Antigenic markers of microsatellite instability

Abstract

The present invention relates to the use of antibodies against Thomsen-Friedenreich (TF) as reagents for in vitro diagnosis of microsatellite instability (MSI). Another aspect of the present invention relates to a method for in vitro diagnosis of MSI, which comprises using a composition of antibodies against TF antigen and measuring the expression of TF antigen in a tissue sample. The present invention also relates to a kit comprising the above-mentioned composition, which is used to detect MSI by implementing the method of the present invention. By sensitively and specifically detecting MSI in patients with gastric cancer, colorectal cancer and cancer of other tissues based on a single marker, the use, composition, method and kit of the present invention can be advantageously used to significantly promote the identification of MSI in clinical situations, thereby being used for diagnosis, prognosis or prediction of response to immunotherapy.

Description

Technical Field

[0001] The present invention relates to the use of antibodies or other agents having affinity for Thomsen-Friedenreich (TF) antigen in a composition for in vitro diagnosis of microsatellite instability (MSI), the in vitro diagnosis being performed by a method comprising the steps of incubating the composition using antibodies against TF antigen in a biological sample and evaluating the expression of TF antigen. A kit is also disclosed, comprising the above composition for detecting MSI by performing the method of the present invention.

[0002] Through rapid, sensitive and specific antibody assays based on a single marker, the uses, compositions, methods and kits of the present invention can be advantageously applied to cancers of gastric tissue, colorectal tissue and other tissues to greatly facilitate the identification of MSI in the clinic, thereby being used for the diagnosis, prognosis or prediction of response to treatment of cancer patients.

[0003] Therefore, the present invention belongs to the technical fields of medicine, pharmacy and biochemistry. Background Art

[0004] Gastric cancer is a heterogeneous disease that requires multidisciplinary treatment [1]. Currently, TCGA (The Cancer Genome Atlas) and ACRG (Asian Cancer Research Group) have proposed a new molecular classification of gastric cancer, in which microsatellite instability (MSI) is described as a unique subgroup [1,2].

[0005] Therefore, MSI is a unique molecular subtype of gastric cancer, and according to TCGA, the MSI subgroup is associated with hypermutation, gastric CpG island methylation phenotype, MLH1 silencing, and mitotic pathways [1].

[0006] In recent years, the clinical consequences of MSI and the therapeutic opportunities targeting this particular cancer subtype have become apparent. Many publications have described MSI from a clinical perspective, finding that it accounts for 5.6% to 33.3% of all gastric cancers and is strongly associated with female sex, older age, intestinal histology, mid / lower gastric location, N0 status, and TNM stage I / II, and with a good overall survival [1-5].

[0007] It is important to note that MSI is not a homogenous group and has been shown to be a prognostic factor due to its association with tumor localization and Lauren classification [6].

[0008] In addition, MSI has been found to be associated with PD-L1 expression, and several specific drugs targeting the PD1 / PD-L1 immune checkpoint pathway are currently used in the clinical management of gastric cancer [7].

[0009] The diagnosis of MSI gastric cancer is crucial, especially before initiating multidisciplinary treatment. It exhibits variable responses to neoadjuvant chemotherapy and requires specific surgical treatment in the presence of synchronous metastases, including possible customized lymphadenectomy, and stratification for the use of targeted therapies [3,8].

[0010] Transformation of epithelial cells is accompanied by several changes in the protein glycosylation machinery, which leads to aberrations in cellular glycosylation, such as truncation of O-linked glycans and expression of sialofucosylated glycan epitopes [9]. Truncation of O-linked glycans has been extensively described in various gastrointestinal cancers and leads to the de novo expression of short O-glycans, such as the Thomsen-Friedenreich (TF or T, Galβ1-3GalNAcα1) antigen, Thomsen-nouvelle (Tn, GalNAcα1) and its sialylated form sialyl Tn (STn, Neu5Acα2-3GalNAcα1) [9]. In addition, the expression of complex sialofucosylated structures, such as sialyl Lewis A (SLea, Neu5Acα2-3Galβ1-3[Fucα1-4]GlcNAcβ-) and sialyl Lewis X (SLex, Neu5Acα2-3Galβ1-4[Fucα1-3])GlcNAcβ-), has been frequently described in gastric tumors, which play an important role in cancer progression and metastasis formation [9].

