Composition for predicting response to immune checkpoint inhibitors in cancer patients

Abstract

One embodiment relates to a composition for predicting the responsiveness of a cancer patient to an immune checkpoint inhibitor and a method for predicting the responsiveness of a cancer patient to an immune checkpoint inhibitor. According to one embodiment, it has been confirmed that the RT-qPCR results for CD274 mRNA in cancer cells of a cancer patient to which an immune checkpoint inhibitor has been administered are significantly correlated with the PD-L1 immunohistochemistry (IHC) results, and are superior in sensitivity, specificity, positive predictive value, negative predictive value, and accuracy to the PD-L1 immunohistochemistry (IHC) results. Thus, the composition for predicting the responsiveness of a cancer patient to an immune checkpoint inhibitor according to one embodiment is superior in predicting the responsiveness of a cancer patient (particularly, a patient with gastric cancer or urothelial cancer) to an immune checkpoint inhibitor than immunohistochemistry for PD-L1, and the composition has the advantage of being able to shorten the time required to predict the responsiveness of an immune checkpoint inhibitor to an immune checkpoint inhibitor by measuring the expression level of CD274 mRNA using RT-qPCR, thereby enabling early diagnosis of cancer.

Description

[Technical field]

[0001] The present invention relates to a composition for predicting the responsiveness of cancer patients to immune checkpoint inhibitors. [Background technology]

[0002] Programmed Death-ligand 1 (PD-L1) immunohistochemistry (IHC) is widely used to predict treatment response to immune checkpoint blockade (ICB). Such tests measure PD-L1 protein levels but vary by antibody clone, staining platform and analysis method. For example, evaluation of PD-L1 expression in advanced gastric or gastroesophageal junction adenocarcinoma is performed using the PD-L1 IHC 22C3 pharmDx assay and combined positive score (CPS), whereas metastatic non-small cell lung cancer (NSCLC) sample testing uses tumor proportion score (TPS) instead of CPS. The Ventana SP142 assay is used in patients with urothelial carcinoma and is used to calculate immune cells (IC) within the tumor microenvironment. Such variability in scoring methods confounds results across clinical trials and clinical practice, which leads to uncertainty about the universal value of PD-L1 expression levels as a biomarker across tumor types. In addition, the use of formalin fixed paraffin embedded (FFPE) archived tumor tissues produced using non-standardized fixation and storage methods may not provide predictable and purposeful results for proper PD-L1 antigen retrieval, which can potentially increase IHC heterogeneity, intensity, and topography of staining. All these factors complicate the use of IHC-assessed PD-L1 levels to predict clinical response to ICB.

[0003] RNA-based analyses on FFPE tissues are currently used in clinical settings to stratify or predict the risk of recurrence in patients affected by various tumor types. RNA-based gene expression analyses include microarray, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR)-based analyses, and RNA sequencing. RT-qPCR analysis is one of the most widely used gene quantification methods because of its wide dynamic range, high sensitivity, high specificity, little or no post-amplification processing, and the ability to increase sample throughput. It also provides high levels of specificity and sensitivity when using highly specific primers targeting stably expressed genes, and can simultaneously measure various targets including genes for sample quality control purposes. Gene expression profiling by RT-qPCR has minimal input requirements and can be much more cost-effective than IHC methods. The strong concordance between RT-qPCR and IHC also validated the analytical validity of RT-qPCR even for challenging FFPE tumor samples. Therefore, implementing gene-specific reverse transcription can greatly increase the success rate of validating molecular fractionators in FFPE sample cohorts.

[0004] Therefore, the inventor aimed to develop an RT-qPCR to measure CD274 mRNA expression levels in correlation with PD-L1 IHC, helping to reduce the testing time and save the tumor tissue archives required for different IHC analyses in the same patient.To this end, three RT-qPCR primers were designed, the results were compared with PD-L1 IHC, and the results were clinically validated in gastric cancer (GC) and urothelial carcinoma patients treated with ICI. Summary of the Invention [Problem to be solved by the invention]

[0005] One aspect is to provide a composition for predicting the responsiveness of a cancer patient to an immune checkpoint inhibitor, comprising a preparation capable of measuring the expression level of the CD274 gene in a sample isolated from the cancer patient.

[0006] Another aspect is to provide a kit for predicting the responsiveness of a cancer patient to an immune checkpoint inhibitor, comprising the composition.

