Application of GLTSCR1 as molecular marker for immunological stratification treatment of MSI-H colorectal cancer

Abstract

The application GLTSCR1 as a MSI-H colorectal cancer immune stratification treatment molecular marker belongs to the field of molecular biology. Specifically, the application finds that low expression or functional deletion of GLTSCR1 in tumor cells can induce primary drug resistance of MSI-H colorectal cancer to PD-1 inhibitors. Through immunohistochemistry, PCR-Sanger sequencing, animal models and clinical cohort verification, it is confirmed that the GLTSCR1 deletion state can be used as an effective stratification marker for MSI-H colorectal cancer immunotherapy, for predicting the efficacy of immune checkpoint inhibitors. The application also provides biological materials required for detecting GLTSCR1 deletion, kits and their use in preparing prediction preparations and diagnostic reagents.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically, it relates to the application of GLTSCR1 as a molecular marker for immune stratification therapy in MSI-H colorectal cancer. Background Technology

[0002] Tumors with DNA mismatch repair deficient (dMMR) / microsatellite instability-high (MSI-H) can produce a large number of tumor neoantigens, activating the body's immune system and forming "hot tumors" characterized by extensive infiltration of immune cells within the tumor tissue. These tumors can significantly benefit from PD-1 / PD-L1 inhibitors, which have been approved by the U.S. Food and Drug Administration (FDA) as the first pan-cancer immunotherapy biomarker. Although dMMR / MSI-H tumor patients have a high immune response rate, multiple studies have found that approximately 20% of dMMR / MSI-H tumors exhibit primary immune resistance to PD-1 inhibitors. However, current guidelines only recommend immunotherapy based on dMMR / MSI-H status, and there is a lack of effective molecular biomarkers to guide clinical stratification of treatment.

[0003] In our previous study, by analyzing the mutation profiles of MSI-H and MSS colorectal cancer patients in the TCGA database, our team discovered a high-frequency mutation in the ncBAF complex core subunit GLTSCR1 in MSI-H colorectal cancer patients. Further analysis of the MSI status and GLTSCR1 frameshift mutations in 657 sporadic colorectal cancer samples revealed a microsatellite site (C8) in exon 6 of the GLTSCR1 gene, consisting of eight tandem repeats of cytosine (C). This site exhibits a deletion (C7) or insertion (C9) mutation at this site, leading to a frameshift and premature stop codon appearance, resulting in the loss of GLTSCR1 protein truncation function. Furthermore, among these 112 patients with MSI-H, the GLTSCR1 frameshift mutation occurred only in MSI-H patients (21.4%, 24 / 112), suggesting that the GLTSCR1 frameshift mutation is a tumor-specific mutation in MSI-H. Several studies have reported a close relationship between the BAF complex and immune regulation and immunotherapy, but no studies have yet evaluated whether the MSI-H tumor-specific mutation GLTSCR1 can serve as a stratification marker for MSI-H tumor immunotherapy. Summary of the Invention

[0004] To further stratify immunotherapy for MSI-H cancer and provide a more accurate method for predicting treatment response, this application provides the following technical solution.

[0005] In a first aspect, the present invention relates to the use of a substance that specifically detects the functional loss or low expression of GLTSCR1 in the preparation of a formulation for predicting the response to MSI-H cancer immunotherapy.

[0006] Furthermore, the functional loss or low expression of GLTSCR1 is caused by frameshift mutations leading to the loss or low expression of GLTSCR1.

[0007] Furthermore, the frameshift mutation occurs at the microsatellite site (C8) of the sixth exon of the GLTSCR1 gene, which consists of eight tandem repeat sequences of cytosine (C), specifically a frameshift mutation caused by the deletion (C7) or insertion (C9) of one C at this site.

[0008] Furthermore, the MSI-H cancer is MSI-H colorectal cancer.

[0009] Furthermore, the immunotherapy is an immune checkpoint inhibitor therapy.

[0010] Furthermore, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

[0011] Furthermore, the PD-1 inhibitor is a PD-1 antibody.

[0012] Furthermore, the substance specifically used to detect GLTSCR1 functional loss or low expression is selected from primers, probes, aptamers, or antibodies.

[0013] Furthermore, the substance specifically used to detect GLTSCR1 functional loss or low expression is PCR primers and / or Sanger sequencing reagent.

