Method for acquiring data on efficacy of CDK4 / 6 inhibitor in lung cancer

Abstract

Provided is a method for acquiring data on the efficacy of a CDK4 / 6 inhibitor in lung cancer cells, the method comprising detecting an EGFR gene mutation and a CDKN2A / B gene deletion or loss-of-function mutation in lung cancer cells derived from a lung cancer patient. The presence of an EGFR gene mutation and a CDKN2A / B gene deletion or loss-of-function mutation indicates the efficacy of the CDK4 / 6 inhibitor.

Description

Method for obtaining data on the efficacy of CDK4 / 6 inhibitors in lung cancer

[0001] The present invention relates to a method for predicting the efficacy of a CDK4 / 6 inhibitor in treating a subject suffering from lung cancer.

[0002] It is estimated that more than 2 million people worldwide develop lung cancer each year. Epidemiological data suggests that approximately 500,000 of these cases are lung adenocarcinomas with EGFR (Epidermal Growth Factor Receptor) gene mutations.

[0003] EGFR gene mutation-positive lung adenocarcinoma is one of the largest subgroups of lung adenocarcinoma. EGFR tyrosine kinase inhibitors, molecular targeted therapies, have been developed for EGFR gene mutation-positive lung adenocarcinoma, and their efficacy has been confirmed in clinical trials. Among these, the third-generation EGFR tyrosine kinase inhibitor osimertinib (trade name Tagrisso) is widely used worldwide due to its efficacy and safety. These EGFR tyrosine kinase inhibitors demonstrate efficacy (response rate) of approximately 70% for EGFR gene mutation-positive lung adenocarcinoma, enabling disease control early in treatment.

[0004] However, there are a certain number of patients who cannot use EGFR tyrosine kinase inhibitors at all due to side effects and complications. Furthermore, many lung cancers show resistance to treatment (initial resistance) early on after starting EGFR tyrosine kinase inhibitor treatment, and many lung cancers acquire resistance (acquired resistance) within 1-2 years of starting treatment. For this reason, there is a need to develop treatment strategies for lung cancer in patients who cannot use EGFR tyrosine kinase inhibitors, as well as for lung cancer that has acquired initial or acquired resistance.

[0005] CDK4 / 6 inhibitors are selective inhibitors of the cyclin-dependent kinases CDK4 and CDK6, and palbociclib and abemaciclib are small molecule compounds used to treat hormone receptor (HR)-positive and HER2-negative breast cancer.

[0006] Non-Patent Document 1 describes that single-agent administration of palbociclib exhibited moderate antitumor activity in non-small cell lung cancer patients with CDKN2A / B gene deletion or loss-of-function mutation.

[0007] Non-patent document 2 describes that 82% of patients with EGFR gene mutations were cyclin D1 positive, and that in a growth inhibition test of non-small cell lung cancer-derived cell lines with EGFR gene mutations, the CDK4 / 6 inhibitor abemaciclib alone was ineffective against both cell lines with and without EGFR gene mutations, but that the combined use of osimertinib and abemaciclib reduced the drug concentration required for 50% growth inhibition.

[0008] Eugene R. Ahn, et al., Precision Oncology no. 4 (2020) 757-766. Published online June 25, 2020. Osoegawa et. al., Investigational New Drugs 41, 183-192 (2023) 2023 Feb 15. doi: 10.1007 / s10637-023-01337-8

[0009] Non-Patent Document 1 does not describe whether or not the patient has an EGFR gene mutation. Furthermore, Non-Patent Document 2 describes that many patients with EGFR gene mutations express cyclin D1, but does not describe a relationship with CDKN2A / B gene deletion or loss-of-function mutation, and does not describe or suggest that combining CDKN2A / B gene deletion or loss-of-function mutation with EGFR gene mutation improves the accuracy of predicting the efficacy of CDK / 4 / 6 inhibitors.

[0010] To date, no biomarkers have been identified that can accurately predict the therapeutic effect of CDK4 / 6 inhibitors on lung cancer.

[0011] The problem to be solved by the present invention is to provide a method for obtaining data on the efficacy of CDK4 / 6 inhibitors in lung cancer.