[0007] Yet another aspect provides a method for predicting the responsiveness of a cancer patient to an immune checkpoint inhibitor, the method comprising the steps of amplifying a CD274 gene in a sample isolated from the cancer patient and measuring the expression level of the CD274 gene.

[0008] Yet another aspect is to provide an application of a preparation capable of measuring the expression level of the CD274 gene in a sample isolated from a cancer patient for predicting the responsiveness of a cancer patient to an immune checkpoint inhibitor. [Means for solving the problem]

[0009] One aspect provides a composition for predicting the responsiveness of a cancer patient to an immune checkpoint inhibitor, the composition comprising a preparation capable of measuring the expression level of the CD274 gene in a sample isolated from the cancer patient.

[0010] The term "cancer patient" may refer to an individual having cancer as a disease, and specifically, the "individual" may be a mammal, such as a mouse, rat, cat, guinea pig, hamster, dog, monkey, chimpanzee, human, etc., and specifically, may be a human.

[0011] The sample may be one or more selected from the group consisting of blood, plasma, serum, tissue, cell, lymphatic fluid, bone marrow fluid, saliva, ocular fluid, semen, brain extract, spinal fluid, synovial fluid, thymic fluid, ascites, amniotic fluid, cell tissue fluid and cell culture fluid, specifically, one or more of blood, plasma, serum, tissue, cell, lymphatic fluid, bone marrow fluid, cell tissue fluid and cell culture fluid, more specifically, one or more selected from the group consisting of tissue, cell, cell tissue fluid and cell culture fluid.

[0012] The term "gene" may refer to any nucleic acid sequence or part thereof that has a functional role in protein coding or transcription or in regulating the expression of other genes. A gene may consist of all the nucleic acid that codes for a functional protein or only a part of the nucleic acid that codes or expresses a protein. A nucleic acid sequence may contain genetic abnormalities within exons, introns, start or end regions, promoter sequences, other regulatory sequences or unique sequences adjacent to the gene.

[0013] In one embodiment, the measurement of the expression level of the CD274 gene may be by measuring the amount of PD-L1 protein (the protein encoded by the CD274 gene) or mRNA, specifically, by measuring the amount of CD274 mRNA.

[0014] When the measurement of the expression level of the CD274 gene is the measurement of the amount of PD-L1 protein, the "measurement of the amount of protein" refers to a process of confirming the presence or absence and the expression level of the protein encoded by the gene in a subject sample. Specifically, the measurement of the amount of protein may be performed by one or more techniques selected from the group consisting of Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, flow cytometry (Fluorescence Activated Cell Sorter, FACS), and protein chip.

[0015] When the measurement of the expression level of the CD274 gene is to measure the amount of CD274 mRNA, the term "measurement of the amount of mRNA" refers to a process of confirming the presence or absence of mRNA of the gene and its expression level in a subject sample. The amount of the mRNA may be measured by one or more techniques selected from the group consisting of reverse transcription polymerase chain reaction (RT-PCR), real-time reverse transcription polymerase chain reaction (Real time RT-PCR), quantitative polymerase chain reaction (qPCR), competitive reverse transcription polymerase chain reaction (Competitive RT-PCR), real-time quantitative polymerase chain reaction (RT-qPCR), inverse polymerase chain reaction (Inverse PCR) and long and accurate polymerase chain reaction (Long and Accurate PCR), RNase protection assay (RPA), Northern blotting and DNA chip, specifically, reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase chain reaction (Competitive RT-PCR), real-time reverse transcription polymerase chain reaction (Real time RT-PCR), real-time quantitative polymerase chain reaction RT-qPCR, more specifically, RT-qPCR.

[0016] In one embodiment, the preparation may be one or more selected from the group consisting of a primer, a probe, an aptamer, an antibody, and a peptide capable of specifically binding to a base sequence containing the CD274 gene or a protein encoded by the base sequence, specifically a primer or a probe capable of specifically binding to a base sequence containing the CD274 gene, more specifically a primer capable of specifically binding to a base sequence containing the CD274 gene.

[0017] In one embodiment, the preparation may measure the expression level of one or more sites selected from the group consisting of the junction of exon 1 and exon 2, the junction of exon 3 and exon 4, and the junction of exon 5 and exon 6 of the CD274 gene.

[0018] Specifically, the formulation may be one or more probes selected from the group consisting of a CD274 TaqMan probe for the junction of exon 1 and exon 2 (assay ID; Hs01125296_m1), a CD274 TaqMan probe for the junction of exon 3 and exon 4 (assay ID; Hs00204257_m1), and a CD274 TaqMan probe for the junction of exon 5 and exon 6 (assay ID; Hs01125301_m1).