[0014] Furthermore, the preparation is a kit or a test reagent composition.

[0015] In a second aspect, the present invention provides a kit for predicting the response to MSI-H cancer immunotherapy, comprising the substance described in the first aspect that specifically detects GLTSCR1 functional loss or low expression.

[0016] Furthermore, it also contains a specific antibody for immunohistochemical detection of GLTSCR1 protein expression levels.

[0017] Furthermore, the state of functional loss or low expression of GLTSCR1 can induce primary immune resistance to PD-1 inhibitors in MSI-H tumors.

[0018] Furthermore, the aforementioned uses or kits can also simultaneously detect the low expression status of GLTSCR1 protein as a supplementary biomarker for stratified prediction of MSI-H tumor immunotherapy.

[0019] The beneficial effects of this invention include:

[0020] 1. This study is the first to discover that functional loss or low expression of GLTSCR1 occurs specifically and frequently in MSI-H tumors, and that this mutation is only found in MSI-H patients, providing a new specific molecular marker for MSI-H tumors.

[0021] 2. It was found that GLTSCR1 deficiency can induce primary immune resistance to PD-1 inhibitors in MSI-H tumors, and the causal relationship was confirmed by gene knockout mouse models and cell line subcutaneous tumor models.

[0022] 3. An assessment system for GLTSCR1 deletion status based on simple detection methods such as immunohistochemistry and PCR-Sanger sequencing was established, which can effectively predict the treatment response of MSI-H tumor patients to immune checkpoint inhibitors. This fills the gap in existing clinical guidelines for stratification markers of MSI-H tumor immunotherapy and provides an important basis for precise stratified treatment and the development of potential therapeutic targets. Attached Figure Description

[0023] Figure 1 The frameshift mutation status of the GLTSCR1 microsatellite sequence (containing 8 cytosine / C atoms) was detected using PCR combined with Sanger sequencing. The results showed that the overall incidence of GLTSCR1 insertion or deletion frameshift mutations was 21.4%. Figure 1 A), and its expression level on tumor cells can be detected by immunohistochemistry. Figure 1 B).

[0024] Figure 2 The regression of target lesions in colorectal cancer patients after immunotherapy was assessed using clinical CT images. Figure 2 A), and divided into three groups (low, medium, and high) based on GLTSCR1 immunohistochemical expression levels. The results showed that the immunotherapy effect in the low GLTSCR1 expression group was worse than that in the other two groups. Figure 2 B).

[0025] Figure 3 A mouse model of colorectal cancer AOM / DSS was constructed using mice with intestinal epithelial-specific Gltscr1 knockout. The results showed that the intestinal epithelial Gltscr1 deletion group exhibited resistance to PD-1 inhibitors.

[0026] Figure 4 By constructing a GLTSCR1 knockdown murine MSI-H colorectal cancer MC38 cell line ( Figure 4A), and was subcutaneously injected into C57 / BL6 mice, and treated with IgG or PD-1 inhibition, respectively. Figure 4 B), the results showed that GLTSCR1 knockdown did not affect tumor volume and weight, but after PD-1 inhibitor treatment, the growth rate, tumor volume, and weight of the tumor in the GLTSCR1 knockdown group were significantly higher than those in the control group. Figure 4 CD indicates that GLTSCR1 deficiency leads to resistance to PD-1 inhibitor treatment.

[0027] Figure 5 By constructing a GLTSCR1 knockdown murine MSI-H colorectal cancer CT26 MLH1 KO cell line ( Figure 5 A), and was subcutaneously injected into Balb / c mice, and treated with IgG or PD-1 inhibition, respectively. Figure 5 B), the results showed that GLTSCR1 knockdown did not affect tumor volume and weight, but after PD-1 inhibitor treatment, the growth rate, tumor volume, and weight of the tumor in the GLTSCR1 knockdown group were significantly higher than those in the control group. Figure 5 CD indicates that GLTSCR1 deficiency leads to resistance to PD-1 inhibitor treatment. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this application clearer, the application will be further described in detail below with reference to the accompanying drawings. The described embodiments should not be regarded as limitations on this application. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0029] Example 1 Immunohistochemical assessment of GLTSCR1 expression

[0030] Materials and methods

[0031] 1. Materials

[0032] This study included 36 colorectal cancer patients who received PD-1 inhibitor therapy, all of whom were from the First Affiliated Hospital of Zhejiang University School of Medicine (Ethics Committee Approval No.: IIT20210185B). We retrospectively collected patients' baseline clinical data and efficacy assessment information, and obtained pre-treatment, untreated primary lesion tissue samples for immunohistochemical experiments. All participants signed written informed consent forms.