[0012] The present invention encompasses the following embodiments: Item 1. A method for obtaining data regarding the effectiveness of a CDK4 / 6 inhibitor in lung cancer cells, the method comprising detecting EGFR gene mutations and CDKN2A / B gene deletions or loss-of-function mutations in lung cancer cells derived from a lung cancer patient. Item 2. The method of Item 1, which indicates the effectiveness of a CDK4 / 6 inhibitor when an EGFR gene mutation and a CDKN2A / B gene deletion or loss-of-function mutation are present. Item 3. A method for obtaining data regarding the effectiveness of a CDK4 / 6 inhibitor in lung cancer cells having an EGFR gene mutation, the method comprising detecting CDKN2A / B gene deletions or loss-of-function mutations in lung cancer cells having an EGFR gene mutation. Item 4. The method of Item 3, which indicates the effectiveness of a CDK4 / 6 inhibitor when a CDKN2A / B gene deletion or loss-of-function mutation is present. Item 5. Item 6. A method for obtaining data regarding the efficacy of a CDK4 / 6 inhibitor in lung cancer cells having a CDKN2A / B gene deletion or loss-of-function mutation, the method comprising detecting an EGFR gene mutation. Item 7. The method of any one of Items 1 to 6, wherein the CDK4 / 6 inhibitor is indicated to be effective when an EGFR gene mutation is present. Item 8. A kit for obtaining data regarding the efficacy of a CDK4 / 6 inhibitor in lung cancer cells, the kit comprising a reagent for measuring an EGFR gene mutation and a reagent for measuring a CDKN2A / B gene deletion or loss-of-function mutation. Item 9. A therapeutic agent for lung cancer having an EGFR gene mutation and a CDKN2A / B gene deletion or loss-of-function mutation, the therapeutic agent comprising a CDK4 / 6 inhibitor as an active ingredient.

[0013] Overview of mouse xenograft experiments. Photographs of various immunohistochemical staining of organoids and xenograft tumors (xenografts). Scale bar: 100 μm. Genotype-phenotype correlation of resistance mechanisms to EGFR-TKIs. Overview of genetic abnormalities in organoids based on whole-exome sequencing analysis. n=31. LUAD; Lung Adenocarcinoma, SQ; Squamous Cell Carcinoma, SCLC; Small Cell Carcinoma, LCNEC; Large Cell Neuroendocrine Carcinoma. The horizontal axis shows the names of organoid lines. Results of cell viability assays using multiple palbociclib concentrations. CDKN2A / B loss: EGFR gene mutant line with CDKN2A / B gene deletion (n=8); CDKN2A / B WT: EGFR gene mutant line with wild-type CDKN2A / B gene (n=5). IC of CDKN2A / B gene deletion line (n=8) and wild-type line (n=5). 50 Values. **p<0.01, t-test. Error bars: standard deviation. Mean tumor volume (mm ) of xenograft tumors treated with palbociclib (n=5) or vehicle (n=5) for CDKN2A / B gene-deficient lines ((A) E-12, (B) E-14, (C) E-17) and (D) CDKN2A / B wild-type line (E-01B) 3). ***p<0.001, **p<0.01, t-test. NS: not significant. Cell viability assay results using multiple concentrations of abemaciclib for EGFR gene mutant cells with CDKN2A / B gene deletion (n=3) and EGFR gene mutant cells with wild-type CDKN2A / B (n=3). CDKN2A / B loss: EGFR gene mutant cells with CDKN2A / B gene deletion, CDKN2A / B WT: EGFR gene mutant cells with wild-type CDKN2A / B. Error bars: standard deviation. Cell viability assay results using multiple concentrations of palbociclib for KRAS gene mutant cells with CDKN2A / B gene deletion (n=3) and KRAS gene mutant cells with wild-type CDKN2A / B (n=1). CDKN2A / B loss: KRAS gene mutant strain with CDKN2A / B gene deletion; CDKN2A / B WT: KRAS gene mutant strain with wild-type CDKN2A / B gene; error bar: standard deviation.

[0014] In this specification, the term "comprise" is a concept that encompasses "consist essentially of" and "consist only of."

[0015] In one embodiment of the present invention, there is provided a method for obtaining data on the efficacy of a CDK4 / 6 inhibitor in lung cancer cells, comprising detecting EGFR gene mutations and CDKN2A / B gene deletion or loss-of-function mutations in lung cancer cells derived from a lung cancer patient. The presence of EGFR gene mutations and CDKN2A / B gene deletion or loss-of-function mutations indicates that a CDK4 / 6 inhibitor is effective in treating lung cancer in the subject.

[0016] The timing of detecting EGFR gene mutations and the timing of detecting CDKN2A / B gene deletions or loss-of-function mutations may be different or simultaneous. For example, the timing of detecting EGFR gene mutations may be earlier or later than the timing of detecting CDKN2A / B gene deletions or loss-of-function mutations.

[0017] In another aspect of the present invention, there is provided a method for obtaining data regarding the efficacy of a CDK4 / 6 inhibitor in lung cancer cells with an EGFR gene mutation, the method comprising detecting a CDKN2A / B gene deletion or loss-of-function mutation in the lung cancer cells with an EGFR gene mutation. The lung cancer cells with an EGFR gene mutation are preferably lung cancer cells with an EGFR gene mutation derived from a lung cancer patient or collected from a lung cancer patient. The presence of a CDKN2A / B gene deletion or loss-of-function mutation indicates that a CDK4 / 6 inhibitor is effective against lung cancer in the subject.

[0018] In another embodiment of the present invention, there is provided a method for obtaining data regarding the efficacy of a CDK4 / 6 inhibitor in lung cancer cells having a CDKN2A / B gene deletion or loss-of-function mutation, the method comprising detecting an EGFR gene mutation in the lung cancer cells, and the presence of an EGFR gene mutation indicates the efficacy of a CDK4 / 6 inhibitor.

