CN116135992A - Marker for identifying tumors sensitive to immune checkpoint inhibitors - Google Patents

Marker for identifying tumors sensitive to immune checkpoint inhibitors Download PDF

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CN116135992A
CN116135992A CN202310366606.6A CN202310366606A CN116135992A CN 116135992 A CN116135992 A CN 116135992A CN 202310366606 A CN202310366606 A CN 202310366606A CN 116135992 A CN116135992 A CN 116135992A
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肖雪
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Sichuan Academy Of Medical Sciences Sichuan Provincial People's Hospital
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Abstract

The invention discloses a biomarker for identifying tumors sensitive to immune checkpoint inhibitors, provides a biomarker, and provides application of a reagent for detecting the expression level of the biomarker and/or HLA Class I in preparation of a product for identifying tumors/cold and hot tumors sensitive to immune checkpoint inhibitors. Experiments prove that the biomarker screened by the invention can effectively identify tumors/cold and hot tumors sensitive to immune checkpoint inhibitor therapy, has higher diagnosis efficiency, is beneficial to realizing personalized treatment of endometrial cancer patients, and has wide application prospect.

Description

Marker for identifying tumors sensitive to immune checkpoint inhibitors
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to a marker for identifying tumors sensitive to immune checkpoint inhibitors.
Background
Immune Checkpoint Inhibitors (ICIs) are recently emerging tumor immunotherapeutic agents, which have achieved significant success in the treatment of a variety of tumors. ICIs, by binding to immune checkpoints located on the cell surface, release the inhibition of cd8+ T cell function, restore cd8+ T cell cytotoxicity, and exert tumor cell killing effect.
Tumors that are sensitive to ICIs have a high level of immune cell infiltration, representing a T cell inflammatory phenotype, commonly referred to as thermal tumors; in contrast, low immune invasive tumors that do not respond to ICIs treatment, which are manifested as T-cell deficiency or T-cell rejection, are often referred to as cold tumors.
The thermal tumor has a large number of tumor infiltrating CD8+T lymphocytes (CD8+TILs), when the ICIs relieve the inhibition of immune checkpoints, the immune response effect of the CD8+TILs on the tumor is restarted, and the viable CD8+TILs kill cancer cells to play an immune treatment role. Thus, thermal tumors with inflammatory phenotypes tend to be more sensitive to ICIs; however, for cold tumors, immune checkpoint inhibitors are also difficult to work with due to the lack of adequate cd8+ TILs.
The separation of tumors into cold and hot tumors is a fundamental method of distinguishing between responses to ICIs treatment, as shown by Wang et al in COX-2-related tumor immune microenvironment in non-small cell lung cancer: a novel signature to predict hot and cold tumor (J Thorac Dis.2022.3), screening non-small cell lung cancer for biomarkers that distinguish between cold and hot tumors, and can predict the response to ICIs treatment.
Searching biomarker to distinguish cold tumor and hot tumor, screening patient benefiting ICIS treatment, and realizing personalized treatment of tumor patient.
Disclosure of Invention
To remedy the deficiencies of the prior art, the present invention provides biomarkers for identifying tumors/cold and hot tumors that are sensitive to immune checkpoint inhibitors.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect the invention provides a biomarker for identifying a tumour/cold and hot tumour susceptible to an immune checkpoint inhibitor, said biomarker comprising one or more of HLA-B, IRF9, PARP9, TNFRSF14, IFI16, HLA-DMA, TYMP, TAPBP, DNMT3A, FCER1G, CD53, CD4, WIPF1, RAC 2.
A second aspect of the invention provides the use of any one of the following:
(1) Use of a reagent for detecting the expression level of a biomarker or HLA Class i according to the first aspect of the present invention for the manufacture of a product for identifying a tumour/cold or hot tumour sensitive to an immune checkpoint inhibitor;
(2) The use of a biomarker or HLA Class i according to the first aspect of the present invention in the construction of a computational model for identifying tumors/cold and hot tumors sensitive to immune checkpoint inhibitors;
(3) The use of a biomarker or HLAClass i according to the first aspect of the invention in the construction of a system for identifying tumors/cold and hot tumors sensitive to immune checkpoint inhibitors.
