Method for obtaining potency data of CDK4 / 6 inhibitor in lung cancer

Abstract

The invention provides a method for acquiring potency data of a CDK4 / 6 inhibitor in lung cancer cells. The method comprises the step of detecting EGFR gene mutation and CDKN2A / B gene deletion or function loss mutation in lung cancer cells from a lung cancer patient. The presence of EGFR gene mutation and CDKN2A / B gene deletion or loss of function indicates that the CDK4 / 6 inhibitor has efficacy.

Description

Technical Field

[0001] This invention relates to a method for predicting the efficacy of CDK4 / 6 inhibitors in treating lung cancer patients. Background Technology

[0002] It is reported that more than 2 million people worldwide are diagnosed with lung cancer each year. According to epidemiological data, approximately 500,000 of these cases are identified as having EGFR (epidermal growth factor receptor) gene mutation-positive lung adenocarcinoma.

[0003] EGFR mutation-positive lung adenocarcinoma is one of the largest subgroups of lung adenocarcinoma. For EGFR mutation-positive lung adenocarcinoma, EGFR tyrosine kinase inhibitors have been developed as molecularly targeted therapies, and their efficacy has been demonstrated in clinical trials. Among them, the third-generation EGFR tyrosine kinase inhibitor osimertinib (trade name: Tagrisso) is widely used globally due to its efficacy and safety. The efficacy (objective response rate) of EGFR tyrosine kinase inhibitors against EGFR mutation-positive lung adenocarcinoma is approximately 70%, and it can control the disease in the early stages of treatment.

[0004] Meanwhile, some patients are completely unable to use EGFR tyrosine kinase inhibitors due to side effects and complications. Furthermore, numerous cancers have demonstrated the development of treatment tolerance (primary tolerance) early in EGFR tyrosine kinase inhibitor treatment, and a significant number of lung cancers develop tolerance approximately 1 to 2 years after treatment initiation (acquired tolerance). Therefore, there is a strong desire to develop treatment strategies for lung cancer patients who cannot use EGFR tyrosine kinase inhibitors, as well as for lung cancers exhibiting either primary or acquired tolerance.

[0005] CDK4 / 6 inhibitors are selective inhibitors of cyclin-dependent kinases CDK4 and CDK6. Palbociclib and abemaciclib are small molecule compounds used as therapeutic agents for hormone receptor (HR)-positive and HER2-negative breast cancer.

[0006] Non-patent literature 1 indicates that palbociclib alone can exhibit moderate antitumor activity in patients with non-small cell lung cancer who have CDKN2A / B gene deletion or loss-of-function mutations.

[0007] Non-patent literature 2 indicates that 82% of patients with EGFR gene mutations are positive for cyclin D1. In a growth inhibition study of cell lines from EGFR gene-mutant non-small cell lung cancer, abecil alone as a CDK4 / 6 inhibitor was ineffective against both EGFR gene-mutant and non-EGFR gene-mutant cell lines. However, when osimertinib was used in combination with abecil, the drug concentration required for half of the growth inhibition was reduced.

[0008] Reference List

[0009] Non-patent literature

[0010] Non-patent literature 1: Eugene R. Ahn, et al., Precision Oncology no. 4 (2020) 757-766. Published online June 25, 2020.

[0011] Non-patent literature 2: Osoegawa et. al., Investigational New Drugs 41, 183-192 (2023) February 15, 2023. doi: 10.1007 / s10637-023-01337-8 Summary of the Invention

[0012] Technical issues

[0013] Non-patent literature 1 does not document the presence or absence of EGFR gene mutations in patients. Non-patent literature 2 indicates that cyclin D1 is expressed in many patients with EGFR gene mutations, but does not describe its association with CDKN2A / B gene deletion or loss-of-function mutations, nor does it suggest or mention improving the accuracy of predicting the efficacy of CDK4 / 6 inhibitors by combining CDKN2A / B gene deletion or loss-of-function mutations with EGFR gene mutations.

[0014] To date, no biomarker has been identified that can accurately predict the therapeutic efficacy of CDK4 / 6 inhibitors in lung cancer.

[0015] One objective of this invention is to provide a method for obtaining efficacy data of CDK4 / 6 inhibitors in lung cancer.

[0016] Solution

[0017] The present invention includes the following embodiments.

[0018] Item 1.

[0019] A method for obtaining efficacy data of CDK4 / 6 inhibitors 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 from lung cancer patients.

[0020] Item 2.

[0021] According to the method of claim 1, the presence of EGFR gene mutation and CDKN2A / B gene deletion or loss-of-function mutation indicates that the CDK4 / 6 inhibitor is effective.

[0022] Item 3.

[0023] A method for obtaining efficacy data of CDK4 / 6 inhibitors in lung cancer cells carrying EGFR gene mutations, the method comprising detecting CDKN2A / B gene deletion or loss-of-function mutations in the lung cancer cells carrying EGFR gene mutations.

[0024] Item 4.

[0025] According to the method of item 3, the presence of a CDKN2A / B gene deletion or loss-of-function mutation indicates that the CDK4 / 6 inhibitor is effective.

