CN115902215A - Non-small cell lung cancer prognosis marker and application thereof - Google Patents
Non-small cell lung cancer prognosis marker and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biomedicine, and particularly relates to a non-small cell lung cancer prognosis marker and application thereof. The invention protects the application of MAIT cells as a prognosis marker of non-small cell lung cancer in the following (A1) or (A2): (A1) Preparing a product for identifying a patient with non-small cell lung cancer who is therapeutically effective against PD-1; (A2) Preparing a product for predicting the resistance of a non-small cell lung cancer patient to an immune checkpoint inhibitor. The present invention finds that MAITs are enriched in tumor tissue, suggesting a poor prognosis, and often express transcriptome profiles associated with the Th-17 cell signaling pathway. In non-small cell lung cancer, an overall decrease in the frequency of circulating MAIT cells and an enrichment at the tumor foci was observed, and different cell subsets suggested different immunotherapeutic prognoses.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a non-small cell lung cancer prognosis marker and application thereof.
Background
Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer and has become the leading cause of cancer-related death worldwide, with a 5-year survival rate of only 4% to 17%. Cancer cells can escape immune surveillance of the host through a variety of pathways (e.g., expression of PD-1 and PD-L1). For patients with advanced non-small cell lung cancer, immune Checkpoint Inhibitors (ICIs) can reactivate T cells by blocking inhibitory interactions between T cells and tumor cells or dendritic cells. General immune checkpoints include programmed death-1 (PD-1), programmed death ligand-1 (PD-L1) [ Doroshow DB, sanmamed MF, hastings K, politi K, rimm DL, chen L, et al. 25:4592-602.].
Previous studies have shown that anti-PD-1/PD-L1 antibodies can improve overall survival in lung cancer patients, but a large number of patients still do not respond to PD-1/PD-L1 blockade. Therefore, it is necessary to find biomarkers that indicate whether a patient is responding to a treatment. Immunohistochemical detection of PD-L1 expression is one of the most commonly used indicators of ICIs therapy approved by the FDA. However, numerous studies have shown that many patients with PD-L1 positive tumors do not benefit from PD1/PD-L1 blocking therapy. Other biomarkers such as tumor mutation burden, tumor infiltrating lymphocytes, mismatch repair defects, and microsatellite instability also have limitations.
Mucosal-associated invariant T cells (MAIT) are an inherently present T cell whose T cell receptor is composed of an invariant α chain and a variable β chain. MAIT cells are enriched in mucosa, peripheral blood and tissues [ Godfrey DI, koay H-F, mcclusky J, gherardin na. The biological and functional importance of MAIT cells. Nat immunol.2019;20:1110-28.]. MAIT cells, once activated, can exert cytotoxicity and produce inflammatory cytokines such as IFN- γ, TNF- α and IL-17A, as well as cytotoxic mediators such as granzyme B and perforin. Studies have shown that MAIT cells are reduced in peripheral blood, abundant in tumor foci in patients with mucosal tumors (gastric, colorectal and lung cancers), and suppress anti-tumor immunity through the production of tumor-promoting cytokines IL-17A and IL-8, which are considered biomarkers for monitoring and predicting clinical benefit of ici in various cancer patients [ Liu C, liu R, wang B, lian J, yao Y, sun H, et al.blocking IL-17 anenhances tumor response to anti-PD-1 immunotherpay in microsatellite tumor stable tumor cancer.2021;9, e001895]. Studies have shown that MAIT Cells rely on IL-17A to Promote Tumor Growth [ Yan J, allen S, mcDonald E, das I, mak JYW, liu L, et al. 10:124-41.]. However, there is no study of MAIT cells in relation to the prognosis of NSCLC and the therapeutic efficacy of ICIs.
Disclosure of Invention
Aiming at the defects of the existing problems, the invention aims to provide a non-small cell lung cancer prognostic marker and application thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows:
in a first aspect, the present invention provides a use of MAIT cells as a prognostic marker for non-small cell lung cancer in (A1) or (A2) as follows:
(A1) Preparing a product for identifying a patient with non-small cell lung cancer who is therapeutically effective against PD-1;
(A2) Preparing a product for predicting the resistance of a non-small cell lung cancer patient to an immune checkpoint inhibitor.