[0019] EGFR gene mutations refer to structural abnormalities in the EGFR (epidermal growth factor receptor) gene, a member of the HER family. The human EGFR genomic sequence is NCBI Gene ID 1956, and the amino acid sequence is UNIProt P00533. The mouse EGFR genomic sequence is NCBI Gene ID: 24329, and the amino acid sequence is UNIProt Q01279.

[0020] Examples of EGFR gene mutations include gene mutations in the region of exons 18-21. Deletion mutations include amino acid deletions and those accompanied by amino acid substitutions. In particular, there are many mutations in the region of codons E746-I759 in exon 18 where one or multiple consecutive amino acids are deleted. For example, there is a simple deletion of five amino acids in codons E746-A750, an S insertion in the L747-E753 deletion, a simple deletion of L747-E751, an insertion of P in the L747-E750 deletion, a point mutation in codon 719 in exon 18 (G719X, where the amino acid may be A, C, or S and is collectively represented as X), a point mutation in codon 709 in exon 18 (E709X, G719X, where the amino acid may be K or A and is collectively represented as X), a deletion mutation in exon 19, and a deletion mutation in exon 19. Examples of EGFR mutations include an insertion mutation in exon 20, a point mutation of S768I in exon 20, a point mutation of L858R in exon 21, and a point mutation of L861Q in exon 21. EGFR gene mutations are registered in the COSMIC (the catalogue of somatic mutations in cancer, https: / / cancer.sanger.ac.uk / cosmic) database. These EGFR gene mutations are publicly known and can be detected using well-known or publicly known detection methods or commercially available kits.

[0021] Among these EGFR gene mutations, those that continue to send phosphorylation signals to downstream growth signal pathways even in the absence of functional stimulation by ligands such as EGF or TGFα are called activating mutations.

[0022] In one embodiment, the EGFR gene mutation is the following EGFR gene mutation whose clinical significance in relation to lung cancer has been clarified in the "Guidelines for EGFR Gene Mutation Testing in Lung Cancer Patients, Version 5.0" (December 16, 2021) of the Japan Lung Cancer Society: (1) Mutations known to be activating: Exon 19 deletion mutation, L858R mutation; G719X mutation, L861Q mutation, S768I mutation; and Exon 20 insertion mutation (A763_Y764insFQEA).

[0023] CDKN2A / B gene deletion or loss-of-function mutation refers to a gene deletion or loss-of-function mutation in at least one of the CDKN2A (cyclin-dependent kinase inhibitor 2A) and CDKN2B (cyclin-dependent kinase inhibitor 2B) genes. The human CDKN2A genomic sequence is NCBI Gene ID: 1029, and its amino acid sequence is UNIProt P42771. The human CDKN2B genomic sequence is NCBI Gene ID: 1030, and its amino acid sequence is UNIProt Q5ZEY8. The mouse CDKN2A genomic sequence is NCBI Gene ID: 12578, and its amino acid sequence is UNIProt Q64364. The mouse CDKN2B genomic sequence is NCBI Gene ID: 12579, and its amino acid sequence is UNIProt Q549R4.

[0024] Both CDKN2A and CDKN2B are tumor suppressor genes involved in cell cycle control, and deletion or loss-of-function mutations in the CDKN2A gene and CDKN2B gene are associated with abnormalities in cell cycle control and cancer progression. Deletion or loss-of-function mutations in the CDKN2A gene and CDKN2B gene can be detected using known detection methods or commercially available kits.

[0025] The CDKN2A / B gene deletion or loss-of-function mutation is not particularly limited as long as it is a mutation that causes loss of function of the gene. The CDK2A gene and the CDK2B gene are located close to each other in the chromosomal region 9p21, and a large region including both the CDK2A gene and the CDK2B gene is often deleted. The CDKN2A / B gene deletion preferably occurs in both alleles.

[0026] CDKN2A / B gene deletions include 9p21 homozygous deletions, which do not necessarily involve deletions of the same region in both alleles, but may involve deletions that result in loss of function of the CDK2A gene and CDK2B gene in each allele.

[0027] Loss-of-function mutations in the CDKN2A / B gene include missense mutations and nonsense mutations. Examples of missense mutations include D84X(H,N,Y) (meaning that D at position 84 of the amino acid sequence is mutated to H, N, or Y) and D108X(H,N,Y). Examples of nonsense mutations include W110*. Loss-of-function mutations in the CDKN2A / B gene include silencing of the CDKN2A / B gene by methylation. For example, methylation of the promoter of the CDKN2A / B gene silences the CDKN2A / B gene.

[0028] Furthermore, CDKN2A / B gene deletions or loss-of-function mutations include cases where one of the two alleles has a deletion, missense mutation, or nonsense mutation, and the other is methylated.

