia treatment.

9. The application according to claim 1, characterized in that, The subjects are either human or non-human animals.

10. A product for diagnosing and differentiating between maintenance intestinal metaplasia and progressive intestinal metaplasia, predicting intestinal metaplasia outcomes, and / or assessing the efficacy of intestinal metaplasia treatment, characterized in that, The product comprises the reagent for detecting the expression level of FOLH1 in the sample as described in any one of claims 1-6.

11. The product according to claim 10, characterized in that, The products include diagnostic kits, diagnostic chips, and / or test strips for diagnosing and differentiating between maintenance and progressive intestinal metaplasia, predicting intestinal metaplasia outcomes, and / or assessing the efficacy of intestinal metaplasia treatment.

12. The product according to claim 10, characterized in that, The samples include tissue samples, blood samples, serum samples, plasma samples, exosome samples, cell samples, fecal samples, and / or urine samples from the subject.

13. The product according to claim 10, characterized in that, The samples were obtained from subjects, who may be human or non-human animals.

14. The use of reagents for detecting FOLH1 expression levels in samples in the preparation of systems and / or devices for diagnosing and differentiating between maintenance and progressive intestinal metaplasia, predicting intestinal metaplasia outcomes, and / or assessing the efficacy of intestinal metaplasia treatment.

15. The application according to claim 14, characterized in that, The reagent is the reagent used to detect the expression level of FOLH1 in the sample as described in any one of claims 1-6.

16. The application according to claim 15, characterized in that, The samples include tissue samples, blood samples, serum samples, plasma samples, exosome samples, cell samples, fecal samples, and / or urine samples from the subject.

17. The application according to claim 15, characterized in that, The samples were obtained from subjects, who may be human or non-human animals.

18. A system and / or device for diagnosing and differentiating between maintenance intestinal metaplasia and progressive intestinal metaplasia, predicting intestinal metaplasia outcomes, and / or assessing the efficacy of intestinal metaplasia treatment, characterized in that, The system and / or device includes a processor, an input module, and an output module; The processor is used to perform logical operations on the input information using bioinformatics methods; the input module is used to input the FOLH1 expression level in the subject sample, and a computer-readable medium containing instructions, which, when executed by the processor, executes an algorithm at the FOLH1 input expression level; the output module is used to output the subject's intestinal metaplasia type, intestinal metaplasia outcome, and / or the therapeutic efficacy of intestinal metaplasia.

19. The system and / or apparatus according to claim 18, characterized in that, The samples include tissue samples, blood samples, serum samples, plasma samples, exosome samples, cell samples, fecal samples, and / or urine samples from the subject.

20. The system and / or apparatus according to claim 18, characterized in that, The subjects are either human or non-human animals.

21. The reagent for detecting FOLH1 expression levels in a sample according to any one of claims 1-6 is used in the diagnosis of maintenance intestinal metaplasia and progressive intestinal metaplasia, the prediction of intestinal metaplasia outcomes, and / or the assessment of the efficacy of intestinal metaplasia treatment.

22. The application according to claim 21, characterized in that, The samples include tissue samples, blood samples, serum samples, plasma samples, exosome samples, cell samples, fecal samples, and / or urine samples from the subject.

23. The application according to claim 21, characterized in that, The samples were obtained from subjects, who may be human or non-human animals.

24. A method for diagnosing and differentiating between maintenance intestinal metaplasia and progressive intestinal metaplasia, predicting intestinal metaplasia outcomes, and / or assessing the efficacy of intestinal metaplasia treatment, characterized in that, The method includes: detecting FOLH1 expression levels in samples from subjects to diagnose and differentiate between maintenance and progressive intestinal metaplasia, predict intestinal metaplasia outcomes, and / or assess the efficacy of intestinal metaplasia treatment.

25. The method according to claim 24, characterized in that, The detection of FOLH1 expression level includes detecting FOLH1 RNA level, FOLH1 DNA level, FOLH1 protein level, or the number of FOLH1-positive cells.

26. The method according to claim 24, characterized in that, The subject-derived samples include subject-derived tissue samples, blood samples, serum samples, plasma samples, exosome samples, cell samples, fecal samples, and / or urine samples.

27. The method according to claim 24, characterized in that, The subjects are either human or non-human animals.

28. A method for inhibiting the development of maintenance intestinal metaplasia into progressive intestinal metaplasia, the method comprising: Administer a therapeutically effective dose of FOLH1 promoter to subjects who require it.

29. The method according to claim 28, characterized in that, The FOLH1 promoter includes substances that increase FOLH1 expression levels, substances that enhance FOLH1 activity, substances that delay FOLH1 metabolism, and / or any combination thereof.

30. The method according to claim 28, characterized in that... The FOLH1 promoter includes a vector expressing FOLH1, nanoparticles carrying the FOLH1 gene, a viral vector carrying the FOLH1 gene, a PEG-modified protein encapsulating the FOLH1 gene or protein, protein microspheres encapsulating the FOLH1 gene or protein, liposomes encapsulating the FOLH1 gene or protein, extracellular vesicles encapsulating the FOLH1 gene or protein, and / or any combination thereof.

31. A biomarker for diagnosing and differentiating between maintenance and progressive intestinal metaplasia, predicting intestinal metaplasia outcomes, and / or assessing the efficacy of intestinal metaplasia treatment, characterized in that, The biomarker is FOLH1.

32. Application of biomarker FOLH1 in diagnosing and differentiating between maintenance and progressive intestinal metaplasia, predicting intestinal metaplasia outcomes, and / or assessing the efficacy of intestinal metaplasia treatment.

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