In some specific embodiments, the use of the MAIT cell is a MAIT-IFNGR cell, in particular a MAIT-IFNGR cell, in the preparation of a product for identifying a non-small cell lung cancer patient for whom anti-PD-1 therapy is effective.
In some specific embodiments, the MAIT cell is a MAIT-IL17 cell, in particular, the use of a MAIT-IL17 cell in the preparation of a product for predicting the resistance of a non-small cell lung cancer patient to an immune checkpoint inhibitor.
In a second aspect, the present invention provides a substance for detecting the amount of MAIT cells, which is used in the following (B1) or (B2): (B1) Preparing a product for identifying a patient with non-small cell lung cancer who is therapeutically effective against PD-1;
(B2) Preparing a product for predicting the resistance of a non-small cell lung cancer patient to an immune checkpoint inhibitor.
In a third aspect, the invention provides a kit for identifying a patient with non-small cell lung cancer who is therapeutically effective against PD-1, said kit comprising a detection reagent and/or a detection apparatus for a prognostic marker as described hereinbefore.
In a fourth aspect, the invention provides a kit for predicting the resistance of a non-small cell lung cancer patient to an immune checkpoint inhibitor, said kit comprising a detection reagent and/or a detection apparatus for a prognostic marker as described hereinbefore.
In some specific embodiments, the detection object of the kit is tumor tissue and/or tissue beside cancer of a patient with non-small cell lung cancer.
Advantageous effects
The invention creatively discloses the distribution, phenotype and function of MAIT cells in non-small cell lung cancer patients and the correlation between different cell phenotypes and clinical prognosis. Our study showed that IL-17A + MAITS is enriched in tumor tissues and expresses a transcriptome profile associated with the Th-17 cellular signaling pathway, suggesting a lower level of response to anti-PD-1 treatment; whereas IFNGR + MAIT cells did not enrich in tumor tissue, suggesting a higher level of response to anti-PD-1 treatment.
Drawings
FIG. 1 t-SNE is a graph showing CD3 in peripheral blood, tumor and paracancerous tissues of a patient with non-small cell lung cancer + Gene expression levels of T cells (note: the subpopulation of cells numbered 6 was confirmed as MAIT cells);
FIG. 2 expression of the immunosuppressive molecules PD-1 and TIM-3 on MAIT cells in tumors and tissues adjacent to tumors and carcinomas in non-small cell lung cancer (NSCLC) patients;
FIG. 3 HE staining (left) immunofluorescence staining (center) and PD-1 of MAIT cells of non-small cell lung carcinoma tumor-infiltrating tissue + Cells and IL-17A + Comparison of cell density information (right);
FIG. 4 HLA-DR and PD-1 expression levels of peripheral blood MAIT cells (control: non-small cell lung cancer patient not subjected to anti-PD-1 therapy, PD: progressive disease patient, SD: stationary disease patient, PR/CR patients fully responsive and partially responsive to anti-PD-1 therapy);
FIG. 5 is a representative graph of MAIT cell IFN- γ R expression in tumor tissue of NSCLC patients receiving anti-PD-1 treatment and summary data thereof;
FIG. 6 is a representative graph and summary data thereof of PD-1 expression by MAIT cells in tumor tissue of a NSCLC patient receiving anti-PD-1 treatment;
figure 7 MAIT cell subpopulation enrichment map (UMAP map) of tumor tissue in response cohort and non-response cohort;
FIG. 8 differentially expressed genes between the two subpopulations MAIT-17 and MAIT-IFNGR;
FIG. 9 Gene enrichment radar profiles of MAIT-IFNGR and MAIT-17 cells;
FIG. 10 IFN-. Gamma.R + granzyme B against tumor infiltration in PD-1 treated NSCLC patients + The ratio between responder and non-responder in MAIT cells and summary data thereof;
FIG. 11 anti-PD-1 treatment of tumor-infiltrated PD-1 in NSCLC patients + IFN-17A + Ratio of responding to non-responding in MAIT cells and summary thereof;
FIG. 12 CT scan of NSCLC patients receiving anti-PD-1 treatment (R: responder, NR: non-responder).
Detailed Description
The present invention will be described in further detail with reference to examples. The reagents or instruments used are not indicated by manufacturers, and are regarded as conventional products which can be purchased in the market.