[0029] We constructed a patient-derived lung cancer organoid library consisting of 31 lines of EGFR mutation-positive lung cancer and investigated the relationship between genetic abnormalities and the efficacy of CDK4 / 6 inhibitors. Unexpectedly, we found that the presence of both EGFR mutation and CDKN2A / B deletion or loss-of-function mutations could predict the efficacy of CDK4 / 6 inhibitors with high specificity. The combination of "EGFR mutation" and "CDKN2A / B deletion or loss-of-function mutation" is an important biomarker that can accurately predict the efficacy of CDK4 / 6 inhibitors in lung cancer patients.

[0030] Lung cancer includes at least one selected from the group consisting of lung adenocarcinoma, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, a mixture of these, and lung cancer that cannot be classified into any of the histological types.

[0031] CDK4 / 6 inhibitors include, but are not limited to, palbociclib, abemaciclib, ribociclib, etc. The CDK4 / 6 inhibitor preferably includes palbociclib or abemaciclib.

[0032] The presence or absence of EGFR gene mutations and CDKN2A / B gene deletions or loss-of-function mutations is an indicator for predicting the therapeutic effect of a CDK4 / 6 inhibitor in a subject suffering from lung cancer, or for predicting the efficacy of a CDK4 / 6 inhibitor in treating a subject suffering from lung cancer.

[0033] EGFR gene mutations and CDKN2A / B gene deletions or loss-of-function mutations are detected in lung cancer cells collected from a subject, and if both EGFR gene mutations and CDKN2A / B gene deletions or loss-of-function mutations are present, it is assessed that a CDK4 / 6 inhibitor is highly likely to be effective in treating lung cancer in the subject.

[0034] EGFR gene mutation testing is already being performed clinically on lung cancer tissue. Furthermore, testing for CDKN2A / B gene deletions or loss-of-function mutations involves immunohistochemistry for p16 in otolaryngological tumors, as well as cancer gene panel tests such as the OncoGuide NCC Oncopanel, FoundationOne CDx Cancer Genomic Profile, and FoundationOne Liquid CDx Cancer Genomic Profile, as well as 9p21 deletion testing using fluorescence in situ hybridization (FISH). By combining these tests already in clinical practice, it is possible to select patients for whom CDK4 / 6 inhibitors are effective.

[0035] Thus, using the method of an embodiment of the present invention, it is possible to easily and accurately select lung cancer patients for whom CDK4 / 6 inhibitors are effective, and to select a treatment method appropriate for the patient.

[0036] In the above detection step, if a subject is detected to have lung cancer cells with both an EGFR gene mutation and a CDKN2A / B gene deletion or loss-of-function mutation, i.e., if both an EGFR gene mutation and a CDKN2A / B gene deletion or loss-of-function mutation are present and data is obtained showing that a CDK4 / 6 inhibitor is effective in treating lung cancer in the subject, a CDK4 / 6 inhibitor can be further administered, thereby enabling treatment of the subject whose lung cancer cells are detected to have both an EGFR gene mutation and a CDKN2A / B gene deletion or loss-of-function mutation.

[0037] In one aspect of the present invention, a kit for obtaining data regarding the efficacy of CDK4 / 6 inhibitors in treating subjects with lung cancer is provided, which kit includes a reagent for measuring EGFR gene mutations and a reagent for measuring CDKN2A / B gene deletions or loss-of-function mutations.

[0038] In one embodiment, the reagent for measuring EGFR gene mutations is a probe for detecting EGFR gene mutations. In another embodiment, the reagent for measuring EGFR gene mutations is a probe for amplifying or detecting EGFR gene. The probe is preferably a nucleic acid probe, more preferably RNA or DNA.

[0039] In one embodiment, the reagent for measuring CDKN2A / B gene deletion or loss-of-function mutation is a probe for detecting CDKN2A / B gene deletion or loss-of-function mutation.

[0040] In another embodiment, the reagent for detecting CDKN2A / B gene deletion or loss-of-function mutation is a probe for amplifying or detecting the CDKN2A / B gene. The probe is preferably a nucleic acid probe, more preferably RNA or DNA.

[0041] Methods are well known in which RNA or cDNA is obtained from lung cancer cells collected from a sample, and a target nucleic acid is detected using a nucleic acid probe by well-known methods such as nucleic acid amplification and nucleic acid hybridization.

[0042] In another embodiment, the reagent for measuring CDKN2A / B gene deletion or loss-of-function mutation is an antibody against the expression product of the CDKN2A / B gene. For example, the presence or absence of CDKN2A / B gene deletion or loss-of-function mutation in lung cancer cells collected from a subject can be detected using an antibody specific to a protein that is the expression product of a wild-type or null mutant form of the CDKN2A / B gene. For example, if the measured level of the CDKN2A / B gene expression product in cells collected from a subject is lower than the level of the CDKN2A / B gene expression product in normal lung cells, this data indicates the presence of a CDKN2A / B gene deletion or loss-of-function mutation.

[0043] The kit may further comprise instructions for using the kit in obtaining data regarding the efficacy of the CDK4 / 6 inhibitor in lung cancer cells.