[0026] Item 5.

[0027] A method for obtaining efficacy data of CDK4 / 6 inhibitors in lung cancer cells carrying CDKN2A / B gene deletion or loss-of-function mutations, the method comprising detecting EGFR gene mutations.

[0028] Item 6.

[0029] According to the method described in item 5, the presence of an EGFR gene mutation indicates that the CDK4 / 6 inhibitor is effective.

[0030] Item 7.

[0031] The method according to any one of items 1 to 6, wherein the CDK4 / 6 inhibitor comprises palbociclib or abeciclib.

[0032] Item 8.

[0033] A kit for obtaining efficacy data of CDK4 / 6 inhibitors in lung cancer cells, the kit comprising reagents for detecting EGFR gene mutations and reagents for detecting CDKN2A / B gene deletion or loss-of-function mutations.

[0034] Item 9.

[0035] A therapeutic agent for lung cancer carrying EGFR gene mutations and CDKN2A / B gene deletion or loss-of-function mutations, said therapeutic agent comprising a CDK4 / 6 inhibitor as an active ingredient. Attached Figure Description

[0036] Figure 1 This shows a summary of the mouse xenotransplantation experiment.

[0037] Figure 2 Photographs of various immunohistochemical stainings of organoids and xenograft tumors are shown, with scale bar: 100 μm.

[0038] Figure 3 This diagram illustrates the genotype-phenotype relationship of EGFR-TKI tolerance mechanisms and provides an overview of organoid genetic abnormalities in whole-exome sequencing analysis, where n=31. LUAD: lung adenocarcinoma; SQ: squamous cell carcinoma; SCLC: small cell carcinoma; LCNEC: large cell neuroendocrine carcinoma. The horizontal axis represents the name of the organoid cell line.

[0039] Figure 4A The results of cell viability assays using different palbociclib concentrations are shown. Among them, CDKN2A / B deletion: EGFR gene variants with CDKN2A / B gene deletion exist (n=8); CDKN2A / B WT: EGFR gene variants with CDKN2A / B gene wild type exist (n=5).

[0040] Figure 4B The IC50 values ​​of the CDKN2A / B gene deletion cell line (n=8) and the wild-type cell line (n=5) were shown. 50 Value, of which, This indicates p < 0.01, and a t-test is performed. Error bars: standard deviation.

[0041] Figure 5 The figures show the mean tumor volume (mm²) of xenografts after treatment with palbociclib (n=5) or solvent (n=5) in CDKN2A / B gene-deleted cell lines ((A) E-12, (B) E-14, (C) E-17) and (D) CDKN2A / B wild-type cell line (E-01B). 3 ),in, This indicates that p < 0.001. This indicates p < 0.01, t-test. NS: No significant difference.

[0042] Figure 6A The results of cell viability assays using different abexiantide concentrations are shown for EGFR gene variants with CDKN2A / B gene deletion (n=3) and EGFR gene variants with wild-type CDKN2A / B gene. CDKN2A / B deletion: EGFR gene variants with CDKN2A / B gene deletion; CDKN2A / B WT: EGFR gene variants with wild-type CDKN2A / B gene; Error bars: Standard deviation.

[0043] Figure 6BThe results show the cell viability assays using different palbociclib concentrations for KRAS gene variants with CDKN2A / B gene deletion (n=3) and wild-type KRAS gene variants with CDKN2A / B gene deletion (n=1). CDKN2A / B deletion: KRAS gene variants with CDKN2A / B gene deletion; CDKN2A / B WT: KRAS gene variants with wild-type CDKN2A / B gene deletion; error bars: standard deviation. Detailed Implementation

[0044] In this specification, the word "including" conceptually encompasses both "consistent with" and "comprises from".

[0045] In one aspect of the invention, a method is provided for obtaining efficacy data of CDK4 / 6 inhibitors 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 from lung cancer patients.

[0046] In the presence of EGFR gene mutations and CDKN2A / B gene deletions or loss-of-function mutations, this indicates that the CDK4 / 6 inhibitor is effective in treating patients with lung cancer.

[0047] The timing for detecting EGFR gene mutations can differ from or coincide with the timing for detecting CDKN2A / B gene deletions or loss-of-function mutations. For example, the timing for detecting EGFR gene mutations can be earlier or later than the timing for detecting CDKN2A / B gene deletions or loss-of-function mutations.

[0048] In another aspect of the invention, a method is provided for obtaining efficacy data of CDK4 / 6 inhibitors in lung cancer cells carrying EGFR gene mutations, the method comprising detecting CDKN2A / B gene deletion or loss-of-function mutations in the lung cancer cells carrying EGFR gene mutations.

[0049] The lung cancer cells carrying the EGFR gene mutation are preferably lung cancer cells with the EGFR gene mutation that are derived from or taken from lung cancer patients.

[0050] In the presence of CDKN2A / B gene deletion or loss-of-function mutations, this indicates that the CDK4 / 6 inhibitor is effective against lung cancer in patients.