Further, the reagents include probes, primers or antibodies that specifically bind to the biomarker or HLA Class i.
Further, the product comprises a kit, test paper, a chip and a nucleic acid membrane strip.
Further, the kit also includes a detectable label.
Further, the detectable label includes a radioisotope, an enzyme, a nanoparticle, a chemiluminescent label, biotin, a dye.
Further, the system includes:
input means for inputting the expression level of the biomarker or HLA Class i of the first aspect of the invention;
and an output device for outputting a result of identifying the tumor/cold-hot tumor sensitive to the immune checkpoint inhibitor.
Further, the tumor is endometrial cancer.
Further, the endometrial cancer is MMRd/MSI-H type endometrial cancer.
In a third aspect the invention provides a kit for identifying a tumour/cold-hot tumour susceptible to an immune checkpoint inhibitor, said kit comprising reagents for detecting the level of expression of a biomarker or HLA Class i according to the first aspect of the invention.
Further, the kit comprises reagents required for detecting the expression level of the biomarker or HLA Class I gene or protein by RT-PCR method, qRT-PCR method, biochip detection method, southern blotting method, in situ hybridization method, immunoblotting method, immunohistochemical method, spatial transcriptome technique.
Further, the tumor is endometrial cancer.
Further, the endometrial cancer is MMRd/MSI-H type endometrial cancer.
The invention has the advantages and beneficial effects that:
the biomarker which can distinguish the tumor/cold and hot tumor which is sensitive to the immune checkpoint inhibitor therapy is screened by the space transcriptome technology, and is verified by adopting a large sample, and the result proves that the biomarker screened by the method can effectively identify the tumor/cold and hot tumor which is sensitive to the immune checkpoint inhibitor, has higher diagnosis efficiency and is beneficial to realizing personalized treatment of tumor patients.
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FIG. 1 is a graph of a DSP spatial transcription assay screening 14 gene expression profile markers for cold and hot MMRd endometrial cancer, wherein 1A is a multiple fluorescence immunohistochemical staining pattern, 1B is a graph of distinct tumor and interstitial regions, 1C is a 14 gene marker heat pattern, 1D is a CD8+ TILs staining pattern, and 1E is a CD8+ TILs density pattern;
FIG. 2 is a spatial analysis diagram of MMRd endometrial cancer immune cell infiltration, wherein 2A is a tumor region CD8+TILs abundance map, 2B is a mesenchymal region CD8+TILs abundance map, 2C is a tumor region macrophage abundance map, 2D is a mesenchymal region macrophage abundance map, 2E is a tumor region Treg cell abundance map, 2F is a mesenchymal region Treg cell abundance map, 2G is a CD4, foxp3 multiple immunohistochemical staining map, 2H is a CD163, CD68 multiple immunohistochemical staining map, 2I is a tumor region Treg cell density map, 2J is a tumor region M1 type macrophage density map, 2K is a tumor region M2 type macrophage density map, 2L is a MSI-H endometrial cancer CD8+TILs map, 2M is a MSI-H endometrial cancer M1 type macrophage abundance map, 2N is a MSI-H endometrial cancer M2 type macrophage abundance map, and 2O is a MSI-H endometrial cancer cell abundance map;
FIG. 3 is a graph showing the analysis of HLAClass I, DNMT3A and CD8 expression in MMRd endometrial cancer, wherein 3A is an immunohistochemical graph of HLA Class 1, DNMT3A and CD8, 3B is a density graph of CD8+ TILs under different HLA Class I expression conditions, and 3C is a density graph of CD8+ TILs under different DNMT3A expression conditions;
FIG. 4 is a ROC graph of HLA Class I, where 4A is a ROC graph of test set HLA Class I and 4B is a ROC graph of validation set HLA Class I.
Detailed Description
The following provides definitions of some of the terms used in this specification. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The invention provides a biomarker for identifying tumors/cold and hot tumors sensitive to immune checkpoint inhibitors, which comprises one or more of HLA-B, IRF9, PARP9, TNFRSF14, IFI16, HLA-DMA, TYMP, TAPBP, DNMT3A, FCER1G, CD53, CD4, WIPF1 and RAC 2.