[0044] The kit may further include a control sample. The control sample may be, for example, the expression level of the CDKN2A / B gene in normal lung cells. In one embodiment, a CDKN2A / B gene deletion or loss-of-function mutation may be present when the expression level of the CDKN2A / B gene in lung cancer cells collected from the subject is lower than the expression level of the CDKN2A / B gene in normal lung cells.

[0045] In one aspect of the present invention, there is provided a therapeutic agent for lung cancer with EGFR gene mutation and CDKN2A / B gene deletion or loss-of-function mutation, which contains a CDK4 / 6 inhibitor as an active ingredient.

[0046] In one embodiment, the therapeutic agent is a therapeutic agent for lung cancer in a subject selected by detecting EGFR gene mutations and CDKN2A / B gene deletion or loss-of-function mutations in lung cancer cells collected from the subject, and determining that both EGFR gene mutations and CDKN2A / B gene deletion or loss-of-function mutations are present in the subject. In such a subject, a CDK4 / 6 inhibitor is likely to be effective in treating the lung cancer.

[0047] The daily dose of a CDK4 / 6 inhibitor varies depending on the patient's age, weight, symptoms, etc., but is typically approximately 0.1 to 5,000 mg, preferably 1 to 1,000 mg, and more preferably 10 to 1,000 mg per day for an adult (50 kg body weight), preferably administered once or in two or three divided doses per day. Administration may be by parenteral administration, such as intravenous injection, intraperitoneal injection, subcutaneous injection, or intramuscular injection, or by oral administration. The administered inhibitor may contain a pharmacologically acceptable carrier, diluent, or excipient. The inhibitor may be administered in any form, including oral administration in the form of tablets, capsules, granules, powders, syrups, etc., or parenteral administration in the form of injections, infusions, suppositories, sprays, etc.

[0048] The administration schedule is selected appropriately depending on conditions such as the type of CDK4 / 6 inhibitor, whether or not concomitant medication is being used, previous treatment history, disease stage, whether or not metastasis is present, and the age and sex of the patient.

[0049] There are many EGFR gene mutation-positive lung cancer patients worldwide, and it is believed that a considerable number of lung cancer patients have both EGFR gene mutations and CDKN2A / B gene deletion or loss-of-function mutations. Therefore, it is expected that treatment using the method of the present invention will be implemented in countries around the world. Being able to treat such patients with drugs other than EGFR tyrosine kinase inhibitors is expected to lead to improved treatment outcomes. The present disclosure further includes the following embodiments: Item 1. A method for obtaining data on the efficacy of a CDK4 / 6 inhibitor in lung cancer cells, the method comprising detecting EGFR gene mutations and CDKN2A / B gene deletion or loss-of-function mutations in lung cancer cells derived from a lung cancer patient. Item 2. The method of Item 1, which demonstrates the efficacy of a CDK4 / 6 inhibitor when both an EGFR gene mutation and CDKN2A / B gene deletion or loss-of-function mutations are present. Item 3. Item 3. A method for obtaining data regarding the effectiveness of a CDK4 / 6 inhibitor in lung cancer cells having an EGFR gene mutation, the method comprising detecting a CDKN2A / B gene deletion or loss-of-function mutation in lung cancer cells having an EGFR gene mutation. Item 4. The method of Item 3, wherein the presence of a CDKN2A / B gene deletion or loss-of-function mutation indicates the effectiveness of a CDK4 / 6 inhibitor. Item 5. A method for obtaining data regarding the effectiveness of a CDK4 / 6 inhibitor in lung cancer cells having a CDKN2A / B gene deletion or loss-of-function mutation, the method comprising detecting an EGFR gene mutation. Item 6. The method of Item 5, wherein the presence of an EGFR gene mutation indicates the effectiveness of a CDK4 / 6 inhibitor. Item 7. The method of any one of Item 1 to 6, wherein the CDK4 / 6 inhibitor comprises palbociclib or abemaciclib. Item 8. A kit for obtaining data on the effectiveness of CDK4 / 6 inhibitors in lung cancer cells, comprising a reagent for measuring EGFR gene mutations and a reagent for measuring CDKN2A / B gene deletion or loss-of-function mutations.Item 9. A therapeutic agent for lung cancer having an EGFR gene mutation and a CDKN2A / B gene deletion or loss-of-function mutation, comprising a CDK4 / 6 inhibitor as an active ingredient. Item 10. The method according to Item 3, wherein the lung cancer cells having an EGFR gene mutation include lung cancer cells having an EGFR gene mutation derived from a lung cancer patient. Item 11. A method for predicting the effectiveness of a CDK4 / 6 inhibitor in treating a subject suffering from lung cancer, comprising detecting an EGFR gene mutation and a CDKN2A / B gene deletion or loss-of-function mutation in lung cancer cells collected from the subject, and assessing that a CDK4 / 6 inhibitor is likely to be effective in treating lung cancer in the subject when an EGFR gene mutation and a CDKN2A / B gene deletion or loss-of-function mutation are present. Item 12. Item 12. A method for predicting the effectiveness of a CDK4 / 6 inhibitor in the treatment of a subject suffering from lung cancer, wherein the subject's lung cancer cells have an EGFR gene mutation, the method comprising: detecting a CDKN2A / B gene deletion or loss-of-function mutation in lung cancer cells collected from the subject, and evaluating, if a CDKN2A / B gene deletion or loss-of-function mutation is present, that a CDK4 / 6 inhibitor is likely to be effective in treating the subject's lung cancer. Item 13. A method for predicting the effectiveness of a CDK4 / 6 inhibitor in the treatment of a subject suffering from lung cancer, wherein the subject's lung cancer cells have a CDKN2A / B gene deletion or loss-of-function mutation, the method comprising: detecting an EGFR gene mutation in lung cancer cells collected from the subject, and evaluating, if an EGFR gene mutation is present, that a CDK4 / 6 inhibitor is likely to be effective in treating the subject's lung cancer. Item 14. Item 14. The method according to any one of Items 11 to 13, wherein the CDK4 / 6 inhibitor comprises palbociclib or abemaciclib.Item 15. A kit for predicting the effectiveness of a CDK4 / 6 inhibitor in the treatment of a subject suffering from lung cancer by a method comprising the following steps (1) and (2), the kit comprising a reagent for measuring EGFR gene mutations and a reagent for measuring CDKN2A / B gene deletion or loss-of-function mutations: (1) detecting EGFR gene mutations and CDKN2A / B gene deletion or loss-of-function mutations in lung cancer cells collected from the subject, and (2) assessing that a CDK4 / 6 inhibitor is likely to be effective in treating lung cancer in the subject when both EGFR gene mutations and CDKN2A / B gene deletion or loss-of-function mutations are present. Item 16. A therapeutic agent for lung cancer with EGFR gene mutations and CDKN2A / B gene deletion or loss-of-function mutations, comprising a CDK4 / 6 inhibitor as an active ingredient.