[0019] The term "primer" refers to a nucleic acid sequence that can form a complementary base pair with a template strand and function as a starting point for copying the template strand, and may be, for example, a nucleic acid sequence of 5 to 50 bases. The primer is usually synthesized, but naturally occurring nucleic acids can also be used. The sequence of the primer does not necessarily have to be exactly the same as the sequence of the template, as long as it is sufficiently complementary and can hybridize with the template.

[0020] The term "probe" refers to a substance that can specifically bind to a target substance to be detected in a sample and can specifically confirm the presence of the target substance in the sample through said binding.

[0021] The term "aptamer" refers to a small single-stranded nucleic acid (DNA or RNA) fragment that has the property of being able to bind with high affinity and specificity to various types of substances, from low molecular weight compounds to proteins, and may be, for example, a single-stranded nucleic acid fragment consisting of 10 to 60 nucleotides.

[0022] The term "antibody" refers to a substance that specifically binds to an antigen to cause an antigen-antibody reaction, and may be a chimeric antibody, a humanized antibody, a human antibody, a synthetic antibody and / or an affinity matured antibody.

[0023] The term "peptide" refers to a polymer of two or more amino acids linked together through amide bonds (or peptide bonds).

[0024] Said primer, aptamer or probe can be chemically synthesized using phosphoramidite solid support method or other widely known methods.Such nucleic acid sequences can also be modified using many means known in the art.Non-limiting examples of such modifications include methylation, capping, substitution of one or more natural nucleotides with homologues and internucleotide modifications, such as uncharged linkages such as methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc. or charged linkages such as phosphorothioates, phosphorodithioates, etc.

[0025] The antibodies or peptides may be naturally derived or wholly or partially synthetically produced and may contain synthetic sequences.

[0026] In one embodiment, the cancer may be one or more selected from the group consisting of lung cancer, gastric cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous melanoma, uterine cancer, ovarian cancer, colon cancer, breast cancer, uterine sarcoma, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, laryngeal cancer, small intestine cancer, thyroid cancer, parathyroid cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, multiple myeloma, chronic or acute leukemia, childhood solid tumors, lymphoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, spinal axis tumor, brain stem glioma, and urothelial cancer, and in particular may be one or more selected from the group consisting of gastric cancer and urothelial cancer.

[0027] The term "cancer" refers to a class of diseases characterized by the development of abnormal cells that have the ability to grow uncontrollably and invade and destroy normal body tissue.

[0028] The term "immune checkpoint inhibitor" refers to compounds, e.g., drugs (including antibodies), that suppress or block proteins expressed by cells of the immune system, e.g., T cells and some types of cancer cells. The proteins can suppress the immune response and block T cells from killing cancer cells, and when the proteins are blocked, the inhibition of the immune system is overcome and T cells can kill cancer cells.

[0029] In one embodiment, the immune checkpoint inhibitor may target one or more immune checkpoints selected from the group consisting of PD-1, CTLA4, KIR, 4-1BB / TNFRS9, TIM3 and LAG3, specifically, the immune checkpoint inhibitor may target one or more immune checkpoints selected from the group consisting of PD-1, CTLA4 and LAG3, more specifically, the immune checkpoint inhibitor may target PD-1, i.e., PD-1 / PD-L1.

[0030] In one embodiment, the immune checkpoint inhibitor may be one or more selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, ipilimumab, atezolizumab, tremelimumab, avelumab, durvalumab, and more particularly, one or more selected from the group consisting of pembrolizumab, nivolumab, atezolizumab, and durvalumab.

[0031] The term "responsiveness" refers to a clinical or therapeutic benefit conferred to a patient at risk of developing or having a disease or disorder, e.g., cancer, including one or more of the following: prolonged survival (including overall survival and progression-free survival); induction of an objective response (including complete or partial remission); or amelioration of signs or symptoms of cancer.

[0032] The term "objective response" refers to a measurable response, such as a complete response (CR) or partial response (PR).

[0033] The term "complete response" refers to disappearance of all signs of cancer (e.g., disappearance of all target lesions) in response to treatment.

[0034] The term "partial response" refers to a decrease in the size or extent of bodily cancer in response to treatment of one or more tumors or lesions.

[0035] The term "prediction" may refer to the prediction of a particular outcome, such as therapeutic response, through the identification of a characteristic, such as the expression level of a particular gene.