HLA-B includes wild-type, mutant or fragments thereof. The term encompasses full length, unprocessed HLA-B, any form of HLA-B derived from processing in a cell, as well as naturally occurring variants (e.g., splice variants or allelic variants) of HLA-B. The term encompasses, for example, human HLA-B as well as HLA-B from any other vertebrate source, including mammals, such as primates and rodents (e.g., mice and rats), gene ID:3106.
IRF9 includes wild-type, mutant or fragments thereof. The term encompasses full length, unprocessed IRF9, any form of IRF9 derived from processing in a cell, as well as naturally occurring variants (e.g., splice variants or allelic variants) of IRF 9. The term encompasses IRF9, e.g., human, as well as IRF9 from any other vertebrate source, including mammals, such as primates and rodents (e.g., mice and rats), gene IDs: 10379.
PARP9 includes wild-type, mutant or fragments thereof. The term encompasses full length, unprocessed PARP9, any form of PARP9 derived from processing in a cell, as well as naturally occurring variants (e.g., splice variants or allelic variants) of PARP 9. The term encompasses, for example, human PARP9 as well as PARP9 from any other vertebrate source, including mammals, such as primates and rodents (e.g., mice and rats), gene IDs: 83666.
TNFRSF14 includes wild type, mutant type, or a fragment thereof. The term encompasses full length, unprocessed TNFRSF14, any form of TNFRSF14 derived from processing in a cell, and naturally occurring variants (e.g., splice variants or allelic variants) of TNFRSF 14. The term encompasses TNFRSF14, e.g., human, as well as TNFRSF14 from any other vertebrate source, including mammals, such as primates and rodents (e.g., mice and rats), gene ID:8764.
IFI16 includes wild-type, mutant or fragments thereof. The term encompasses full length, unprocessed IFI16, any form of IFI16 derived from processing in a cell, as well as naturally occurring variants (e.g., splice variants or allelic variants) of IFI 16. The term encompasses IFI16, e.g., human, as well as IFI16 from any other vertebrate source, including mammals, such as primates and rodents (e.g., mice and rats), gene ID:3428.
HLA-DMA includes wild-type, mutant or fragments thereof. The term encompasses full length, unprocessed HLA-DMA, any form of HLA-DMA derived from processing in a cell, as well as naturally occurring variants (e.g., splice variants or allelic variants) of HLA-DMA. The term encompasses, for example, human HLA-DMA as well as HLA-DMA from any other vertebrate source, including mammals such as primates and rodents (e.g., mice and rats), gene ID:3108.
TYMP includes wild type, mutant or fragments thereof. The term encompasses full-length, unprocessed TYMP, any form of TYMP derived from processing in a cell, as well as naturally occurring variants (e.g., splice variants or allelic variants) of TYMP. The term encompasses, for example, human TYMP as well as TYMP from any other vertebrate source, including mammals such as primates and rodents (e.g., mice and rats), gene ID:1890.
TAPBP includes wild-type, mutant or fragments thereof. The term encompasses full length, unprocessed TAPBPs, any form of TAPBP derived from processing in a cell, as well as naturally occurring variants (e.g., splice variants or allelic variants) of TAPBP. The term encompasses, for example, human TAPBP as well as TAPBP from any other vertebrate source, including mammals such as primates and rodents (e.g., mice and rats), gene ID:6892.
DNMT3A comprises wild-type, mutant or fragments thereof. The term encompasses full length, unprocessed DNMT3A, any form of DNMT3A derived from processing in a cell, and naturally occurring variants (e.g., splice variants or allelic variants) of DNMT 3A. The term encompasses, for example, human DNMT3A as well as DNMT3A from any other vertebrate source, including mammals, such as primates and rodents (e.g., mice and rats), gene ID:1788.
FCER1G includes wild-type, mutant, or fragments thereof. The term encompasses full-length, unprocessed FCER1G, any form of FCER1G derived from processing in a cell, and naturally occurring variants (e.g., splice variants or allelic variants) of FCER 1G. The term encompasses FCER1G, e.g., human, as well as FCER1G from any other vertebrate source, including mammals, such as primates and rodents (e.g., mice and rats), gene ID:2207.