[0050] 1. Methods: Establishment of EGFR gene mutation-positive lung cancer organoids. This study was approved by the Institutional Review Boards of Keio University Hospital, Kawasaki Municipal Kawasaki Hospital, and Saiseikai Central Hospital. Written informed consent was obtained from all patients enrolled in this study. Clinical specimens from EGFR gene mutation-positive lung cancer patients, whose EGFR gene mutations were identified using known clinical methods, were collected sequentially from May 2018 to April 2022. Clinical specimens used in this study included surgical resection, bronchoscopic biopsy, CT-guided needle biopsy, ascites, pleural effusion, or autopsy specimens. Lung cancer organoids were established using a method we previously reported (Cell Rep. 2023 Mar 28;42(3):112212). Briefly, tissue samples were cut into 1mm pieces using surgical scissors. 3 The cells were minced into fragments. The fragments were digested with Liberase TH (Roche) at 37°C for 30 minutes. The digested pellet was treated with TrypLE Express (Thermo Fisher Scientific) at 37°C for 10 minutes. The isolated cells were embedded in a droplet of Matrigel Growth Factor Reduced (Corning) and overlaid with medium. Organoids from pleural effusion were established as previously described (Cell Rep. 2023 Mar 28;42(3):112212).

[0051] Immunohistochemical staining. Immunohistochemical staining was performed as previously described (Cell Rep. 2023 Mar 28;42(3):112212). Briefly, patient specimens and isolated xenografts were fixed in 4% paraformaldehyde. Organoid tissue blocks were prepared using iPgell (GSPG20-1, Nippon Genetics Co., Ltd.) according to the manufacturer's instructions. Paraffin-embedded tissues were sectioned and stained with hematoxylin and eosin (H&E) using standard protocols. Antibodies used for immunohistochemical staining included rabbit anti-synaptophysin (Proteintec, 17785-1-AP, 1:1500), mouse anti-CD56 (DAKO, M7304, 1:200), rabbit anti-chromogranin A (Abcam, ab15160, 1:1000), mouse anti-CDKN2A / p16INK4a (GeneTex, GTX01783, 1:100), mouse anti-TTF1 (DAKO, M3575, 1:100), mouse anti-p63 (DAKO, M7317, 1:100), mouse anti-p40 (Nichirei, bc28, 1:2), and rabbit anti-AQP5 (Abcam, ab92320, 1:20).

[0052] Whole-exome sequencing (WES) analysis. WES analysis was performed as previously described (Cell Rep. 2023 Mar 28;42(3):112212). Briefly, DNA was extracted using the QIAamp Blood Mini Kit (QIAGEN). DNA quality was assessed by agarose gel electrophoresis. 150-bp paired-end libraries were prepared using the SureSelect Human All Exon V6 kit (Agilent) according to the manufacturer's protocol. Sequencing was performed on a IIIumina Novaseq instrument. The purified fastq files were mapped to the human reference genome version GRCh37 (hg19) using BWA-MEM version 0.7.17 (http: / / bio-bwa.sourceforge.net / ) [Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754-1760 (2009)]. Somatic mutations, single nucleotide variations (SNVs), insertions, and deletions were detected using the Genome Analysis Toolkit version 4.1.9.0 (https: / / gatk.broadinstitute.org). Variant detection was performed using Mutect2. To remove germline variants, detected variants were filtered by removing variants detected with an allele frequency of 0.1% or higher in dbSNP or the Japanese Germline Variant Database (a human gene mutation database). Furthermore, variants were filtered by excluding variants detected in at least two of the 23 normal organoids and 35 blood samples sequenced in our previous study (Cell Rep. 2023 Mar 28;42(3):112212). Data were deposited in the DNA Data Bank of Japan (DDBJ) database under accession numbers JGAS000610 (study) and JGAD000739 (dataset).