The study cohort contained 158 non-small cell lung cancer patients, 14 colorectal cancer patients, 20 esophageal cancer patients, 16 gastric adenocarcinoma patients treated in the Jiangsu tumor hospital during the years 2018 to 2022. Also, all human specimens were collected following the hellinnki. The study procedure passed the approval of the human research committee of the cancer hospital, jiangsu, and the procedure was performed according to standard entries.
Example 1
We have studied tumor-infiltrating MAIT cells, which included organizing patients for a cohort of patients. With informed consentPeripheral blood samples were subjected to density gradient centrifugation using Ficoll solution (Amersham Biosciences, uppsala, sweden) according to standard procedure (1) to isolate Peripheral Blood Mononuclear Cells (PBMCs). Fresh tumor tissues and peritumoral tissues are taken in a tumor hospital in Jiangsu province immediately after the operation of a patient is finished, and a severe necrotic area is avoided. Paratumoral tissue was collected 5cm from the tumor margins and confirmed by 2 specialized pathologists. In addition, a portion of the tissue specimens was fixed with neutral buffered formalin and embedded in paraffin for immunofluorescent staining. For flow cytometry analysis, single cell suspensions of tissue samples were prepared. Briefly, fresh tissue is cut to about 1mm 3 Was digested in RPMI1640 (HyClone Laboratories, south Logan, UT, USA) and 0.05% collagenase I (Sigma-Aldrich, st. Louis, MO, USA) and 0.002% DNase I (Sigma-Aldrich, st. Louis, MO, USA) was added for 30 minutes at 37 ℃. Cells were filtered through a 70 μm grid (BD Biosciences, heidelberg, germany) and centrifuged with Percoll. After two washes, the cells were resuspended in RPMI1640 supplemented with 10% fetal bovine serum for subsequent experiments. As shown in FIG. 1, cells were grouped by gene expression level, and MAIT cells were identified. Single cell suspensions extracted from blood and tissue samples were stained with fluorescently labeled antibodies and then analyzed by multicolor flow cytometry. After the above flow analysis, the study found that MAIT cells infiltrated in tumors had higher CD38 levels than in the para-carcinoma tissues, however, the level of HLA-DR was not different. As shown in fig. 2, the levels of PD-1 and TIM-3 of MAIT cells derived from cancer tissue were significantly elevated compared to the para-cancerous tissue. As shown in FIG. 3, granzyme B and IFN-. Gamma.production was inhibited in tumor tissues, whereas IL-17A was elevated. There was no difference in the levels of CTLA-4 and IL-8 present in MAIT cells in cancerous and paracancerous tissues.
Example 2
We hypothesized that peripheral circulating MAIT cells can be considered as a biomarker to predict the success of PD-1 blocking therapy and that clinically relevant assays can be performed prior to the use of ICIs. Thus, patients can be divided into control groups (non-small cell lung cancer patients who have not received ICIs) and non-responsive groups (in the disease stable and progressive stages) and responsive groups (again divided into complete response and partial response), and solid cancers assessed using the response assessment criteria (rest version 1.1). As shown in FIG. 4, MAIT cells were examined for phenotype and function by flow cytometry, including CD3, CD4, CD8, TCR V α 7.2, CD161, HLA-DR, CD38, PD-1, CTLA-4, TIM-3, IFN- γ, granzyme B, IL-17A, IL-8, annexin V, ki-67, and IFN- γ R, among others. In terms of cytokine detection, MAIT cells were activated for 4h by PM (Biolegend, USA), ionomycin (Biolegend, USA), brefeldin a (Biolegend, USA) and monensin (Biolegend, USA). Following cell fixation using an immobilization/permeation buffer (eBioscience, thermoFisher) according to the manufacturer's instructions, intracellular staining was performed for IFN-. Gamma., granzyme B, IL-17A and IL-8. Flow cytometry data were compensated in FlowJo (TreeStar, USA) using a single staining control. Samples were collected using a CytoFLEX LX flow cytometer (Beckman Coulter, break, CA, USA) and FACS Aira II SORP (BD) and analyzed using FlowJo software. HLA-DR was upregulated in the responders, while SD and control groups did not.
As shown in FIG. 5, granzyme B and IFNG were elevated in the responder and stationary phase groups compared to the control group after anti-PD-1 treatment. As shown in FIG. 6, MAIT cells positive for PD-1 were significantly reduced in responders (compared to SD, PD and control). These results indicate that peripheral MAIT cells block high expression of PD-1.