CD53 includes wild-type, mutant or fragments thereof. The term encompasses full length, unprocessed CD53, any form of CD53 derived from processing in a cell, as well as naturally occurring variants (e.g., splice variants or allelic variants) of CD 53. The term encompasses, for example, human CD53 as well as CD53 from any other vertebrate source, including mammals, such as primates and rodents (e.g., mice and rats), gene IDs: 963.
CD4 includes wild-type, mutant or fragments thereof. The term encompasses full length, unprocessed CD4, any form of CD4 derived from processing in a cell, as well as naturally occurring variants (e.g., splice variants or allelic variants) of CD 4. The term encompasses, for example, human CD4 as well as CD4 from any other vertebrate source, including mammals, such as primates and rodents (e.g., mice and rats), gene IDs: 920.
WIPF1 includes wild type, mutant or fragments thereof. The term encompasses full length, unprocessed WIPF1, any form of WIPF1 derived from processing in a cell, as well as naturally occurring variants (e.g., splice variants or allelic variants) of WIPF 1. The term encompasses, for example, human WIPF1 as well as WIPF1 from any other vertebrate source, including mammals, such as primates and rodents (e.g., mice and rats), gene ID:7456.
RAC2 comprises wild type, mutant or fragment thereof. The term encompasses full length, unprocessed RAC2, any form of RAC2 derived from processing in a cell, and naturally occurring variants (e.g., splice variants or allelic variants) of RAC 2. The term encompasses, for example, human RAC2 as well as RAC2 from any other vertebrate source, including mammals such as primates and rodents (e.g., mice and rats), gene IDs: 5880.
the invention provides the use of any one of the following:
(1) The use of a reagent for detecting the expression level of the biomarker or HLA Class I in the preparation of a product for identifying tumors/cold and hot tumors sensitive to an immune checkpoint inhibitor;
(2) The application of the biomarker or HLA Class I in constructing a calculation model for identifying tumors/cold and hot tumors sensitive to immune checkpoint inhibitors;
(3) The use of the above biomarker or HLAClass i for the construction of a system for identifying tumors/cold and hot tumors sensitive to immune checkpoint inhibitors.
HLAClass I (HLAClass I ABC), also known as HLA-class I, contains a range of subtypes, HLA-A/HLA-B/HLA-C, respectively.
The reagents include probes, primers, or antibodies that specifically bind to the biomarker or HLAClass i.
In the present invention, a primer refers to a short nucleic acid molecule, such as a DNA oligonucleotide, e.g., a sequence of at least 15 nucleotides, that can anneal to a complementary target nucleic acid molecule by nucleic acid hybridization to form a hybrid between the primer and the target nucleic acid strand. The primer can be extended along the target nucleic acid molecule by a polymerase. Thus, primers can be used to amplify a target nucleic acid molecule, wherein the sequence of the primer is specific for the target nucleic acid molecule, so that, for example, the primer will hybridize to the target nucleic acid molecule under very high stringency hybridization conditions.
The specificity of the primer increases with its length. Thus, for example, a primer containing 30 consecutive nucleotides will anneal to the target sequence with a higher specificity than a corresponding primer of only 15 nucleotides. Thus, probes and primers containing at least 15, 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides can be selected for higher specificity. In specific embodiments, the primer is at least 15 nucleotides in length, e.g., at least 15 consecutive nucleotides that are complementary to the target nucleic acid molecule. Specific lengths of primers useful in practicing the methods disclosed herein include primers having at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 45, at least 50 or more consecutive nucleotides complementary to a target nucleic acid molecule to be amplified, such as 15-60 nucleotides, 15-50 nucleotides, 20-40 nucleotides, 25-50 nucleotides or 15-30 nucleotides.
In the present invention, a probe refers to an oligonucleotide capable of hybridizing to a target nucleic acid of interest. One of skill in the art will appreciate that depending on the stringency of the hybridization conditions, probes will typically substantially bond target sequences lacking complete complementarity to probe sequences. The probe may be associated with an appropriate tag or reporter moiety (reporter moiety) so that the probe (and its target) may be detected, visualized, measured and/or assayed.
The probes or primers may be modified in any manner as long as the modifications given are compatible with the desired function of the given oligonucleotide. One of skill in the art can readily determine whether a given modification is appropriate or desirable for any given oligonucleotide of the invention.