[0053] In vitro cell viability assay. Cell viability assays were performed using CellTiter-Glo (Promega) according to the manufacturer's protocol, as previously reported by the inventors (Cell Rep. 2023 Mar 28;42(3):112212). Organoids established in the "Establishment of EGFR-mutant lung cancer organoids" section were seeded in 96-well plates (1500 cells / well) and treated with palbociclib or abemaciclib. Control cells were treated with the same concentration of vehicle, DMSO. Absorbance was measured on day 7 after treatment using Cytation 5 (BioTek, #MO1210). IC50 values ​​were calculated using Prism software (GraphPad Prism 7.04). At 1 μM of palbociclib or abemaciclib, cell viability ( / DMSO) of ≥ 75% was considered resistant, and < 75% was considered sensitive.

[0054] Organoid xenotransplantation and animal experiments. All animal experiments were approved by the Keio University School of Medicine Laboratory Animal Center. Xenotransplantation experiments were performed as previously reported (Cell Rep. 2023 Mar 28;42(3):112212). Female NOG (NOD / Shi-scid, IL-2RγKO Jic) mice (5 weeks old, approximately 20 g / mouse) were purchased from Charles River Japan. For each transplant, organoid clusters (approximately 1 × 10 5 The cells were suspended in 50 μL of Matrigel and subcutaneously implanted into mice. Mice were euthanized 2 or 3 months after implantation.

[0055] In vivo efficacy studies of the CDK4 / 6 inhibitor palbociclib were performed using organoid clusters. The xenograft tumor volume (TV) was 100 mm 3Once the tumor volume reached this level, drug administration was initiated (Figure 1). Tumor volume (TV) was calculated by measuring the length (L), width (W), and height (H) of each tumor with a vernier caliper, as follows: TV = (L × W × H / 2). Mice were randomly assigned to a vehicle-treated group (n = 5) or a palbociclib (150 mg / kg / day)-treated group (n = 5). The drug was administered orally daily for 3 weeks, and TV and body weight were measured 2–3 times weekly. After 3 weeks of drug administration, mice were sacrificed and tumors were isolated.

[0056] Statistical Analysis: IC50, tumor volume, or immunohistochemistry values ​​were determined using the Wilcoxon rank-sum test or chi-square test. The significance level is indicated by the P value for each experiment. Asterisks in the figures indicate the following: *P value < 0.05, **P value < 0.01, ***P value < 0.001, and nsP value > 0.05. Statistical analysis was performed using GraphPad Prism version 7.04 statistical software (GraphPad Prism Corp., California, USA).

[0057] 2. Results: Establishment of a lung cancer organoid library. We established 31 lung cancer organoid lines using various clinical specimens obtained by surgical resection, bronchoscopic biopsy, CT-guided needle biopsy, ascites, pleural effusion, or autopsy. We also performed immunohistochemical staining of the established organoids and xenograft tumors (lung adenocarcinoma (E-16, E-05, E-01B), lung squamous cell carcinoma (E-12), small cell lung cancer (E-02B), and large cell endocrine carcinoma (E-15), all of which harbor EGFR gene mutations) to confirm that the organoids are an experimental model that reflects clinical practice (Figure 2).

[0058] Genotype and phenotype Whole exome sequencing (WES) was performed on the 31 established organoid lines to identify EGFR gene mutations and various genetic abnormalities related to resistance (Figure 3).

[0059] Therapeutic effect of CDK4 / 6 inhibitors on EGFR gene-mutated lung cancer with CDKN2A / B gene deletion or loss-of-function mutation. According to the in vitro cell viability assay (Figure 4A), cell viability decreased at a high concentration of 10 μM palbociclib in both lung cancer organoid lines, EGFR gene mutation-positive lines with CDKN2A / B gene deletion (lines with Homo deletion in the CDKN2A / B row in Figure 3), and EGFR gene mutation-positive lines with wild-type CDKN2A / B. However, at 1 μM palbociclib, cell viability was reduced to less than 75% in all EGFR gene mutation-positive lines with CDKN2A / B gene deletion, while cell viability was higher than 75% in all EGFR gene mutation-positive lines with wild-type CDKN2A / B.

[0060] Referring to Figure 4B, the EGFR gene mutation-positive cell line with CDKN2A / B gene deletion had a higher IC than the EGFR gene mutation-positive cell line with wild-type CDKN2A / B. 50 The values ​​were significantly lower, indicating a high therapeutic effect of palbociclib.