[0011] TF antigen, also known as core 1 structure, is an intermediate structure during the maturation of mucin-type O-glycans in the Golgi apparatus. Under physiological conditions, TF antigen is below the detection limit because it is modified by additional sugars and becomes inaccessible due to the surrounding larger glycans [10,11]. It is only found on the luminal surface of the pancreatic duct, distal renal tubules, and renal collecting ducts

[11] . In addition, macrophages of the thymus, spleen, and lymph nodes carry TF antigen, suggesting a potential immunological role

[11] . Importantly, TF antigen is not found in healthy gastric epithelium and in pre-malignant conditions

[12] . In gastric cancer, TF antigen is expressed in significant amounts in approximately 21% of tumors, associated with a higher immune response

[12] .

[0012] As molecules secreted into the circulation and due to their expression specificity to malignant cells, aberrant glycans and glycoconjugates have long been used as cancer biomarkers

[13] .

[0013] The TF antigen has several additional properties that make it useful as a serological biomarker, as it is rarely expressed in human tissues, either in the healthy state or under other pathological conditions.[11,14] Second, the basal level of exposed TF in the blood is very low due to naturally circulating anti-TF IgM and IgG antibodies and rapid clearance of terminally galactosylated glycoconjugates by the liver.

[15] Finally, as a mucin-type O-glycosylation, it can be carried by a variety of secreted glycoproteins (e.g., MUC1 or CD44) that are overexpressed in gastric cancer.

[0014] Traditionally, MSI / dMMR status is also tested in colorectal cancer samples to identify patients at high risk for Lynch syndrome and for prognostic stratification. However, recent data suggest that MSI / dMMR has predictive value for immune checkpoint inhibitor therapy regardless of the tissue of origin of the cancer

[16] . In fact, the US Food and Drug Administration (FDA) has approved pembrolizumab (anti-programmed cell death protein 1 (PD-1)) for use in any solid tumor that has progressed on MSI / dMMR after prior treatment.

[0015] At present, a method for carrying out MSI determination is to extract by tumor and constitutive DNA.Use instruments such as ABI PRISM from Applied Biosystems, the mononucleotide repeatability of BAT-26, NR-21, BAT-25, NR-27 and NR-24 is evaluated with fluorescently labeled polymerase chain reaction (PCR) primers.Use the multiple PCR operating procedures disclosed by Qiagen, in five-plex PCR, the tumor of each patient and the germline DNA of matching are carried out to the co-amplification of repeated sequences.Allele map is detected by the automatic DNA sequencer ABI PRISM 3100Genetic Analyzer of Applied Biosystems, and the MSI state is determined according to the guidelines of the National Cancer Institute for the MSI for cancer detection and familial susceptibility: whenever there are more than 2 markers in 5 loci to show instability, it is believed that tumor is MSI high.On the contrary, when a locus or no locus is unstable, it is diagnosed as MSI low or microsatellite stable respectively.

[0016] Mismatch repair deficiency (dMMR) testing can also be performed by evaluating the expression of the four MMR proteins (MLH1, PMS2, MSH2, and MSH6) in tumor cell nuclei by immunohistochemistry in paraffin sections using commercially available antibodies.

[0017] Therefore, current MSI / dMMR analysis is a resource-intensive procedure for assessing the tumor PCR profile for five MSI markers and comparing it to a matched normal DNA profile or for assessing the absence of at least one of the four nuclear-expressed markers in tumor sections.