In the present invention, antibodies refer to immunoglobulin molecules and immunologically active portions (fragments) of immunoglobulin molecules, i.e., molecules that contain an antibody binding site or paratope. The term includes, but is not limited to, monoclonal antibodies, recombinant antibodies, single chain antibodies, chimeric antibodies and fragments thereof that are immunoreactive with the biomarker.
The product comprises a kit, test paper, a chip and a nucleic acid membrane strip.
In the present invention, a kit refers to a kit of parts or a set of components, a combination or mixture of which can be used to perform the measurement/detection of one or more analytes or markers.
The kit of the invention also includes a detectable label.
Such detectable labels include, but are not limited to, radioisotopes, enzymes, nanoparticles, chemiluminescent labels, biotin, dyes.
Wherein the radioisotope includes, but is not limited to 32 P、 33 P、 35 S、 3 H and 125 I. these radioisotopes have different half-lives, decay types and energy levels, which can be tailored to match the needs of a particular protocol. For example, the number of the cells to be processed, 3 h is a low energy emitter that produces low background levels, however the low energy also causes autoradiography over long periods of time. Radiolabeled ribonucleotides, deoxyribonucleotides and amino acids are commercially available. The radiolabeled nucleotide at the first or alpha phosphate group, or the third or gamma phosphate group, is available. For example, [ alpha-32P ]]dATP and [ gamma-32P]dATP is commercially available. In addition, radiolabeled nucleotides with different specific activities are also commercially available and can be adapted for use in different protocols.
Nanoparticles range in size from 1 to 1000nm and include different chemical structures such as gold and silver particles and quantum dots. Silver or gold nanoparticles in the range of 40-120nm will scatter monochromatic light with high intensity when irradiated with angled incident white light. The wavelength of the scattered light depends on the size of the particles. 4-5 different particles in close proximity will each scatter monochromatic light, which when superimposed will give a specific unique color. The particles are manufactured by companies such as Genicon Sciences (Carlsbad, calif.). Derivatized silver or gold particles can be attached to a wide range of molecular arrays, including proteins, antibodies, small molecules, receptor ligands, and nucleic acids. For example, the particle surface may be chemically derivatized to allow attachment to nucleotides.
Dyes generally have one or more types of emitted light, which may be visible or invisible, such as ultraviolet or infrared light. In exemplary embodiments, the dye may be a Fluorescence Resonance Energy Transfer (FRET) dye; dyes having an amino group at the α or β position (such as naphthylamine dye, 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalenesulfonate, and 2-p-toluidinyl-6-naphthalenesulfonate); a dye having 3-phenyl-7-isocyanatocoumarin; acridines such as 9-isothiocyanacridine and acridine orange; pyrene, oxadiazole and stilbene; a dye having 3- (epsilon-carboxypentyl) -3 '-ethyl-5, 5' -dimethoxycarbocyanine (CYA); 6-carboxyfluorescein (FAM); 6-carboxy-4 ',5' -dichloro-2 ',7' -dimethoxy fluorescein (JOE); ALEXAFLUORTM; cy2; 6-carboxy-2 ',4, 7' -tetrachlorofluorescein (TET); 6-carboxy-2 ',4',5',7' -Hexachlorofluorescein (HEX); 5-carboxy-2 ',4',5',7' -tetrachlorofluorescein (ZOE); NAN; NED; cy3; cy3.5; cy5; cy5.5; cy7; and cy7.5; IR800CW, ICG, alexa Fluor 350; alexa Fluor 488; alexa Fluor 532; alexa Fluor 546; alexa Fluor 568; alexa Fluor 594; alexa Fluor 647; alexa Fluor680 or Alexa Fluor 750.
The kit also includes other components, such as buffers and solutions (e.g., pretreatment reagents) for separating and/or processing the test sample. The kit may additionally include one or more other controls. One or more components of the kit may be lyophilized, and the kit may further comprise reagents suitable for reconstitution of the lyophilized components.
The various components of the kit are optionally provided in a suitable container. The one or more containers may be microtiter plates. The kit may also include a container for holding or storing a sample (e.g., a container or cartridge for a blood or urine sample). The kit may optionally also include reaction vessels, mixing vessels, and other components to facilitate preparation of reagents or test samples, as appropriate. The kit may also include one or more instruments for assisting in obtaining the test sample, such as syringes, pipettes, forceps, and a measuring spoon.