[0036] In one example, a comparative analysis was performed on the results of PD-L1 immunohistochemistry (IHC) in cancer cells of patients with gastric cancer or urothelial cancer and the results of RT-qPCR for CD274 mRNA, and it was found that there was a significant correlation between the results of PD-L1 immunohistochemistry and the results of RT-qPCR for CD274 mRNA (see Example 1).

[0037] Furthermore, in one example, a comparative analysis was performed between the results of PD-L1 immunohistochemistry (IHC) in cancer cells of patients with gastric cancer or urothelial cancer treated with pembrolizumab, nivolumab, atezolizumab, or durvalumab, which are anti-PD-1 / PD-L1 preparations used as immune checkpoint inhibitors, and the results of RT-qPCR for CD274 mRNA. As a result, it was confirmed that there was a significant correlation between the results of PD-L1 immunohistochemistry and the results of RT-qPCR for CD274 mRNA (see Example 2).

[0038] In addition, in one example, it was confirmed that there was a significant correlation between the survival of gastric cancer or urothelial cancer patients and the results of PD-L1 immunohistochemistry (IHC) or RT-qPCR results for CD274 mRNA in the cancer cells of gastric cancer or urothelial cancer patients (see Example 3).

[0039] In addition, in one example, it was confirmed that RT-qPCR results for CD274 mRNA in cancer cells of patients with gastric cancer or urothelial cancer were superior in sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy to PD-L1 immunohistochemistry (IHC) results (see Example 4).

[0040] Looking at Examples 1 to 4, it was confirmed that the composition for predicting the responsiveness of cancer patients to immune checkpoint inhibitors according to one embodiment has superior predictive power for the responsiveness of cancer patients (particularly, gastric cancer or urothelial cancer patients) to immune checkpoint inhibitors compared to immunohistochemistry for PD-L1.

[0041] Another aspect provides a kit for predicting the responsiveness of a cancer patient to an immune checkpoint inhibitor, comprising the composition.

[0042] The "cancer patient", "sample", "preparation", "cancer", "immune checkpoint inhibitor", etc. may be within the scope described above.

[0043] The kit may include primers, probes, or antibodies that selectively recognize markers for measuring the expression levels of the genes whose expression increases or decreases in response to a therapeutic response to an immune checkpoint inhibitor, as well as one or more other component compositions, solutions, or devices suitable for an analytical method.

[0044] The kit for measuring the mRNA expression level of the gene may be a kit containing essential elements for performing RT-PCR. The RT-PCR kit may contain, in addition to the respective primers specific to the gene, test tubes or other suitable containers, reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitor, DEPC-water, sterile water, etc.

[0045] Yet another aspect provides a method for predicting the responsiveness of a cancer patient to an immune checkpoint inhibitor, comprising the steps of amplifying a CD274 gene in a sample isolated from the cancer patient and measuring the expression level of the CD274 gene.

[0046] The "cancer patient", "sample", "cancer", "immune checkpoint inhibitor", etc. may be within the scope described above.

[0047] In one embodiment, the CD274 gene may be CD274 mRNA.

[0048] In one embodiment, the CD274 gene may also comprise one or more sites selected from the group consisting of the junction of exon 1 and exon 2, the junction of exon 3 and exon 4, and the junction of exon 5 and exon 6 of the CD274 gene.

[0049] Measuring the expression level of the CD274 gene may involve measuring the expression level of the CD274 gene using one or more probes selected from the group consisting of a CD274 TaqMan probe for the junction of exon 1 and exon 2 (assay ID; Hs01125296_m1), a CD274 TaqMan probe for the junction of exon 3 and exon 4 (assay ID; Hs00204257_m1), and a CD274 TaqMan probe for the junction of exon 5 and exon 6 (assay ID; Hs01125301_m1).

[0050] In one embodiment, the amplification may be performed using one amplification method selected from the group consisting of reverse transcription polymerase chain reaction (RT-PCR), real time reverse transcription polymerase chain reaction (Real time RT-PCR), quantitative polymerase chain reaction (qPCR), competitive reverse transcription polymerase chain reaction (Competitive RT-PCR), real time quantitative polymerase chain reaction (RT-qPCR), inverse polymerase chain reaction (Inverse PCR) and long and accurate polymerase chain reaction (Long and Accurate PCR), specifically, using one amplification method selected from the group consisting of RT-PCR, RT-qPCR and Real time PCR, more specifically, using RT-qPCR.