[0051] In another aspect of the invention, a method is provided for obtaining efficacy data of CDK4 / 6 inhibitors in lung cancer cells carrying CDKN2A / B gene deletion or loss-of-function mutations, the method comprising detecting EGFR gene mutations in the lung cancer cells.

[0052] In the presence of EGFR gene mutations, this indicates that the CDK4 / 6 inhibitor is effective.

[0053] The EGFR gene mutation refers to an abnormality in the gene structure of EGFR (epidermal growth factor receptor), a member of the HER family. The human EGFR genome sequence is represented by NCBI Gene ID 1956, and its amino acid sequence is represented by UNIProtP00533. The mouse EGFR genome sequence is represented by NCBI Gene ID: 24329, and its amino acid sequence is represented by UNIProtQ01279.

[0054] Examples of EGFR gene mutations include mutations in the exon 18-21 region. Examples of deletion mutations include amino acid deletions, and more specifically, amino acid deletions accompanied by amino acid substitutions. In particular, mutations involving the deletion of one or more consecutive amino acids in the E746-I759 region of exon 18 are common. Examples of such mutations include: simple deletion of five amino acids in codons E746-A750; deletion of L747-E753 accompanied by insertion of S; simple deletion of L747-E751; deletion of L747-E750 accompanied by insertion of P; point mutation at codon 719 of exon 18 (G719X, which is collectively represented as X because the amino acids can be A, C, or S); point mutation at codon 709 of exon 18 (E709X and G719X, which are collectively represented as X because the amino acids can be K or A); deletion mutation in exon 19; insertion mutation in exon 20; point mutation at S768I in exon 20; point mutation at L858R in exon 21; and point mutation at L861Q in exon 21. EGFR gene mutations are listed in the COSMIC (Cancer Somatic Mutation Catalogue, https: / / cancer.sanger.ac.uk / cosmic) database. These EGFR gene mutations are all well-known and can be detected using publicly known or widely recognized detection methods or commercially available kits.

[0055] Among these EGFR gene mutations, those that continue to transmit phosphorylation signals to downstream growth signaling pathways even when stimulation by ligands (such as EGF and TGFα) does not occur functionally are called activating mutations.

[0056] In one implementation, EGFR gene mutation refers to the following EGFR gene mutations that are clinically significant for lung cancer, as defined in the Japanese Lung Cancer Society's "Guidelines for EGFR Gene Mutation Detection in Lung Cancer Patients, Version 5.0" (December 16, 2021):

[0057] (1) EGFR gene mutations known to be activating mutations

[0058] • Deletion mutation of exon 19, L858R mutation

[0059] • G719X mutation, L861Q mutation, S768I mutation

[0060] • Insertion mutation in exon 20 (A763_Y764insFQEA)

[0061] CDKN2A / B gene deletion or loss-of-function mutations refer to gene deletions or loss-of-function mutations in at least one of CDKN2A (cyclin-dependent kinase inhibitor 2A) and CDKN2B (cyclin-dependent kinase inhibitor 2B). The genomic sequence of human CDKN2A is represented by NCBI Gene ID: 1029, with an amino acid sequence of UNIProt P42771; the genomic sequence of human CDKN2B is represented by NCBI Gene ID: 1030, with an amino acid sequence of UNIProt Q5ZEY8; the genomic sequence of mouse CDKN2A is represented by NCBI Gene ID: 12578, with an amino acid sequence of UNIProt Q64364; and the genomic sequence of mouse CDKN2B is represented by NCBI Gene ID: 12579, with an amino acid sequence of UNIProt Q549R4.

[0062] CDKN2A and CDKN2B are both tumor suppressor genes involved in cell cycle control. Deletion or loss-of-function mutations in the CDKN2A and CDKN2B genes are associated with abnormal cell cycle control and cancer progression. Deletion or loss-of-function mutations in the CDKN2A and CDKN2B genes can be detected using well-known detection methods or commercially available kits.

[0063] There are no particular restrictions on the CDKN2A / B gene deletion or loss-of-function mutation, as long as the CDKN2A / B gene deletion or loss-of-function mutation leads to loss of gene function. The CDKN2A and CDKN2B genes are adjacent in the 9p21 region of chromosome 2, and large segments containing both CDKN2A and CDKN2B genes are frequently deleted. Preferably, both alleles of the CDKN2A / B gene are deleted.

[0064] The CDKN2A / B gene deletion includes a 9p21 homozygous deletion. In a 9p21 homozygous deletion, the two alleles may not necessarily have the same region deleted; the deletion may be a loss of function of the CDKN2A and CDKN2B genes at each allele.

[0065] The loss-of-function mutations in the CDKN2A / B gene include missense mutations and nonsense mutations. Missense mutations are, for example, D84X(H, N, Y) (meaning the D at position 84 of the amino acid sequence is mutated to H, N, or Y) or D108X(H, N, Y). Nonsense mutations are, for example, W110. The loss-of-function mutations of the CDKN2A / B gene include CDKN2A / B gene silencing caused by methylation, for example, silencing of the CDKN2A / B gene caused by methylation of the CDKN2A / B gene promoter.

[0066] CDKN2A / B gene deletion or loss-of-function mutations include the following states: one of the two alleles is deleted, missense, or nonsense, and the other of the two alleles is methylated.