[0033] 2. Method

[0034] 1) Collect 3 μm thick sections, place the sections in a 60 ℃ oven overnight to remove residual moisture, and then store them in a -40 ℃ refrigerator for long-term storage;

[0035] 2) Before starting the experiment, place the slices back into the 60 ℃ oven to dry for 30 min, and then place the slices into three xylene tanks for 10 min in sequence to fully remove the paraffin.

[0036] 3) After removing the residual liquid, place the tissue sections in a series of ethanol solutions (100%-95%-75%-50% ethanol) for 5 minutes each time. After hydration, rinse the tissue sections three times with PBS on a slow shaker for 5 minutes each time.

[0037] 4) After aspirating the residual liquid, place the sections in 3% hydrogen peroxide methanol for 15 min to block endogenous peroxidase. After the block is complete, rinse the tissue sections with PBS 3 times on a slow shaker for 5 min each time.

[0038] 5) Prepare Tris-EDTA alkaline repair solution (pH 9.0), mix well and pour into a pressure cooker. Place the slices horizontally in the pressure cooker and ensure the liquid fully covers the slices. Place the pressure cooker on an induction cooker and heat it. Start timing 3 minutes after the pressure cooker valve starts to turn and release steam.

[0039] 6) After the timer is finished, turn off the induction cooker, open the gas valve to release the gas completely, then open the pressure cooker lid and let it cool naturally to room temperature before taking out the sections. Rinse the tissue sections with PBS 3 times on a slow shaker for 5 minutes each time.

[0040] 7) Prepare a blocking solution containing 10% fetal bovine serum using PBS. Use a histochemical pen to draw circles around the tissue periphery, being careful to avoid touching the tissue. Carefully add 100 μL of blocking solution to each section and incubate at room temperature for 30 min.

[0041] 8) After blocking, discard the blocking solution and add 100 μL of primary antibody (diluted with immunohistochemistry primary antibody dilution buffer), MLH1 1:500, GLTSCR1 1:100, and incubate overnight at 4 ℃.

[0042] 9) The next day, remove the tissue sections, equilibrate at room temperature for 15 minutes, and then rinse the tissue sections three times with PBS on a slow shaker for 5 minutes each time.

[0043] 10) After aspirating the liquid, add the secondary antibody enhancement solution and incubate at room temperature for 10 min. Then, rinse the tissue sections three times with PBS on a slow shaker for 5 min each time.

[0044] 11) After aspirating the liquid, add the secondary antibody working solution and incubate at room temperature for 30 min. Then, rinse the tissue sections three times with PBS on a slow shaker.

[0045] 12) Dilute the DAB concentrate with DAB substrate diluent at a ratio of 1:20 in a fume hood. Mix well with a pipette and filter with filter paper. Add the filtered DAB working solution to the tissue immediately, start timing, and place the tissue in tap water as soon as it turns brownish-yellow. Rinse the tissue section with running water for 40 minutes to stop the color development.

[0046] 13) Filter the hematoxylin into a beaker with filter paper in advance, place the tissue section in the hematoxylin and make sure the tissue is fully submerged, soak for about 1 minute, then rinse the tissue section with running water for 10 minutes to remove unbound hematoxylin and perform bluing.

[0047] 14) After bluing, the slices were placed in a gradient of ethanol for dehydration (50%-75%-90%-100% ethanol) for 5 min each time;

[0048] 15) Pass the dehydrated tissue sections through two xylene tanks sequentially for 5 minutes each time to achieve full transparency;

[0049] 16) Mount the slides with resin, observe the tissue under a microscope after the slides have dried, and scan the slides using a slide scanner. Figure 1 B. Immunohistochemical assessment of GLTSCR1 expression changes was observed, and the predictive efficacy of this result was validated in a clinical cohort. Figure 2 AB).