[0051] According to one embodiment of the method, by measuring the expression level of CD274 mRNA, the time required to predict the response to immune checkpoint inhibitors can be shortened, and cancer (particularly gastric cancer or urothelial cancer) can be diagnosed early.

[0052] Yet another aspect is to provide an application of a preparation capable of measuring the expression level of the CD274 gene in a sample isolated from a cancer patient for predicting the responsiveness of a cancer patient to an immune checkpoint inhibitor.

[0053] The "cancer patient", "sample", "cancer", "immune checkpoint inhibitor", etc. may be within the scope described above. Effect of the Invention

[0054] According to one embodiment, it was confirmed that the RT-qPCR results for CD274 mRNA in cancer cells of cancer patients administered with an immune checkpoint inhibitor were significantly correlated with the PD-L1 immunohistochemistry (IHC) results, and were superior in sensitivity, specificity, positive predictive value, negative predictive value, and accuracy to the PD-L1 immunohistochemistry (IHC) results. Thus, the composition for predicting the responsiveness of a cancer patient to an immune checkpoint inhibitor according to one embodiment is superior in predicting the responsiveness of a cancer patient (particularly, a patient with gastric cancer or urothelial cancer) to an immune checkpoint inhibitor than immunohistochemistry for PD-L1, and the composition has the advantage of being able to shorten the time required to predict the responsiveness of an immune checkpoint inhibitor by measuring the expression level of CD274 mRNA using RT-qPCR, thereby enabling early diagnosis of cancer. [Brief description of the drawings]

[0055] [Figure 1] FIG. 1 shows a schematic representation of CD274 and TaqMan probes spanning the exon 1-2, exon 3-4 and exon 5-6 boundaries. [Diagram 2] FIG. 1 shows the results of PD-L1 immunohistochemistry staining in gastric cancer and results using SP142 analysis in urothelial cancer. [Figure 3A] FIG. 1 shows the correlation between PD-L1 score and CD274 mRNA expression level in gastric cancer and urothelial cancer. [Figure 3B] FIG. 1 shows the correlation between PD-L1 score and CD274 mRNA expression level in gastric cancer and urothelial cancer. [Figure 3C] FIG. 1 shows the correlation between PD-L1 score and CD274 mRNA expression level in gastric cancer and urothelial cancer. [Figure 4A] FIG. 1 shows the cutoff of PD-L1 binding positive score in gastric cancer and the cutoff of PD-L1 immune cells in urothelial cancer. [Figure 4B]FIG. 1 shows the cutoff of PD-L1 binding positive score in gastric cancer and the cutoff of PD-L1 immune cells in urothelial cancer. [Figure 4C] FIG. 1 shows the cutoff of PD-L1 binding positive score in gastric cancer and the cutoff of PD-L1 immune cells in urothelial cancer. [Diagram 5] FIG. 1 shows response results to anti-PD-1 / PD-L1 inhibitors in gastric cancer and urothelial cancer. [Figure 6] FIG. 1 shows the PD-L1 expression and mRNA expression results of CD274 in gastric cancer and urothelial cancer patients. [Figure 7] FIG. 1 shows patient survival outcomes in response to RT-qPCR and PD-1 / PD-L1 treatment in patients with urothelial cancer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] The present invention will be described in more detail below with reference to examples. However, these examples are merely for illustrative purposes and the scope of the present invention is not limited to these examples.

[0057] Reference Examples Reference Example 1. Patients and data collection Data were retrospectively collected from 100 patients with severe gastric cancer (GC) (n=59) or urothelial carcinoma (n=41) treated with palliative chemotherapy (n=100) and additional anti-programmed death 1 (PD-1) / PD-L1 immunotherapy (n=49) at Samsung Seoul Medical Center from December 2016 to January 2020. The mean age was 61.0 years (range, 33-81 years), and 30 patients (61.2%) were male. Treatment response for 49 patients treated with immunotherapy was evaluated every 6-12 weeks by Immune Response Evaluation Criteria in Solid Tumors (iRECIST). Patients with a follow-up period of ≥6 weeks were included. The primary clinical endpoint of this study was the objective response rate (ORR), defined as complete response (CR) or partial response (PR). Patients with progressive disease (PD) or stable disease (SD) were classified as non-responders. Clinicopathological data were retrospectively extracted from electronic medical records. The study was conducted in accordance with the Institutional Review Board guidelines for data analysis and research treatment (IRB No. 2018-09-041-001), and patient consent was obtained.