[0067] The inventors of this invention constructed a patient-derived organoid library of EGFR gene mutation-positive lung cancer from 31 systems and investigated the association between gene abnormalities and the efficacy of CDK4 / 6 inhibitors. During the study, the inventors unexpectedly discovered that the presence of CDKN2A / B gene deletion or loss-of-function mutations in addition to EGFR gene mutation positivity could specifically predict the efficacy of the CDK4 / 6 inhibitors. The combination of "EGFR gene mutation" and "CDKN2A / B gene deletion or loss-of-function mutation" is important as a biomarker, as it can improve the accuracy of predicting the efficacy of CDK4 / 6 inhibitors in lung cancer patients.

[0068] Examples of lung cancer include at least one selected from lung adenocarcinoma, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, mixed types thereof, and lung cancer that cannot be classified into any one histological type.

[0069] Examples of CDK4 / 6 inhibitors include, but are not limited to, palbociclib, abeciclib, and ribociclib. The CDK4 / 6 inhibitor preferably contains palbociclib or abeciclib.

[0070] The presence or absence of EGFR gene mutations and CDKN2A / B gene deletions or loss-of-function mutations are indicators used to predict the therapeutic effect of the CDK4 / 6 inhibitor on lung cancer patients, or to predict the efficacy of the CDK4 / 6 inhibitor in treating lung cancer patients.

[0071] EGFR gene mutations and CDKN2A / B gene deletion or loss-of-function mutations were detected in lung cancer cells taken from the patient. In the presence of both EGFR gene mutations and CDKN2A / B gene deletion or loss-of-function mutations, the CDK4 / 6 inhibitor was deemed highly likely to be effective in treating the patient's lung cancer.

[0072] EGFR gene mutation testing in lung cancer tissues has been performed clinically. Furthermore, p16 immunostaining has been used in fields such as otolaryngology tumors to detect CDKN2A / B gene deletions or loss-of-function mutations. Additionally, oncogene testing panels (such as the "OncoGuide NCC Tumor Gene Panel," "FoundationOne CDx Tumor Genome Atlas," and "FoundationOne Liquid CDx Tumor Genome Atlas") and 9p21 region deletion detection via fluorescence in situ hybridization (FISH) can be employed. By combining these testing methods already performed in the clinical setting, it is possible to screen for groups of patients to whom the CDK4 / 6 inhibitors are effective.

[0073] Therefore, by employing the method according to embodiments of the present invention, lung cancer patients who exhibit efficacy with the CDK4 / 6 inhibitor can be screened with high accuracy and simplicity, thereby enabling the selection of appropriate treatment methods for patients.

[0074] In the above detection steps, both EGFR gene mutations and CDKN2A / B gene deletion or loss-of-function mutations are detected in the patient's lung cancer cells. This provides data indicating the presence of both EGFR gene mutations and CDKN2A / B gene deletion or loss-of-function mutations, and that the CDK4 / 6 inhibitor is effective in treating the patient's lung cancer. In this case, the CDK4 / 6 inhibitor can be further administered. Therefore, patients with both EGFR gene mutations and CDKN2A / B gene deletion or loss-of-function mutations detected in their lung cancer cells can be treated.

[0075] In one aspect of the invention, a kit is provided for obtaining efficacy data of CDK4 / 6 inhibitors in the treatment of lung cancer patients, the kit comprising reagents for detecting EGFR gene mutations and reagents for detecting CDKN2A / B gene deletion or loss-of-function mutations.

[0076] In one embodiment, the reagent for detecting EGFR gene mutations is a probe for detecting EGFR gene mutations. In another embodiment, the reagent for detecting EGFR gene mutations is a probe for amplifying or detecting the EGFR gene. The probe is preferably a nucleic acid probe, and more preferably RNA or DNA.

[0077] In one embodiment, the reagent for detecting CDKN2A / B gene deletion or loss-of-function mutations is a probe for detecting CDKN2A / B gene deletion or loss-of-function mutations.

[0078] In another embodiment, the reagent for detecting CDKN2A / B gene deletion or loss-of-function mutations is a probe for amplifying or detecting the CDKN2A / B gene. The probe is preferably a nucleic acid probe, and more preferably RNA or DNA.

[0079] The methods of obtaining RNA or cDNA from lung cancer cells collected from samples, and the methods of detecting target nucleic acids using nucleic acid probes through known methods (such as nucleic acid amplification or nucleic acid hybridization), are known to the public.

[0080] In another embodiment, the reagent used to detect CDKN2A / B gene deletion or loss-of-function mutations is an antibody against the expression product of the CDKN2A / B gene. For example, an antibody specific to the protein (the expression product of a wild-type or null mutant of the CDKN2A / B gene) can be used to detect the presence or absence of CDKN2A / B gene deletion or loss-of-function mutations in lung cancer cells taken from a patient. For example, if the detection value of the CDKN2A / B gene expression product in the patient's cells is lower than the level in normal lung cells, the detection value indicates the presence of a CDKN2A / B gene deletion or loss-of-function mutation.