[0018] Overall, despite the importance of MSI for patient stratification, the time and resources required to diagnose MSI remain a barrier, and single biomarker-based assays are known in the art for detecting this signature. Summary of the invention

[0019] The present invention relates to the use of an antibody having affinity for Thomsen-Friedenreich (TF) antigen as a reagent for in vitro diagnosis of microsatellite instability (MSI).

[0020] Another embodiment of the present invention relates to a reagent composition for in vitro diagnosis of MSI, which comprises an antibody having affinity for TF antigen.

[0021] In one embodiment, the composition comprises the anti-TF antigen antibody 3C9.

[0022] In another embodiment, the composition comprises the anti-TF antigen antibody SPM320.

[0023] In another embodiment, the composition comprises the anti-TF antigen antibody A68 / B-A11.

[0024] In another embodiment, the composition comprises the anti-TF antigen antibody A78 / G-A7.

[0025] In another embodiment, the composition comprises the anti-TF antigen antibody B79-H / B8.

[0026] In another embodiment, the composition comprises the anti-TF antigen antibody C80-I / C9.

[0027] In another embodiment, the composition comprises the anti-TF antigen antibody A68-B / A11.

[0028] In another embodiment, the composition comprises a fragment of the variable region of the aforementioned anti-TF antibody.

[0029] In another embodiment, the composition comprises a lectin having affinity for the TF antigen, such as Jacalin.

[0030] In another embodiment, the composition further comprises at least one of the following components: a buffer, a blocking agent, a preservative, or a mixture thereof.

[0031] Another embodiment of the present invention relates to a kit for in vitro diagnosis of MSI, comprising at least one of the above-mentioned compositions.

[0032] The kit may also contain control and / or calibrator tissue or cell biological samples expressing the TF antigen.

[0033] Another embodiment of the present invention relates to a method for in vitro diagnosis of MSI of a biological sample, characterized by comprising the following steps:

[0034] a) biological samples obtained in vitro (e.g. formalin-fixed paraffin-embedded (FFPE) tissue blocks) were cut into 3 μm sections, mounted on slides, dewaxed with 100% xylene, washed with 100%, 95%, 70%, 50% ethanol and immersed in distilled water for rehydration;

[0035] b) 3% hydrogen peroxide in methanol (H 2 O 2 ) inactivates endogenous peroxidase;

[0036] c) Block with normal rabbit serum in PBS containing 10% bovine serum albumin (BSA) for 30 minutes.

[0037] d) contacting with the composition of claims 2-12 and incubating the slices at a temperature between 25°C and 4°C for 1 hour or overnight, respectively, most preferably at 4°C overnight;

[0038] e) incubating with biotin-labeled secondary antibody for 30 minutes;

[0039] f) Incubate for another 30 minutes using Vector Labs' ABC kit or other similar products;

[0040] g) staining the sections with 3,3′-diaminobenzidine tetrahydrochloride (DAB) and counterstained with Gill’s hematoxylin solution, and applying a coverslip over the mounting medium;

[0041] h) Examining the sections under a microscope and estimating the proportion of positive cancer cells within the tumor to classify as a positive or negative MSI diagnosis, with a cut-off value of 5% to 25% for cancer cell positivity for TF staining, and most preferably 5%.

[0042] In another embodiment, the in vitro biological sample tested by the above method includes cells in frozen tissue or liquid cytology samples, gastric washings, bladder washings or urine, ascites, pleural effusion or cerebrospinal fluid, or diluted stool samples.

[0043] In another embodiment, the method for in vitro diagnosis of MSI in an in vitro biological sample is characterized by comprising the following steps:

[0044] a) obtaining an in vitro serum or plasma biological sample;

[0045] b) using one of the compositions of claims 2 to 12 to perform an enzyme-linked immunosorbent assay (ELISA) to detect TF antigen.

[0046] In another embodiment, the ELISA is characterized in that it is used to detect the level of autoantibodies against TF antigen.