Example 3
As shown in fig. 7, to clarify the overall appearance of MAIT cells in the microenvironment of non-small cell lung cancer from ICIs, we measured immune cells from non-small cell lung cancer before and after anti-PD-1 treatment using single cell sequencing data omics (GEO access GSE 176022). The MAIT cells were divided into two subsets, and the reactive cohort was found to be enriched predominantly in the MAIT-IFNGR subset, whereas the non-responder subset was enriched predominantly in the MAIT-17 subset.
The transcriptional profiles of the two subpopulations were compared as shown in figure 8. We found a MAIT subset that highly expressed IL-17A immune checkpoint receptors, such as PDCD1 and ENTPD1 (CD 39). As shown in fig. 9, the MAIT-IFNG subpopulation is predisposed to gene expression genes associated with immune activation (e.g., ripo 2, CD6, TYROBP). The MAIT-IFNGR subgroup has certain cytotoxicity function, and the pathways such as a T cell activation pathway, a lymphocyte adhesion pathway and the like are highly enriched, which is consistent with the good diagnosis effect on ICIs therapy. The MAIT-17A subset functions in contrast to MAIT-IFNGR, and this can be used to illustrate clinical resistance to anti-PD-1 therapy.
Example 4
We used flow analysis (see the above examples for details) to evaluate the clinical cohort for MAIT-IFNGR (using IFGR markers) and the MAIT-17 subtype (using PD-1). The responder group CD3 compared to the non-responder group + IFNGR of + CD3 + T cells were largely elevated in the response cohort, however PD-1 + The number of MAIT cells decreased. As shown in fig. 10 and 11, ifngr + Granzyme B + The subtype of MAIT cells is increased, PD-1 + IL-17A + There was a reduction in MAIT cells. With informed consent, we sampled the tumor tissue of this patient and also tracked clinical data such as patient CT. We have specifically found that the infiltration of MAIT-IFNGR cells is elevated in responsive neoplastic lesions, whereas MAIT17A + The cells are reduced. At the same time, there was an increase in MAIT-17 cells in unresponsive tumor lesion tissue, whereas the markers of MAIT-IFNGR were reduced. The data indicate that MAIT-IFNGR cells with cytotoxic function can be used to identify non-small cell lung cancer patients who are therapeutically effective against PD-1; similarly, MAIT-IL17 cells can be used to predict patient resistance to ICIs (immune checkpoint inhibitors).
The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept and the scope of the appended claims is intended to be protected.
Claims (7)
- The application of MAIT cells as a prognosis marker of non-small cell lung cancer in the following (A1) or (A2):(A1) Preparing a product for identifying a patient with non-small cell lung cancer who is therapeutically effective against PD-1;(A2) Preparing a product for predicting the resistance of a non-small cell lung cancer patient to an immune checkpoint inhibitor.
- 2. Use according to claim 1, wherein the MAIT cells are MAIT-IFNGR cells, in particular MAIT-IFNGR cells, for the preparation of a product for identifying non-small cell lung cancer patients for whom a treatment against PD-1 is effective.
- 3. Use according to claim 1, wherein the MAIT cells are MAIT-IL17 cells, in particular MAIT-IL17 cells, for the preparation of a product for predicting the resistance of a non-small cell lung cancer patient against an immune checkpoint inhibitor.
- 4. Use of a substance for detecting the amount of MAIT cells in (B1) or (B2) as follows:(B1) Preparing a product for identifying a patient with non-small cell lung cancer who is therapeutically effective against PD-1;(B2) Preparing a product for predicting the resistance of a non-small cell lung cancer patient to an immune checkpoint inhibitor.
- 5. A kit for identifying a patient with non-small cell lung cancer who is therapeutically effective against PD-1, said kit comprising a detection reagent and/or a detection apparatus for the prognostic marker set forth in claim 1.
- 6. A kit for predicting the resistance of a non-small cell lung cancer patient to an immune checkpoint inhibitor, said kit comprising a detection reagent and/or a detection apparatus for a prognostic marker as defined in claim 1.
- 7. The kit according to claim 4 or 5, wherein the detection object of the kit is tumor tissue and/or tissue beside cancer of the patient with non-small cell lung cancer.
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