[0081] The kit may further include instructions for use when obtaining efficacy data of CDK4 / 6 inhibitors in lung cancer cells.

[0082] The kit may further include a control sample. The control sample exhibits, for example, the gene expression level in normal lung cells. In one embodiment, if the expression level of the CDKN2A / B gene in lung cancer cells taken from a patient is lower than that in normal lung cells, a CDKN2A / B gene deletion or loss-of-function mutation may be present.

[0083] In one aspect of the invention, a therapeutic agent is provided for lung cancer carrying EGFR gene mutations and CDKN2A / B gene deletion or loss-of-function mutations, the therapeutic agent comprising a CDK4 / 6 inhibitor as an active ingredient.

[0084] In one embodiment, the therapeutic agent is a treatment for lung cancer in a patient selected because both EGFR gene mutation and CDKN2A / B gene deletion or loss-of-function mutation are present in lung cancer cells taken from the patient. In this patient, the CDK4 / 6 inhibitor is highly likely to be effective in treating the patient's lung cancer.

[0085] The daily dosage of the CDK4 / 6 inhibitor depends on the patient's age, weight, symptoms, etc., but typically for adults (weighing 50 kg), the dosage can be approximately 0.1 mg to 5000 mg daily, preferably 1 mg to 1000 mg, and more preferably 10 mg to 1000 mg. This dose of CDK4 / 6 inhibitor is preferably administered once daily, or divided into approximately two to three daily doses. Administration can be parenteral, such as intravenous, intraperitoneal, subcutaneous, or intramuscular injection; or oral. The inhibitor to be administered may contain a pharmaceutically acceptable carrier, diluent, or excipient. There are no limitations on the dosage form of the inhibitor; for example, the inhibitor can be administered orally in tablets, capsules, granules, powders, syrups, etc., or parenterally in injections, drops, suppositories, sprays, etc.

[0086] The dosage regimen should be selected appropriately based on factors such as the type of CDK4 / 6 inhibitor, whether there is concomitant medication, previous treatment history, disease stage, presence of metastasis, and the patient's age and gender.

[0087] The number of lung cancer patients with EGFR gene mutations worldwide is enormous, and the number of lung cancer patients carrying EGFR gene mutations as well as CDKN2A / B gene deletion or loss-of-function mutations is also considered quite large. Therefore, it is believed that treatment using the method according to the present invention can be implemented in countries around the world. This treatment is expected to improve treatment outcomes in cases where such patients can be treated with drugs of a different type than EGFR tyrosine kinase inhibitors.

[0088] This disclosure further includes the following embodiments.

[0089] Item 1.

[0090] A method for obtaining efficacy data of CDK4 / 6 inhibitors in lung cancer cells, the method comprising:

[0091] Detect EGFR gene mutations and CDKN2A / B gene deletion or loss-of-function mutations in lung cancer cells from lung cancer patients.

[0092] Item 2.

[0093] According to the method of claim 1, the presence of EGFR gene mutation and CDKN2A / B gene deletion or loss-of-function mutation indicates that the CDK4 / 6 inhibitor is effective.

[0094] Item 3.

[0095] A method for obtaining efficacy data of CDK4 / 6 inhibitors in lung cancer cells carrying EGFR gene mutations, the method comprising:

[0096] The CDKN2A / B gene deletion or loss-of-function mutation was detected in the lung cancer cells carrying the EGFR gene mutation.

[0097] Item 4.

[0098] According to the method of item 3, the presence of a CDKN2A / B gene deletion or loss-of-function mutation indicates that the CDK4 / 6 inhibitor is effective.

[0099] Item 5.

[0100] A method for obtaining efficacy data of CDK4 / 6 inhibitors in lung cancer cells carrying CDKN2A / B gene deletion or loss-of-function mutations, the method comprising:

[0101] Detect EGFR gene mutations.

[0102] Item 6.

[0103] According to the method described in item 5, the presence of an EGFR gene mutation indicates that the CDK4 / 6 inhibitor is effective.

[0104] Item 7.

[0105] The method according to any one of items 1 to 6, wherein the CDK4 / 6 inhibitor comprises palbociclib or abeciclib.

[0106] Item 8.

[0107] A kit for obtaining efficacy data of CDK4 / 6 inhibitors in lung cancer cells, the kit comprising reagents for detecting EGFR gene mutations and reagents for detecting CDKN2A / B gene deletion or loss-of-function mutations.

[0108] Item 9.

[0109] A therapeutic agent for lung cancer carrying EGFR gene mutations and CDKN2A / B gene deletion or loss-of-function mutations, said therapeutic agent comprising a CDK4 / 6 inhibitor as an active ingredient.

[0110] Item 10.

[0111] According to the method of item 3, the lung cancer cells carrying EGFR gene mutations include lung cancer cells carrying EGFR gene mutations from lung cancer patients.

[0112] Item 11.

[0113] A method for predicting the efficacy of CDK4 / 6 inhibitors in the treatment of lung cancer patients, the method comprising:

[0114] The test detected EGFR gene mutations and CDKN2A / B gene deletion or loss-of-function mutations in lung cancer cells taken from the patient; and

[0115] When EGFR gene mutations and CDKN2A / B gene deletions or loss-of-function mutations are present, the CDK4 / 6 inhibitor is deemed highly likely to be effective in treating the patient's lung cancer.