[0050] Example 2: Detection of mutations in GLTSCR1 microsatellite sequences

[0051] 1. Method

[0052] 1) Scraping of tissue sections

[0053] Colorectal cancer paraffin tissue was cut into 5 μM thick paraffin sections using a microtome and observed under a microscope. The tumor area was marked with a pen, and an appropriate number of paraffin sections were cut according to the size of the tumor. The tumor tissue in the marked area was carefully scraped off with a disposable scalpel and collected into a 1.5 mL centrifuge tube. Tumor tissue from the same paraffin tissue section was scraped off and collected into the same 1.5 mL centrifuge tube.

[0054] 2) DNA dewaxing and digestion

[0055] Add 1 mL of xylene to the centrifuge tube, vortex vigorously for 10 s, then centrifuge at 14000 rpm for 2 min at room temperature (15-25℃). Carefully discard the supernatant, being careful not to aspirate the precipitate. Next, add 1 mL of anhydrous ethanol to the centrifuge tube and vortex thoroughly to remove the xylene. After mixing, centrifuge at 14000 rpm for 2 min at room temperature (15-25℃), carefully discarding the supernatant, being careful not to aspirate the precipitate. Open the centrifuge tube cap and air dry at room temperature or 37℃ for about 10 min, until the anhydrous ethanol has completely evaporated. Then add 180 μL of Buffer ATL and 20 μL of Proteinase K to the centrifuge tube, vortex thoroughly to mix, and place the centrifuge tube in a metal bath at 56℃ overnight for digestion until the sample is completely lysed.

[0056] 3) Extract tissue DNA using the QIAamp DNA FFPE Tissue Kit.

[0057] The lysed sample was incubated at 90°C for 1 h to reverse some of the formaldehyde-modified nucleic acids. After the centrifuge tube cooled to room temperature, the liquid on the tube wall was collected by centrifugation. 200 μL of Buffer AL was added and mixed thoroughly, followed by 200 μL of anhydrous ethanol, and the mixture was mixed thoroughly again. A white flocculent precipitate may form during this process. The liquid on the tube wall was then collected by centrifugation, and the supernatant was carefully transferred to a QIAamp adsorption column, avoiding the aspiration of the precipitate. The adsorption column was then centrifuged at 8000 rpm for 1 min, and the QIAamp adsorption column was placed in a new 2 mL collection tube. 500 μL of Buffer AW was added, and the column was centrifuged at 18000 rpm for 1 min. 500 μL of Buffer AW2 was added, and the column was centrifuged at 8000 rpm for 1 min. The waste liquid was discarded, and the QIAamp adsorption column was placed in a clean 2 mL collection tube and centrifuged at 14000 rpm for 3 min. Finally, place the QIAamp adsorption column into a new 1.5 mL centrifuge tube, add 100 μL of Buffer ATE to the center of the QIAamp adsorption column, incubate at room temperature for 5 min, then centrifuge at 14000 rpm for 1 min and collect the centrifuged product. The DNA is stably stored at -40°C.

[0058] 4) Perform PCR sequencing on the obtained DNA and commission a company to complete the Sanger sequencing. Figure 1 A shows the frameshift mutations of GLTSCR1 after Sanger sequencing. These mutations are high-frequency mutations of GLTSCR1 in human samples.

[0059] Example 3: Constructing a gene-modified mouse AOM / DSS model to evaluate the effect of intestinal epithelium-specific knockout of GLTSCR1 on the efficacy of PD-1 inhibitors.

[0060] 1. Method

[0061] 1) Gene mouse construction:

[0062] Using CRISPR / Cas9 technology, the GLTSCR1 gene was modified using flux through homologous recombination. The process was as follows: gRNA was designed and transcribed in vitro, and a homologous recombination vector (Donor vector) was constructed. Cas9, gRNA, and Donor vector were simultaneously injected into mouse zygotes. Cas9 protein, guided by gRNA, bound to the target site, causing a DNA double-strand break. The Donor vector repaired the broken double strand through homologous recombination, thus achieving flux modification of the target gene. When these flux mice were mated with villin-Cre mice, GLTSCR1-specific knockout model mice could be obtained from intestinal villi and crypt epithelial cells.