[0061] 5 , in the squamous cell carcinoma-derived cell line E-12 with an EGFR gene mutation (exon 19 deletion (19del)) and a CDKN2A / B gene deletion, the lung adenocarcinoma-derived cell line E-14 with an EGFR gene mutation (19del) and a CDKN2A / B gene deletion, and the lung adenocarcinoma-derived cell line E-17 with an EGFR gene mutation (G719A) and a CDKN2A / B gene deletion, the mean tumor volume of xenograft tumors on day 21 was significantly reduced in the palbociclib-treated group compared with the vehicle-treated group ((A)-(C)). In contrast, in the cell line E-01B with an EGFR gene mutation (L858R) and wild-type CDKN2A / B genes, there was no significant difference in the mean tumor volume of xenograft tumors between the palbociclib-treated group and the vehicle-treated group ((D)).

[0062] Referring to FIG. 6A, all EGFR gene mutant cells with CDKN2A / B gene deletion were sensitive to abemaciclib, while all EGFR gene mutant cells with wild-type CDKN2A / B gene were resistant to abemaciclib.

[0063] As shown in Figure 6B, KRAS mutant cells with CDKN2A / B gene deletion or loss-of-function mutations coexisted in both palbociclib-sensitive and -resistant cells, indicating that they are not useful as biomarkers. The KRAS gene is located downstream of EGFR signaling, and like mutant EGFR, its gene mutation promotes constitutive cell proliferation. The combination of CDKN2A / B gene deletion or loss-of-function mutations and KRAS gene mutations was not useful as a biomarker for CDK4 / 6 inhibitor sensitivity.

[0064] Table 1 shows the relationship between the presence or absence of mutations in each gene (EGFR gene, KRAS gene, CDKN2A / B gene) and sensitivity / resistance to palbociclib in 25 lung cancer organoid lines. The 25 lines include some that overlap with the organoid lines used in the aforementioned experiment. When the biomarkers used were "positive for CDKN2A / B gene deletion or loss-of-function mutation" and "positive for EGFR gene mutation," all 10 biomarker-positive lines were sensitive to palbociclib, while of the 15 biomarker-negative lines, 6 were sensitive and 9 were resistant, resulting in a specificity of 100%.

[0065]

[0066] Four organoid lines of lung squamous cell carcinoma were established and their correlation with palbociclib sensitivity / resistance was investigated.When the biomarkers used were "positive for CDKN2A / B gene deletion or loss-of-function mutation" and "positive for EGFR gene mutation," one biomarker-positive line was sensitive to palbociclib, and of the three negative lines, one line was palbociclib-resistant and two lines were sensitive to palbociclib, with a specificity of 100%.

[0067] These data provide preclinical evidence supporting the use of CDK4 / 6 inhibitors for the treatment of lung cancer harboring CDKN2A / B deletion or loss-of-function mutations and EGFR mutations. Our results suggest that CDK inhibitors, particularly CDK4 / 6 inhibitors, may be effective in treating lung cancer harboring CDKN2A / B deletion or loss-of-function mutations and EGFR mutations.

Claims

1. A method for obtaining data regarding the efficacy of a CDK4 / 6 inhibitor in lung cancer cells, comprising detecting EGFR gene mutations and CDKN2A / B gene deletions or loss-of-function mutations in lung cancer cells derived from a lung cancer patient.

2. The method according to claim 1, which shows the effectiveness of a CDK4 / 6 inhibitor when an EGFR gene mutation and a CDKN2A / B gene deletion or loss-of-function mutation are present.

3. A method for obtaining data regarding the efficacy of a CDK4 / 6 inhibitor in lung cancer cells having an EGFR gene mutation, the method comprising detecting CDKN2A / B gene deletion or loss-of-function mutation in lung cancer cells having an EGFR gene mutation.

4. The method according to claim 3, which indicates the efficacy of a CDK4 / 6 inhibitor in the presence of a CDKN2A / B gene deletion or loss-of-function mutation.

5. A method for obtaining data regarding the efficacy of a CDK4 / 6 inhibitor in lung cancer cells having a CDKN2A / B gene deletion or loss-of-function mutation, the method comprising detecting an EGFR gene mutation.

6. The method according to claim 5, which indicates the efficacy of a CDK4 / 6 inhibitor when an EGFR gene mutation is present.

7. The method of any one of claims 1 to 6, wherein the CDK4 / 6 inhibitor comprises palbociclib or abemaciclib.

8. A kit for obtaining data regarding the efficacy of a CDK4 / 6 inhibitor in lung cancer cells, comprising a reagent for measuring EGFR gene mutations and a reagent for measuring CDKN2A / B gene deletion or loss-of-function mutations.

9. A therapeutic agent for lung cancer with EGFR gene mutations and CDKN2A / B gene deletions or loss-of-function mutations, containing a CDK4 / 6 inhibitor as the active ingredient.

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