In the present invention, tumors include, but are not limited to, adrenocortical carcinoma, anal carcinoma, appendicular carcinoma, astrocytomas (cerebellar astrocytomas, cerebral astrocytomas, childhood astrocytomas, pineal astrocytomas), basal cell carcinoma, cholangiocarcinomas, bladder carcinoma, bone tumors, brain cancer, breast cancer, bronchial adenomas, carcinoid tumors, primary unknown carcinomas, cervical cancer, chronic myeloproliferative disorders, desmoplastic small round cell tumors, endometrial cancer, ependymomas, epithelial vascular endothelial tumors (EHE), esophageal cancer, ewing tumor sarcoma family, extracranial germ cell tumors, extragonadal germ cell tumors, eye cancer, gall bladder cancer, gestational trophoblastoma, glioma, colorectal cancer, head and neck cancer, heart cancer, liver cancer, islet cell carcinoma, kaposi's sarcoma renal cancer (renal cell carcinoma), laryngeal carcinoma, leukemia, lip cancer, liposarcoma, lung cancer (non-small cell lung cancer, small cell lung cancer), lymphomas (non-hodgkin's lymphoma, aids-related lymphoma, burkitt's lymphoma, cutaneous T cell lymphoma), megalobemia, bone malignant fibrous histiocytoma/osteosarcoma, medulloblastoma, melanoma, meckel cell carcinoma, multiple endocrine tumor syndrome, bone marrow cancer, mycotic tumor, myelodysplastic syndrome, myxoma, nasal and paranasal cavity cancer, neuroblastoma, ovarian cancer, pancreatic cancer, penile cancer, pharyngeal cancer (hypopharyngeal carcinoma, nasopharyngeal carcinoma, oropharynx cancer, pheochromocytoma, pineal blastoma, supratentorial neuroectodermal tumor, pituitary adenoma, pleural pneumoblastoma, pneumoblastoma, prostate cancer, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, szary syndrome, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, testicular cancer, thymoma, thyroid cancer, urinary tract cancer, and vaginal cancer.
In an embodiment of the invention, the tumor is selected from endometrial cancer.
The endometrial cancer comprises MMRd/MSI-H (mismatch repair deficiency/microsatellite instability) endometrial cancer, POLE mutant (POLEmut) endometrial cancer, p53 protein abnormal (p 53 abn) endometrial cancer, and nonspecific molecular profiling (NSMP) endometrial cancer.
In a specific embodiment of the invention, the endometrial cancer is selected from MMRd/MSI-H (mismatch repair deficiency/microsatellite instability).
In the present invention, cd8+ TILs, i.e. cd8+ tumor infiltrating T lymphocytes, may be abbreviated as cd8+ T cells.
The invention is further illustrated below in connection with specific embodiments. It should be understood that the particular embodiments described herein are presented by way of example and not limitation. The principal features of the invention may be used in various embodiments without departing from the scope of the invention.
Examples
1 Experimental materials
Tissue chips made from 45 MMRd endometrial cancer samples (2 mm diameter per TMA): pathological tissue samples diagnosed with endometrial cancer in the hospitals of people in Sichuan province from 2019 to 2021;
82 MMRd endometrial cancer whole-section samples: paraffin Embedded (FFPE) pathological tissue samples diagnosed as endometrial cancer in the people hospitals of the province of Sichuan in 2013 to 2021. 82 samples were all mismatch repair deficient (MMRd/MSI).