[0047] Through simple, rapid, sensitive and specific antibody assays based on a single marker, the uses, compositions, methods and kits of the present invention can be advantageously applied to cancer patients, including but not limited to gastric cancer and colorectal cancer, to greatly facilitate the in vitro diagnosis of MSI in the clinic, thereby being used for diagnosis, prognosis or prediction of response to immunotherapy. DETAILED DESCRIPTION

[0048] The present invention discloses that the expression of TF antigen in five cancer-associated glycan epitopes was analyzed in cancers with MSI high (positive) and MSI low or stable state (negative), revealing a novel, highly significant association between the expression of TF antigen and MSI status (Tables 1 and 2), while it is not undesirably detected in normal or non-malignant lesions (Table 3). Figure 1 ).

[0049] Therefore, the present invention discloses the use of antibodies against TF antigen or other agents having affinity for TF antigen in compositions for use in the following methods or kits for detecting microsatellite instability (MSI) in tumor tissue samples, including but not limited to gastric cancer.

[0050] Possible specific antibodies that can be used for this purpose include TF antigen antibodies 3C9, SPM320, A68 / B-A11, A78 / G-A7, B79-H / B8, C80-I / C9, A68-B / A11, all of which are characterized by having affinity for TF antigen and can be obtained from commercial channels, such as Novus Biologicals, Invitrogen, Cell Sciences, Creative Diagnostics, Creative Biolabs the Abcam, Santa Cruz, Histosearch, Life Span Biosciences, Creative Diagnostics, or other companies, such as Abnova, Raybiotech, NSJ Reagents, My Biosource, American Research Products Inc, Fitzgerald Industries International and Progen.

[0051] In other embodiments of the present invention, fragments of the variable regions of the above antibodies can be used for the purposes of the present invention.

[0052] In other embodiments of the present invention, lectins with high affinity for TF antigen (eg, Jacalin) can be used for the purpose of the present invention.

[0053] The antibody, antibody fragment or lectin is used in a composition for detecting MSI.

[0054] In one embodiment of the present invention, the composition for detecting MSI is prepared as follows: a specific antibody, antibody fragment or lectin having affinity for the TF antigen is diluted in phosphate buffered saline (PBS) with 2% (w / v) bovine serum albumin (BSA) in an amount of 5 to 50 μg, most preferably 33 μg.

[0055] Using the composition, an embodiment of the present invention can be developed, which relates to a method for in vitro diagnosis of MSI based on the expression of TF antigen ( Figure 1 ), the method comprising the following steps:

[0056] a) biological samples obtained in vitro (e.g. formalin-fixed paraffin-embedded (FFPE) tissue blocks) were cut into 3 μm sections, mounted on slides, dewaxed with 100% xylene, washed with 100%, 95%, 70%, 50% ethanol and immersed in distilled water for rehydration;

[0057] b) 3% hydrogen peroxide in methanol (H2 O 2 ) inactivates endogenous peroxidase;

[0058] c) Tissue sections were blocked with normal rabbit serum in PBS containing 10% bovine serum albumin (BSA) for 30 minutes.

[0059] d) contacting the above composition comprising an antibody, antibody fragment or lectin specific for the TF antigen with the tissue section and incubating at a temperature between 25°C and 4°C for 1 hour or overnight, respectively, most preferably at 4°C overnight;

[0060] e) applying biotin-labeled secondary antibody for 30 minutes;

[0061] f) Apply Vector Labs' ABC kit or other similar products for 30 minutes;

[0062] g) staining the sections with 3,3′-diaminobenzidine tetrahydrochloride (DAB) and counterstained with Gill’s hematoxylin solution;

[0063] h) Examining the slides microscopically and estimating the proportion of positive cancer cells within the tumor to classify into either MSI-positive or MSI-negative categories, with a cut-off value of 5% to 25% for cancer cell positivity for TF, and most preferably 5%.