[0058] Reference Example 2. RNA extraction and RT-qPCR execution Total RNA was analyzed by ReliaPrep TMRNA was isolated from formalin fixed paraffin embedded (FFPE) tumor tissues using the FFPE Total RNA Miniprep System (Promega, WI, USA). Reverse transcription polymerase chain reaction (RT-PCR) was performed using a high-dose cDNA reverse transcription kit (Thermo Fisher Scientific, CA, USA) according to the manufacturer's instructions. Each target gene was analyzed using gene expression assays including forward and reverse primers and FAM-labeled MGB TaqMan probes from Applied Biosystems (Thermo Fisher Scientific) according to previous studies. By finding a high correlation between PD-L1 (Programmed Death-Ligand 1) IHC (immunohistochemistry) and NanoString nCounter assay, we selected CD274 TaqMan probes (assay ID; Hs01125296_m1) spanning exon 1-2 boundary, CD274 TaqMan probes (assay ID; Hs00204257_m1) spanning exon 3-4 boundary, and CD274 TaqMan probes (assay ID; Hs01125301_m1) spanning exon 5-6 boundary to perform real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) (Figure 1). PCR (polymerase chain reaction) amplification was performed in triplicate using the following conditions for QuantStudio 6 (Thermo Fisher Scientific): 50°C for 2 min and 94°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. The mRNA expression levels of each gene were analyzed by 20-fold RT-qPCR. -ΔCt (ΔCt = ΔCt target gene -ΔCt GUSB2 ) method was used for the measurements.

[0059] Reference example 3.PD-L1 IHC (immunohistochemistry) Gastric FFPE tissue blocks were sectioned at 4 μm and stained on an Autostainer Link 48 system (Agilent Technologies) using the Dako PD-L1 IHC 22C3 pharmDx kit (Agilent Technologies). For urothelial carcinoma, rabbit anti-human PD-L1 monoclonal antibody (clone SP142; Ventana Medical Systems, Tucson, AZ, USA) was used identically as previously described. The combined positive score (CPS) of PD-L1 expression in gastric cancer was calculated by dividing the number of PD-L1-stained tumor and immune cells by the total number of viable tumor cells, multiplied by 100. A combined positive score (CPS) cutoff of 1 and 10 was used to evaluate the concordance between RT-qPCR and IHC for gastric cancer. For urothelial carcinoma, tumor-infiltrating immune cells (IC) covering ≥5 tumor areas were defined as PD-L1 positive.

[0060] Reference Example 4. Statistical analysis of immunohistochemistry (IHC) and RT-qPCR To compare IHC and RT-qPCR, we used CPS ≥ 1 and CPS ≥ 10 for gastric cancer and IC ≥ 5 for urothelial cancer. To calculate sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy, a positive IHC result was considered as CPS ≥ 1 or CPS ≥ 10 for gastric cancer and IC ≥ 5 for urothelial cancer. Logistic regression was also performed to evaluate response prediction based on tumor type, IHC results and RT-qPCR results.

[0061] Objective response rates (ORR; CR / PR) and disease control rates (DCR; CR / PR / SD) were compared with CD274 mRNA RT-qPCR results using two-tailed unpaired Student's t-tests. Receiver operating characteristic (ROC) curves were generated, and the diagnostic value of the different panels was evaluated by calculating the area under the ROC curve (AUC). Kaplan-Meier estimates of progression-free survival (PFS) and overall survival (OS) were compared using the log-rank test. All graphs were generated using GraphPad Prism version 9.0 (GraphPad Software Inc., San Diego, USA). Statistical significance was set at P < 0.05. All statistical analyses were performed using SPSS software version 27.0 (IBM Corp., Armonk, New York, USA).

[0062] Working Example Example 1. Analytical comparison of PD-L1 immunohistochemistry (IHC) and CD274 RT-qPCR results PD-L1 positivity with CPS ≥ 1 was found in 32 (54.2%) cases using the 22C3 pharmDx assay, and CPS ≥ 10 was confirmed in 13 (22%) of 59 gastric cancers. PD-L1 CPS ranged from 0 to 95, with a mean CPS of 9.24. PD-L1 positivity with IC ≥ 5 was confirmed in 12 (29.3%) of 41 urothelial cancers using the Ventana SP142 assay. PD-L1 IC in urothelial cancers ranged from 0 to 95, with a mean IC of 10.46. PD-L1 IHC in gastric cancer using the 22C3 pharmDx and urothelial cancer using the Ventana SP142 assay are shown (Figure 2).