[0063] 2) AOM / DSS model:

[0064] Five-week-old male GLTSCR1fl / fl Villin-Cre mice were intraperitoneally injected with AOM at a concentration of 10 mg / kg, followed by three rounds of DSS treatment one week later. Each round of DSS treatment lasted three weeks. In the first week, 1.5% wt / vol DSS was added to the mice's drinking water, and normal drinking water was provided for the following two weeks. After the three rounds, mice were treated with IgG or anti-mouse PD-1 monoclonal antibody at 1 mg / kg every 3 days starting from week 18. After the three rounds of administration, colorectal tissue was collected from the mice, washed with PBS, and then preserved in formalin for subsequent embedding. Figure 3 AB demonstrated that specific knockout of GLTSCR1 in the intestinal epithelium can induce resistance to PD-1 inhibitors.

[0065] Example 4: Evaluation of the effect of GLTSCR1 deficiency on the efficacy of PD-1 inhibitors using a subcutaneous tumor model

[0066] Materials and methods

[0067] 1. Materials

[0068] Colorectal cancer cell lines HT29 and SW620 were provided by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. RPMI 1640, 0.05% Trypsin, and fetal bovine serum were purchased from Gibco. Transfection reagent Genemut was purchased from SignaGen. Transwell double-layer plates were purchased from Costa, Matrigel gel from BD, and crystal violet from Beyotime. Other reagents were domestically produced analytical grade.

[0069] 2. Methods

[0070] Mice were anesthetized with 3% isoflurane, and 1×10 6 One MC38 Scramble cell and one MC38 ShGltscr1 stably knocked-down cell were resuspended in 100 μL PBS and injected subcutaneously into 6-week-old male C57 / BL6 mice using a 1 mL syringe; or 1.5 × 10⁻⁶ cells were injected into the MC38 Scramble cell line and one MC38 ShGltscr1 stably knocked-down cell line. 6 One CT26 MLH1 KO Scramble cell and one CT26 MLH1 KO ShGltscr1 stably knocked-down cell were resuspended in 100 μL PBS and injected subcutaneously into 6-week-old male Balb / c mice using a 1 mL syringe. When the subcutaneous tumor volume became palpable (approximately 30-50 mm3), the mice were treated with 1 mg / kg of IgG or anti-mouse PD-1 monoclonal antibody via intraperitoneal injection every 3 days. Simultaneously, the diameter of the subcutaneous tumor was measured every 2 days using calipers, and the tumor volume was calculated using the following formula: length × width × width / 2. After 3-4 doses, all mice were euthanized, and the subcutaneous tumor tissue was fixed in 4% paraformaldehyde for at least 48 hours for subsequent pathological examination. Figure 4 AD and Figure 5 AD demonstrated that GLTSCR1 deficiency in a mouse subcutaneous tumor model can induce resistance to PD-1 inhibitors.

[0071] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention.

Claims

1. Use of a substance that specifically detects functional loss or low expression of GLTSCR1 in the preparation of a formulation for predicting the response to immunotherapy in MSI-H colorectal cancer.

2. The use according to claim 1, characterized in that, The loss or low expression of GLTSCR1 function is caused by frameshift mutations in the coding gene.

3. The use according to claim 2, characterized in that, The frameshift mutation occurs at a microsatellite site (C8) in the sixth exon of the GLTSCR1 gene, which consists of eight tandem repeats of cytosine (C). The mutation is a frameshift mutation caused by the deletion (C7) or insertion (C9) of one C at this site, which leads to a loss or reduction in the function of GLTSCR1.

4. The use according to any one of claims 1 to 3, characterized in that, The MSI-H cancer mentioned is MSI-H colorectal cancer.

5. The use according to any one of claims 1 to 4, characterized in that, The immunotherapy mentioned is immune checkpoint inhibitor therapy.

6. The use according to claim 5, characterized in that, The immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

7. The use according to claim 6, characterized in that, The PD-1 inhibitor is a PD-1 antibody.

8. The use according to any one of claims 1 to 7, characterized in that, The substance specifically used to detect the loss or low expression of GLTSCR1 is selected from primers, probes, aptamers, or antibodies.

9. The use according to claim 8, characterized in that, The substances specifically used to detect GLTSCR1 functional loss or low expression are PCR primers and / or Sanger sequencing reagents.

10. The use according to any one of claims 1 to 9, characterized in that, The preparation is a kit or a test reagent composition.

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