[0116] Item 12.

[0117] A method for predicting the efficacy of CDK4 / 6 inhibitors in the treatment of lung cancer patients whose lung cancer cells carry EGFR gene mutations, the method comprising:

[0118] The detection of CDKN2A / B gene deletion or loss-of-function mutations in lung cancer cells taken from the patient; and

[0119] When CDKN2A / B gene deletion or loss-of-function mutations are present, the CDK4 / 6 inhibitor is deemed highly likely to be effective in treating the patient's lung cancer.

[0120] Item 13.

[0121] A method for predicting the efficacy of CDK4 / 6 inhibitors in the treatment of lung cancer patients whose lung cancer cells carry CDKN2A / B gene deletion or loss-of-function mutations, the method comprising:

[0122] Detection of EGFR gene mutations in lung cancer cells taken from the patient; and

[0123] When an EGFR gene mutation is present, the CDK4 / 6 inhibitor is deemed highly likely to be effective in treating the patient's lung cancer.

[0124] Item 14.

[0125] The method according to any one of items 11 to 13, wherein the CDK4 / 6 inhibitor comprises palbociclib or abeciclib.

[0126] Item 15.

[0127] A kit for predicting the efficacy of CDK4 / 6 inhibitors in the treatment of lung cancer patients by means of steps (1) and (2) below, the kit comprising reagents for detecting EGFR gene mutations and reagents for detecting CDKN2A / B gene deletion or loss-of-function mutations:

[0128] (1) Detection of EGFR gene mutations and CDKN2A / B gene deletion or loss-of-function mutations in lung cancer cells taken from the patient; and

[0129] (2) When there is an EGFR gene mutation and a CDKN2A / B gene deletion or loss-of-function mutation, the CDK4 / 6 inhibitor is deemed to be highly likely to be effective in treating the patient’s lung cancer.

[0130] Item 16.

[0131] A therapeutic agent for lung cancer carrying EGFR gene mutations and CDKN2A / B gene deletion or loss-of-function mutations, said therapeutic agent comprising a CDK4 / 6 inhibitor as an active ingredient.

[0132] Example

[0133] 1. Methods

[0134] Construction of organoids for lung cancer with EGFR gene mutation

[0135] This study was approved by the Institutional Review Committees of Keio University Hospital, Kawasaki City Hospital, and Saiseikai Central Hospital. Written informed consent was obtained from all patients participating in this study. Clinical specimens from lung cancer patients carrying EGFR gene mutations were collected consecutively from May 2018 to April 2022, and the EGFR gene mutations were identified using clinically known methods. The clinical specimens used in this study included surgical resection specimens, bronchoscopic biopsy specimens, CT-guided puncture biopsy specimens, ascites, pleural effusion, and autopsy specimens. Lung cancer organoids were constructed using our previously reported method (Cell Rep. 2023 Mar 28;42(3):112212), which is briefly described below. For tissues, the samples were finely cut to 1 mm using surgical scissors. 3The fragments were digested with Liberase TH (Roche) at 37°C for 30 minutes. The digested clumps were then treated with TrypLEExpress (Thermo Fisher Scientific) at 37°C for 10 minutes. The isolated cells were embedded in Matrigel Growth Factor Reduced (Corning) droplets and then culture medium was added. Organoids derived from pleural effusion were constructed according to the above report (Cell Rep. 2023 Mar 28;42 (3):112212).

[0136] Immunohistochemical staining

[0137] Immunohistochemical staining was performed according to the aforementioned report (Cell Rep. 2023 Mar 28;42 (3):112212). Briefly, patient specimens and isolated xenografts were fixed with 4% paraformaldehyde. Organoid tissue blocks were prepared according to the manufacturer's instructions using iPgell reagent (GSPG20-1, Japan Gene Co., Ltd.). Paraffin-embedded tissue sections and hematoxylin-eosin (H&E) staining were performed using standard procedures. Antibodies used for immunohistochemical staining include: rabbit anti-synaptophysin (Proteintech, 17785-1-AP, 1:1500), mouse anti-CD56 (DAKO, M7304, 1:200), rabbit anti-chromogranin A (abcam, ab15160, 1:1000), mouse anti-CDKN2A / p16INK4a (Gene Tex, 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).