[0063] The CD274 mRNA expression levels (relative quantification) of spanning exons 1-2, 3-4 and 5-6 are 0-2.4897 with a mean relative quantification (RQ) of 0.1004, 0-7.5214 with a mean relative quantification of 0.2371 and 0-3.7064 with a mean relative quantification of 0.0928, respectively. The CD274 mRNA expression levels within these three exon-exon junctions showed a high correlation (Spearman correlation analysis r for exons 1-2 and 3-4). 2 = 0.92; for exons 1-2 and 5-6, r 2 = 0.89; for exons 3-4 and 5-6, r 2 = 0.99) (Figure 3A). In all 100 samples evaluated, the PD-L1 score was significantly higher in exons 1-2 (r 2 =0.81), exon 3-4 (r 2 =0.65) and exons 5-6 (r 2 = 0.59) was highly correlated with CD274 mRNA expression levels (Figure 3A). PD-L1 CPS score with 22C3 pharmDx in gastric cancer was correlated with CD274 exon 1-2 (r 2 =0.81), exon 3-4 (r 2 =0.67) and exons 5-6 (r 2 = 0.62) junction (Figure 3B). In urothelial carcinoma, the Ventana SP142 PD-L1 IC score was significantly associated with CD274 exon 1-2 (r 2 =0.93), exon 3-4 (r 2 =0.82) and exons 5-6 (r 2 = 0.76) was significantly associated with junction (Figure 3C).

[0064] Relative quantification cutoffs of CD274 mRNA expression at exon 1-2, exon 3-4, and exon 5-6 junctions were evaluated by area under the curve (AUC) analysis based on PD-L1 CPS cutoffs of 1 and 10 for gastric cancer and PD-L1 IC cutoff of 5 for urothelial cancer (Table 1 and Figures 4A-4C). At a CPS cutoff of 10 in gastric cancer, the highest area under the curve (AUC) value was 0.783 obtained from CD274 mRNA expression at exon 1-2 junction with a cutoff of 0.0722 (P<0.0001). In urothelial cancer, area under the curve (AUC) analysis based on a PD-L1 IC cutoff of IC 5 showed the highest area under the curve (AUC) value of 0.781 obtained from CD274 mRNA expression at exon 1-2 junction with a cutoff value of 0.0480 (P<0.0001).

[0065] [Table 1]

[0066] Example 2. Predicting patient response to anti-PD-1 / PD-L1 inhibitors From May 2018 to October 2020, 49 patients were treated with anti-PD-1 / PD-L1 agents and treatment response was evaluated with a follow-up period of more than 6 weeks (pembrolizumab [n = 16], nivolumab [n = 16], atezolizumab [n = 13] and durvalumab [n = 4]) (Table 2). The median number of cycles to PD-1 / PD-L1 agents was 8.9 (range, 1-37) as of May 20, 2021, and the median median follow-up period was 11.3 months. In addition, the clinicopathological characteristics of patients treated with anti-PD-1 / PD-L1 therapy were summarized (Table 3).

[0067] Anti-PD-1 / PD-L1 responders (CR / PR, n=16) and non-responders (PD / SD, n=33) were identified using the iRECIST category of objective response rate (ORR). Results confirmed that PD-L1 expression (P=0.010) and high CD274 mRNA expression (P<0.001) were significantly associated with response to immunotherapy (Figure 5A). The predictive performance of PD-L1 IHC and receiver operating characteristics (ROC) for CD274 exon 1-2 mRNA expression showed high discriminatory ability. The areas under the curves (AUCs) and their 95% confidence intervals (CIs) were 0.76 (0.61-0.91) for PD-L1 and 0.75 (0.59-0.91) for CD274 exon 1-2 mRNA expression (Figure 5B). Similar associations using iRECIST categories of disease control rate (CR / PR / SD, n=30 and PD, n=19) were observed for PD-L1 expression (P=0.015) and high CD274 mRNA expression (P=0.038), with areas under the curve (AUC) predicting response to immunotherapy of 0.70 (0.55-0.86) and 0.68 (0.53-0.83), respectively (Figures 5C and 5D).