2 Experimental methods
2.1 Spatial transcription sequencing analysis of MMRd endometrial cancer tissue chip
The RNA hybridization probe of Nanostring (detecting the expression of nearly 2000 mrnas related to immune response, tumor microenvironment, tumor biology, tumor inflammation) was used to incubate with TMA pathological sections overnight to hybridize in situ with the target RNA in the sample (the RNA hybridization probe has a linker group that can be uv-hydrolyzed in addition to the binding site of the target RNA for ligation to DNA sequences with specific tags). After hybridization, the sample is morphologically labeled with a polychromatic fluorescent antibody and scanned for imaging, thereby distinguishing tumor cells, interstitium or immune regions. Then, tumor centers, invasion margin, and interstitial immune cell regions were selected for each sample in TMA sections, and uv irradiation was performed, respectively. At this point the photolyzable linker on the probe breaks and the attached DNA sequence is released and absorbed by the capillary into the 96-well plate. These collected DNA sequences consist of several parts: RNA target specific markers, specific molecular tags UMI, and primer binding sites. Thus can be used for RNA identification, second generation sequencing (next generation sequencing, NGS) and sample discrimination (sample demultiplexing). The DNA sequence was then pooled, the PCR product purified and sequenced. The NGS data information is then traced back to the specific location selected on the TMA pathology section, enabling in situ target abundance analysis for each selected region of each sample.
2.2 immunohistochemistry
Each tumor pathology sample was serially sectioned and then incubated at 60 ℃ for 1 hour. Dewaxing with xylene, rehydrating with gradient alcohol, repairing antigen, and adding 3% H 2 O 2 Blocking was performed and then staining was performed on MLH1 (clone ES 05), MSH2 (clone RED 2), MSH6 (clone EP 49), PMS2 (clone EP 51), CD8 (clone SP 16) and HLAClass I ABC (clone EMR 8-5). After overnight incubation with primary antibody at 4 ℃, incubation with secondary antibody was performed for 30 min followed by DAB development (Dako REALTMEnVisionTM). Finally, the slide was stained with hematoxylin and blocked.
2.3 immunohistochemical interpretation criteria
MMRd: the MMR status of each sample was determined by immunohistochemistry of MMR proteins (MLH 1, MSH2, MSH6 and PMS 2). Lymphocytes, mesenchymal cells or normal endometrial nuclei were stained as positive internal controls. Mismatch repair deficiency (MMRd) is defined as the complete absence of nuclear staining of any MMR protein in tumor cells, with a positive internal control.
Cd8+tils density: the density of cd8+ TILs was evaluated as the number of cd8+ TILs infiltrated into the tumor cell area. For each sample, cd8+ TILs were counted from five randomly selected high power fields and averaged to obtain the cd8+ TILs density for that sample.
HLAClass I expression: HLAClass I positive (+) is defined as >90% of tumor cells being expressed in the membrane and/or cytoplasm; HLAClass I partial deletion loss (. + -.) is defined as 10-90% of tumor cells expressing the protein; HLAClass I negative (-) is defined as <10% of tumor cells having HLA Class I expression.
2.4 Cold and hot tumor differentiation criteria
MMRd endometrial cancer was divided into three immune subtypes, "cold (cold)", "hot)", and "intermediate" (intermediate) according to the expression profile of the 14 gene in a spatial transcriptional sequencing analysis, representing different degrees of immune infiltration, respectively. Cd8+ TILs density was highest in hot tumors and lowest in cold tumors.
In immunohistochemical experiments, cd8+ TILs were high in density as "hot" tumors and low in density as "cold" tumors.
2.5 statistical analysis
WGCNA and Lasso regression analysis screened for markers of core gene expression profile associated with cd8+ TILs infiltration in tumor areas.
Of 82 MMRd endometrial cancer samples, 18 MMRd endometrial cancer samples were randomly selected as a test set, 64 MMRd endometrial cancer samples as a validation set, and ROC curves were plotted.
3 results of experiments
After multiplex fluorescence immunohistochemical staining of TMA containing 45 MMRd endometrial cancers, tumor and interstitial regions were marked (fig. 1a,1 b), followed by DSP spatial transcription sequencing, and the results of the tumor and interstitial regions were analyzed separately. The 14 gene expression profile of the tumor region was found to be able to divide MMRd endometrial cancer into three immune subtypes, "cold", "hot" and "intermediate" representing different degrees of immune infiltration, respectively (fig. 1C). By subjecting the samples to CD8 immunohistochemical staining, the numbers of cd8+ TILs infiltrated in the tumor area were calculated, and the cd8+ TILs density of the "hot" tumors was found to be significantly higher than that of the other two types of tumors among the three types separated by 14 gene expression profile (fig. 1d,1 e).