[0138] Whole exome sequencing (WES) analysis

[0139] WES analysis was performed according to the aforementioned report (Cell Rep. 2023 Mar 28;42 (3):112212), and is briefly described below. For DNA extraction, the QIAamp Blood Micro-extraction Kit (QIAGEN) was used. DNA quality was checked by agarose gel electrophoresis. A 150 bp paired-end sequencing library was prepared using the SureSelect Human Whole Exome V6 Kit (Agilent Technologies) according to the manufacturer's protocol. Sequencing was performed using an Illumina Novaseq sequencer. The pruned fastq file was mapped to the human reference genome GRCh37 version (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. Bioinformatics25, 1754-1760 (2009)]. Somatic mutations, single nucleotide variants (SNVs), insertions, and deletions were detected using the Genomics Analysis Toolkit version 4.1.9.0 (https: / / gatk.broadinstitute.org). Mutect2 was used to detect mutants. To eliminate germline variation, detected variants were filtered by removing variants with an allele frequency of 0.1% or higher detected by dbSNP or the Japanese Germline Variation Database (Human Genome Mutation Database). In addition, variants were filtered by removing variants detected in at least two of 35 blood samples and 23 normal organoids from the inventors' previous study (Cell Rep. 2023 Mar 28;42 (3):112212). The data were stored in the Japanese DNA Database (DDBJ) with accession numbers JGAS000610 (research) and JGAD000739 (dataset).

[0140] In vitro cell viability assay

[0141] As reported by the inventors of this invention (Cell Rep. 2023 Mar 28; 42 (3):112212), the cell viability assay was performed using CellTiter-Glo (promega) according to the manufacturer's instructions. The various organoids constructed in "Construction of Organoids from EGFR Mutation-Positive Lung Cancer" were seeded into 96-well plates (1500 cells / well) and treated with palbociclib and abexianb. Control cells were treated with an equal concentration of DMSO as the solvent. Absorbance was measured using Cytation5 (BioTek, #MO1210) on day 7. IC50 was calculated using Prism software (GraphPad Prism 7.04). 50 Value. Using 1 μM palbociclib or abeciclib, cell viability ( / DMSO group) ≥75% was considered tolerable, and cell viability <75% was considered sensitive.

[0142] Organoid xenotransplantation and animal experiments

[0143] All animal experiments were approved by the Laboratory Animal Center of Keio University School of Medicine. Xenotransplantation experiments were conducted according to the aforementioned report (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 Laboratories, Japan. For each transplantation, organoid clusters (approximately 1 × 10⁻⁶) were used. 5 Cells were suspended in 50 μL of matrix gel and subcutaneously transplanted into mice. The mice were euthanized 2 or 3 months after transplantation.

[0144] In in vivo efficacy studies of palbociclib as a CDK4 / 6 inhibitor, the aforementioned organoid cluster was transplanted, and the tumor volume (TV) of the xenograft reached 100 mm. 3 Or start administration when older ( Figure 1 The length (L), width (W), and height (H) of each tumor were measured using calipers, and the tumor volume (TV) was calculated as: TV = (L × W × H) / 2. Mice were randomly divided into a solvent control group (n=5) and a palbociclib (150 mg / kg / day) treatment group (n=5). The mice were administered oral medication daily for 3 consecutive weeks, with TV and body weight measured 2-3 times per week. After 3 weeks of treatment, the mice were sacrificed and the tumors were isolated.

[0145] Statistical analysis

[0146] IC 50Tumor volume or immunohistochemical values ​​were determined using the Wilcoxon rank-sum test or chi-square test. In each experiment, the significance level was expressed by the p-value. Asterisks in the figure indicate the following: This indicates a p-value < 0.05. This indicates that the p-value is < 0.01. ns indicates a p-value < 0.001, and ns indicates a p-value > 0.05. Statistical analysis was performed using GraphPad Prism version 7.04 statistical software (GraphPad Prism Corporation, California, USA).

[0147] 2. Results

[0148] Construction of lung cancer organoid library

[0149] The inventors of this invention constructed 31 series of lung cancer organoids using various clinical specimens obtained through surgical resection, bronchoscopic biopsy, CT-guided puncture biopsy, ascites, pleural effusion, or autopsy. Furthermore, immunohistochemical staining was performed on the constructed organoids and xenograft tumors (lung adenocarcinoma (E-16, E-05, E-01B lineages), squamous cell lung cancer (E-12 lineage), small cell lung cancer (E-02B lineage), and large cell neuroendocrine carcinoma (E-15), all of which were EGFR gene mutation positive) to confirm that the organoids were experimental models reflecting actual clinical practice. Figure 2 ).

[0150] Genotype and phenotype

[0151] Whole-exome sequencing (WES) of the 31 constructed organoid series confirmed multiple genetic abnormalities associated with EGFR gene mutations and tolerance. Figure 3 ).

[0152] CDK4 / 6 inhibitors target EGFR gene mutations caused by CDKN2A / B gene deletions or loss-of-function mutations. Treatment efficacy of lung cancer

[0153] according to Figure 4A In vitro cell viability assays were performed on EGFR gene mutation-positive lines with CDKN2A / B gene deletion. Figure 3In lung cancer organoid lines (both homozygous deletions in the CDKN2A / B line and wild-type EGFR mutation-positive lines), a high concentration of 10 μM palbociclib reduced cell viability. However, at a palbociclib concentration of 1 μM, palbociclib resulted in cell viability below 75% in all examples of EGFR mutation-positive lines with CDKN2A / B deletions, while cell viability was above 75% in all examples of EGFR mutation-positive lines with wild-type CDKN2A / B.