[0064] By employing this approach in 30 gastric cancer cases (13 MSI-positive and 17 MSI-negative by current PCR-based analysis), a surprisingly highly significant association was found between the expression of TF and the MSI status (p<0.001; Fisher's exact test). Of the 10 TF-positive cases, 9 were MSI-positive, indicating that the TF antigen has an unprecedented specificity for this gastric cancer molecular subtype (Table 1). The results using this approach showed that the TF antigen was also excellent in terms of sensitivity, as 16 of the 20 TF-negative cases were MSI-negative (Table 1). This resulted in sensitivity and specificity values ​​of 69.2% (9 / 13) and 94.1% (16 / 17), respectively. The positive and negative predictive factor values ​​were 90% (9 / 10) and 80% (16 / 20), respectively.

[0065] Another advantage of this approach is that the mucosa adjacent to the tumor (including normal glandular and highly inflamed areas, as well as intestinal metaplasia and dysplasia) was completely negative, indicating that this glycan marker is not present in non-malignant and pre-malignant states ( Figure 1 ac). In positive cases, staining is usually membranous and is found in about 30% of all cancer cells in the tumor on average. In well-differentiated gastric cancers, staining is usually at the apical membrane and includes secretions ( Figure 1df). In poorly differentiated gastric cancer, subcellular localization usually shifts to the cytoplasm ( Figure 1 gj). TF expression appears to be associated with a favorable prognosis in patients, as reflected by an increased median survival (88 months vs. 31.5 months) and a higher proportion of patients alive 5 years after diagnosis (70% vs. 30%) (Table 2). This improved prognosis is in line with expectations, as it is known that patients with MSI experience better survival on average.

[0066] In other embodiments of the method for detecting MSI of the present invention, the biological sample may include tumor cells in frozen tissue or liquid cytology samples, gastric washings, bladder washings or urine, ascites, pleural effusion or cerebrospinal fluid, or diluted stool samples.

[0067] The present invention also relates to a kit comprising the above composition, which is used for detecting MSI by implementing the method of the present invention.

[0068] In one embodiment of the present invention, the kit further comprises a control and / or calibration tissue or cell biological sample expressing the TF antigen.

[0069] In summary, through rapid, sensitive and specific antibody assays based on a single marker, the uses, compositions, methods and kits of the present invention can be advantageously applied to the in vitro diagnosis of MSI of cancers of gastric tissue, colorectal tissue and other tissues, thereby significantly improving the identification of this unique cancer subtype in the clinic, thereby being used for the prognosis of cancer patients or the prediction of response to treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Table 1: Describes the analysis of the association between aberrant glycan epitopes and MSI status. A) The expression of five markers of aberrant glycosylation, namely Thomsen-Friedenreich (TF, Galβ1-3GalNAcα1) antigen, Thomsen-nouvelle (Tn, GalNAcα1) and its sialylated form sialyl Tn (STn, Neu5Acα2-3GalNAcα1), sialyl-Lewis A (SLea, Neu5Acα2-3Galβ1-3[Fucα1-4]GlcNAcβ-), and sialyl-Lewis X (SLex, Neu5Acα2-3Galβ1-4[Fucα1-3]GlcNAcβ-), was evaluated in 13 MSI-positive and 17 MSI-negative gastric cancers. B) MSI and markers were statistically analyzed using Fisher's exact test, and TF was found to be highly significantly associated with MSI (**). Missing values ​​are indicated by ( / ).

[0071] Table 2: Describes the clinicopathological analysis of gastric cancer patients according to TF antigen status. A total of 30 patients were included in the cohort, including TF-positive patients (10) and TF-negative patients (20), and the TF status (negative / positive) was compared with the clinicopathological characteristics. Fisher's exact test (for both categories of characteristics) and χ 2 The association between the clinicopathological group and the two TF marker status groups was calculated using the Mann-Whitney-Wilcoxon test (for features with more than three categories). The differences in age and median survival (in months) after diagnosis between the TF status groups were statistically evaluated using the Mann-Whitney-Wilcoxon test. P values ​​≤ 0.05 were considered statistically significant (**).