Immunoinfiltration analysis of tumor and interstitial regions after DSP spatial transcriptional sequencing found that the abundance of cd8+ TILs, macrophages and Treg cells was significantly higher in both tumor and interstitial regions for "hot" tumors than in both other subgroups (fig. 2A-2F). Subsequent validation experiments with multiplex fluorescence immunohistochemistry showed that the density of Tregs in "hot" tumors was significantly higher than in "cold" tumors in the tumor area; however, in the interstitial region, the difference in Tregs density was not significant between "cold" and "hot" tumors. The infiltration of M1 and M2 macrophages in the tumor region "hot" tumors was significantly higher than in the other two groups of tumors (FIGS. 2G-2K). Immunocytoinfiltration analysis of 158 MSI-H endometrial cancer samples in the TCGA database revealed that the results of immunocompetence support the results of DSP and multiplex fluorescence immunohistochemistry after "cold" and "hot" classification of these 158 tumors with 14 gene Panel (FIG. 2L-2O).
In MMRd endometrial cancer samples, the density of the regions cd8+ TILs highly expressed by HLA Class I was high, the density of the regions cd8+ TILs low, on the contrary, the density of the regions cd8+ TILs low in DNMT3A was high, and the density of the regions cd8+ TILs high in DNMT3A was low (fig. 3A). In 82 MMRd endometrial cancer samples, the cd8+ TILs density of HLA Class i+ tumors was significantly higher than that of HLA Class I "(fig. 3B), and in 82 MMRd endometrial cancer the cd8+ TILs density of the DNMT3A high-expressing region was significantly lower than that of the regions (++, strongly expressed ++, mid-expressed +/-, weakly expressed or deleted) that were either less or negative to DNMT3A expression (fig. 3C). It can be seen that at the protein level HLA Class I expression and cd8+ TILs infiltration are positively correlated, whereas DNMT3A expression and cd8+ TILs infiltration are negatively correlated (p <0.05; p <0.01; p <0.001; p < 0.0001).
HLA Class I had a high diagnostic efficacy with an AUC in test set of 0.7083 and an AUC in validation set of 0.7320 (FIG. 4).
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims (10)

1. A biomarker for identifying a tumor/cold and hot tumor sensitive to an immune checkpoint inhibitor, wherein the biomarker comprises one or more of HLA-B, IRF9, PARP9, TNFRSF14, IFI16, HLA-DMA, TYMP, TAPBP, DNMT3A, FCER1G, CD53, CD4, WIPF1, RAC 2.
2. The use of any one of the following:
(1) Use of an agent that detects the expression level of a biomarker or HLA Class i according to claim 1 for the manufacture of a product for identifying tumors/cold and hot tumors sensitive to immune checkpoint inhibitors;
(2) Use of the biomarker or HLA Class i of claim 1 for constructing a computational model for identifying tumors/cold-hot tumors sensitive to immune checkpoint inhibitors;
(3) Use of a biomarker or HLA Class i according to claim 1 for the construction of a system for identifying tumors/cold-hot tumors sensitive to immune checkpoint inhibitors.
3. The use according to claim 2, wherein the agent comprises a probe, primer or antibody that specifically binds to the biomarker or HLAClass i.
4. The use according to claim 2, wherein the product comprises a kit, a test paper, a chip, a nucleic acid membrane strip.
5. The use of claim 4, wherein the kit further comprises a detectable label.
6. The use according to claim 5, wherein the detectable label comprises a radioisotope, an enzyme, a nanoparticle, a chemiluminescent label, biotin, a dye.
7. The use according to claim 2, wherein the tumour is endometrial cancer.
8. The use according to claim 7, wherein the endometrial cancer is an MMRd/MSI-H type endometrial cancer.
9. A kit for identifying a tumor/cold-hot tumor sensitive to an immune checkpoint inhibitor, said kit comprising reagents for detecting the biomarker or HLA Class i expression level of claim 1.
10. The kit of claim 9, wherein the kit comprises reagents required for detecting the level of expression of the biomarker or HLAClass i gene or protein by RT-PCR, qRT-PCR, biochip assay, southern blotting, in situ hybridization, immunoblotting, immunohistochemistry, spatial transcriptome techniques.
CN202310366606.6A 2023-04-07 2023-04-07 Marker for identifying tumors sensitive to immune checkpoint inhibitors Pending CN116135992A (en)

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