[0154] Reference Figure 4B Compared with wild-type EGFR gene mutation-positive lines with CDKN2A / B, the IC50 of EGFR gene mutation-positive lines with CDKN2A / B gene deletion was significantly lower. 50 The value was significantly lower, indicating that palbociclib was more effective.

[0155] Reference Figure 5 In the squamous cell carcinoma derivative line E-12 carrying an EGFR gene mutation (exon 19 deletion (19del)) and a CDKN2A / B gene deletion, the lung adenocarcinoma derivative line E-14 carrying an EGFR gene mutation (19del) and a CDKN2A / B gene deletion, and the lung adenocarcinoma derivative line E-17 carrying an EGFR gene mutation (G719A) and a CDKN2A / B gene deletion, the mean tumor volume of xenograft tumors was significantly lower in the palbociclib group compared to the solvent-based administration group ((A) to (C)). Meanwhile, in the series carrying an EGFR gene mutation (L858R) and a wild-type CDKN2A / B gene (E-01B), there was no significant difference in mean tumor volume of xenograft tumors between the palbociclib group and the solvent-based administration group (D).

[0156] Reference Figure 6A EGFR gene mutants with CDKN2A / B gene deletion were abecidine-sensitive in all examples, while EGFR gene mutants with wild-type CDKN2A / B gene were abecidine-tolerant in all examples.

[0157] Reference Figure 6B KRAS gene variants with CDKN2A / B gene deletions or loss-of-function mutations were found to be unusable as biomarkers because palbociclib sensitivity and palbociclib tolerance coexist. The KRAS gene is downstream of EGFR signaling, and mutations in it promote constitutive cell proliferation, similar to mutant EGFR. Even when combined with CDKN2A / B gene deletions or loss-of-function mutations, KRAS gene mutations cannot be used as biomarkers for CDK4 / 6 inhibitor sensitivity.

[0158] Table 1 shows the relationship between the presence or absence of mutations in various genes (EGFR, KRAS, and CDKN2A / B genes) and palbociclib sensitivity / tolerance in 25 lung cancer organoid series. These 25 series include those overlapping with the organoid lines used in the experiments described above. When "positive for CDKN2A / B gene deletion or loss-of-function mutations" and "positive for EGFR gene mutations" were considered valid biomarkers, all 10 series showed palbociclib sensitivity, and of the 15 series showing negative biomarkers, 6 series were palbociclib sensitivity and 9 series were palbociclib tolerance. Specificity was 100%.

[0159] [Table 1]

[0160]

[0161] Four organoid lines for squamous cell lung cancer were constructed, and their relationship with palbociclib sensitivity / tolerance was investigated. When "CDKN2A / B gene deletion or loss-of-function mutation positivity" and "EGFR gene mutation positivity" were considered effective biomarkers, one positive biomarker series was palbociclib sensitive, and among the three negative biomarker series, one series was palbociclib tolerant and two series were palbociclib sensitive. Specificity was 100%.

[0162] This data provides preclinical evidence supporting the use of CDK4 / 6 inhibitors for the treatment of lung cancer harboring CDKN2A / B gene deletion or loss-of-function mutations and EGFR gene mutations. The results obtained by the inventors of this invention demonstrate that CDK inhibitors, particularly CDK4 / 6 inhibitors, may be effective in treating lung cancer harboring CDKN2A / B gene deletion or loss-of-function mutations and EGFR gene mutations.

Claims

1. A method for obtaining efficacy data of CDK4 / 6 inhibitors in lung cancer cells, the method comprising: Detect EGFR gene mutations and CDKN2A / B gene deletion or loss-of-function mutations in lung cancer cells from lung cancer patients.

2. The method according to claim 1, wherein, The presence of EGFR gene mutations, as well as CDKN2A / B gene deletions or loss-of-function mutations, indicates that the CDK4 / 6 inhibitor is effective.

3. A method for obtaining efficacy data of CDK4 / 6 inhibitors in lung cancer cells carrying EGFR gene mutations, the method comprising: The CDKN2A / B gene deletion or loss-of-function mutation was detected in the lung cancer cells carrying the EGFR gene mutation.

4. The method according to claim 3, wherein, The presence of CDKN2A / B gene deletions or loss-of-function mutations indicates that the CDK4 / 6 inhibitor is effective.

5. A method for obtaining efficacy data of CDK4 / 6 inhibitors in lung cancer cells carrying CDKN2A / B gene deletion or loss-of-function mutations, the method comprising: Detect EGFR gene mutations.

6. The method according to claim 5, wherein, The presence of an EGFR gene mutation indicates that the CDK4 / 6 inhibitor is effective.

7. The method according to any one of claims 1 to 6, wherein, The CDK4 / 6 inhibitors include palbociclib or abeciclib.

8. A kit for obtaining efficacy data of CDK4 / 6 inhibitors in lung cancer cells, the kit comprising reagents for detecting EGFR gene mutations and reagents for detecting CDKN2A / B gene deletion or loss-of-function mutations.

9. A therapeutic agent for lung cancer carrying EGFR gene mutations and CDKN2A / B gene deletion or loss-of-function mutations, said therapeutic agent comprising a CDK4 / 6 inhibitor as an active ingredient.

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