[0072] Figure 1 : Representative staining images of Thomsen-Friedenreich (TF) antigen expression in human gastric tissue samples. (a) Gastric mucosa is TF-negative, represented here by histologically normal mucosa adjacent to MSI-positive and TF-positive tumors. (b) Representative staining images of intestinal metaplasia, showing that even goblet cells, which are typically enriched in truncated O-glycan carriers, are TF-negative. (c) TF-negative dysplasia adjacent to TF-positive carcinoma, demonstrating the specificity of TF for malignant cells. (df) Well-differentiated gastric carcinoma shows typical membranous TF staining located on the apical surface, including mucinous secretions. (gi) Poorly differentiated gastric carcinoma shows predominantly cytoplasmic staining. (i) TF-positive cancer cells within infiltrating blood vessels.

[0073] Example

[0074] Example 1 - Preparation of a composition for detecting MSI comprising antibodies against TF antigen

[0075] In one embodiment of the present invention, 5 μg to 100 μg of an antibody against TF antigen is diluted with phosphate buffered saline (PBS) containing 2% (w / v) bovine serum albumin (BSA) to prepare a composition for in vitro diagnosis of MSI.

[0076] Example 2—Preparation of a composition comprising an antibody or antigen receptor fragment for detecting MSI

[0077] In one embodiment of the present invention, the composition for in vitro diagnosis of MSI comprises a variable region fragment targeting the TF antigen, including but not limited to a variable chain of an immunoglobulin heavy chain or light chain, and a variable chain of a T cell receptor.

[0078] Example 3—Preparation of a composition comprising lectin for detecting MSI

[0079] In one embodiment of the present invention, the composition for in vitro diagnosis of MSI comprises a lectin having a high affinity for the TF antigen, including but not limited to Jacalin and a mixture thereof.

[0080] Example 4—Methods for in vitro diagnosis of MSI in FFPE tissue samples

[0081] In one embodiment, a method using a composition prepared as described in the previous embodiments comprises the following steps:

[0082] a) biological samples obtained in vitro (e.g. formalin-fixed paraffin-embedded (FFPE) tissue blocks) were cut into 3 μm sections, mounted on slides, dewaxed with 100% xylene, washed with 100%, 95%, 70%, 50% ethanol and immersed in distilled water for rehydration;

[0083] b) 3% hydrogen peroxide in methanol (H 2 O 2 ) inactivates endogenous peroxidase;

[0084] c) Block with normal rabbit serum in PBS containing 10% bovine serum albumin (BSA) for 30 minutes.

[0085] d) contacting with the composition of claims 2-12 and incubating the slices at a temperature between 25°C and 4°C for 1 hour or overnight, respectively, most preferably at 4°C overnight;

[0086] e) incubating with biotin-labeled secondary antibody for 30 minutes;

[0087] f) Incubate for another 30 minutes using Vector Labs' ABC kit or other similar products;

[0088] g) staining the sections with 3,3′-diaminobenzidine tetrahydrochloride (DAB) and counterstained with Gill’s hematoxylin solution, and applying a coverslip over the mounting medium;

[0089] h) Examine the slides microscopically and estimate the proportion of positive cancer cells within the tumor to classify as a positive or negative MSI diagnosis, with a cut-off value of greater than 5% for cancer cell positivity for TF antigen staining.

[0090] The advantage of this approach is that by expressing TF antigen as a single marker, a highly reliable predictor of MSI status in cancer is obtained, and therefore, the application of TF antigen measurement has great potential for stratifying patients by MSI in a time- and cost-effective manner.