[0068] [Table 2-1] [Table 2-2]

[0069] [Table 3]

[0070] Example 3. Correlation of PD-L1 IHC (immunohistochemistry) and CD274 RT-qPCR results with patient survival In gastric cancer patients, progression-free survival (PFS) was strongly associated with PD-L1 expression (P=0.018) and high mRNA expression of CD274 across the exon 1-2 junction (P=0.010) (Figure 6A). A similar association was observed between overall survival (OS) and PD-L1 expression (P=0.047), but overall survival was not significantly associated with mRNA expression (P=0.134) (Figure 6B). For urothelial cancer patients, PD-L1 expression was significantly associated with both progression-free survival (P=0.016) and overall survival (P=0.009). However, mRNA expression of the CD274 exon 1-2 junction did not significantly correlate with progression-free survival and overall survival of patients (Figure 7).

[0071] Example 4. Clinical utility of PD-L1 IHC (immunohistochemistry) and CD274 RT-qPCR results The clinical utility of PD-L1 assessment was compared with IHC and RT-qPCR using standard parameters of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy (Table 4). Two values ​​(CPS ≥ 1% and 10%; RQ ≥ 0.0276 and ≥ 0.0772) were used in gastric cancer samples to obtain the best performance for predicting response to immunotherapy. PD-L1 CPS ≥ 1% showed the highest sensitivity (90%), and RT-qPCR with a cutoff of mean relative quantification of 0.0722 showed the highest specificity (100%) in gastric cancer. The highest sensitivity was achieved with a combined positive score of ≥ 1% in gastric cancer samples (90%), despite a very low positive predictive value (50%). In urothelial cancer, we observed even higher sensitivity (66.7%) and specificity (90%) with RT-qPCR with an even higher area under the curve (AUC) value compared to PD-L1 IHC.

[0072] [Table 4]

Claims

1. A composition for predicting the responsiveness of a cancer patient to an immune checkpoint inhibitor, comprising a preparation capable of measuring the expression level of the CD274 gene in a sample isolated from the cancer patient.

2. 2. The composition of claim 1, wherein the sample is one or more selected from the group consisting of blood, plasma, serum, tissue, cells, lymphatic fluid, bone marrow fluid, saliva, ocular fluid, semen, brain extract, spinal fluid, synovial fluid, thymic fluid, peritoneal fluid, amniotic fluid, cell tissue fluid and cell culture fluid.

3. The composition according to claim 1 , wherein the measurement of the expression level of the CD274 gene is measurement of the amount of CD274 mRNA.

4. The composition according to claim 1 , wherein the preparation is a primer or a probe capable of specifically binding to a base sequence comprising the CD274 gene.

5. The composition of claim 1, wherein the preparation is for measuring the expression level of one or more sites selected from the group consisting of the junction of exon 1 and exon 2, the junction of exon 3 and exon 4, and the junction of exon 5 and exon 6 of the CD274 gene.

6. 2. The composition of claim 1, wherein the cancer is one or more selected from the group consisting of lung cancer, gastric cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous melanoma, uterine cancer, ovarian cancer, colon cancer, breast cancer, uterine sarcoma, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, laryngeal cancer, small intestine cancer, thyroid cancer, parathyroid cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, multiple myeloma, chronic or acute leukemia, childhood solid tumors, lymphoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, spinal axis tumor, brain stem glioma, and urothelial cancer.

7. 2. The composition of claim 1, wherein the immune checkpoint inhibitor targets one or more immune checkpoints selected from the group consisting of PD-1, CTLA4, KIR, 4-1BB / TNFRS9, TIM3, and LAG3.

8. 2. The composition of claim 1, wherein the immune checkpoint inhibitor is one or more selected from the group consisting of pembrolizumab, nivolumab, atezolizumab, and durvalumab.

9. A kit for predicting the responsiveness of a cancer patient to an immune checkpoint inhibitor, comprising the composition described in claim 1.

10. amplifying the CD274 gene in a sample isolated from a cancer patient; A method for predicting the responsiveness of a cancer patient to an immune checkpoint inhibitor, comprising: measuring the expression level of the CD274 gene.

11. The method of claim 10, wherein the CD274 gene is CD274 mRNA.

12. The method of claim 10, wherein the CD274 gene comprises one or more sites selected from the group consisting of the junction of exon 1 and exon 2, the junction of exon 3 and exon 4, and the junction of exon 5 and exon 6 of the CD274 gene.

13. The method according to claim 10, wherein the amplification is performed using real-time quantitative polymerase chain reaction (RT-qPCR).

14. Use of a formulation capable of measuring the expression level of the CD274 gene in samples isolated from cancer patients to predict the responsiveness of cancer patients to immune checkpoint inhibitors.

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