[0091] Example 5 - Method for in vitro diagnosis of MSI in a cell sample

[0092] In another embodiment, the biological sample to be used in the methods described in the previous embodiments can be frozen tissue, or contain cells imprinted and fixed on a slide from a liquid cytology sample, gastric washings, bladder washings or urine, ascites, pleural effusion or cerebrospinal fluid, or a diluted stool sample. The advantage of such cell samples is that they can be obtained from minimally invasive procedures that do not require surgical procedures for obtaining tissue biopsies or surgical specimens.

[0093] Example 6—Serological Methods for In Vitro Diagnosis of MSI

[0094] Another example concerns the applicability of a serological assay based on ELISA (enzyme-linked immunosorbent assay) detecting TF antigen in the diagnosis of MSI subtypes, which has potential clinical applications.

[0095] Therefore, in another embodiment, a method for in vitro diagnosis of MSI can be developed, the method comprising the following steps:

[0096] a) obtaining an in vitro serum or plasma biological sample;

[0097] b) using one of the compositions of claims 2 to 12 to perform ELISA to detect TF antigen.

[0098] Example 7—Serological Methods for In Vitro Diagnosis of MSI

[0099] In another embodiment, the serological methods described in the previous examples can follow a strategy such as detecting elevated levels of autoantibodies against TF, which can be indicative of MSI status.

[0100] References

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[0117] Lisbon, May 3, 2019

Claims

1. Use of a reagent having affinity for Thomsen-Friedenreich (TF) antigen in the preparation of a kit for diagnosing microsatellite instability (MSI) gastric cancer, wherein the reagent is selected from the group consisting of: an antibody against TF antigen, a variable region of the antibody, and a lectin having affinity for TF antigen, in, The anti-TF antigen antibody is selected from the group consisting of 3C9, SPM320, A68 / B-A11, A78 / G-A7, B79-H / B8, C80-I / C9 and A68-B / A11, and Wherein, the lectin having affinity for TF antigen is Jacalin.

2. The use according to claim 1, characterized in that The kit further comprises at least one of the following components: a buffer, a blocking agent, a preservative, or a mixture thereof.

3. The use according to claim 1 or 2, wherein: Diagnosis of MSI is performed by a method comprising the following steps: a) biological samples obtained in vitro were cut into 3 μm sections, mounted on slides, dewaxed with 100% xylene, washed with 100%, 95%, 70%, and 50% ethanol, and immersed in distilled water for rehydration; b) inactivation of endogenous peroxidase using 3% hydrogen peroxide (H2O2) in methanol; c) blocking with normal rabbit serum in PBS containing 10% bovine serum albumin (BSA) for 30 minutes; d) contacting with a composition comprising the reagent of claim 1 and incubating the slice at a temperature between 25° C. and 4° C. for 1 hour or overnight, respectively; e) incubating with biotin-labeled secondary antibody for 30 minutes; f) Incubate for 30 minutes using Vector Labs' ABC kit or other similar products; g) staining the sections with 3,3′-diaminobenzidine tetrahydrochloride (DAB) and counterstained with Gill’s hematoxylin solution, and applying a coverslip over the mounting medium; h) Examine the sections using light microscopy and estimate the proportion of positive cancer cells within the tumor to classify as a positive or negative MSI diagnosis, with a cut-off value of 5% to 25% for TF staining.

4. The use according to claim 3, wherein: The biological sample obtained in vitro is a formalin-fixed paraffin-embedded (FFPE) tissue block.

5. The use according to claim 3, wherein: The biological sample includes cells in frozen tissue or liquid cytology samples, gastric washings, bladder washings or urine, ascites, pleural effusions or cerebrospinal fluid, or diluted stool samples.

6. The use according to claim 1 or 2, wherein the diagnosis of MSI is performed by a method comprising the following steps: a) obtaining an in vitro serum or plasma biological sample; b) using the composition comprising the reagent according to claim 1 or 2 to perform enzyme-linked immunosorbent assay (ELISA) to detect TF antigen.

7. The use according to claim 6, wherein: The ELISA is characterized by including the detection of the level of autoantibodies against TF.

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