Selection method for patient who has short effective period of epidermal growth factor receptor tyrosine kinase inhibitor with respect to non-small cell lung cancer having EGFR activating mutations

Abstract

To select a patient who has a short effective period of an epidermal growth factor receptor tyrosine kinase inhibitor with respect to non-small cell lung cancer having EGFR activating mutations.SOLUTION: A method for selecting a patient who has an effective period of an epidermal growth factor receptor tyrosine kinase inhibitor with respect to non-small cell lung cancer having EGFR activating mutations shorter than a predetermined standard period includes the steps of: calculating a CIN score of tumor tissue collected from the patient; classifying the patient into a CIN-High group or a CIN-Low group by comparing the CIN score with a median value of the CIN score; and determining that the effective period of the epidermal growth factor receptor tyrosine kinase inhibitor with respect to the non-small cell lung cancer having the EGFR activating mutation is shorter than the predetermined standard period if the CIN score is in the CIN-High group.SELECTED DRAWING: Figure 1-C

Description

[Technical Field]

[0001] The present invention provides a method for selecting patients with EGFR-mutated NSCLC who will have a short effective period of EGFR inhibitor treatment. [Background technology]

[0002] Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are the standard first-line treatment for patients with non-small cell lung cancer (NSCLC) harboring EGFR-activating mutations (Non-Patent Documents 1-3).

[0003] EGFR-TKIs result in tumor regression in many cases of EGFR-mutant NSCLC. However, ultimately, all tumors acquire resistance to EGFR-TKIs due to various mechanisms, such as secondary mutations in the EGFR gene (e.g., missense mutations T790M or C797S) and MET gene amplification (Non-Patent Documents 4-6). Despite intensive research to date, the mechanisms of resistance have not yet been fully identified (Non-Patent Document 7).

[0004] In addition, although the majority of patients respond to EGFR-TKI treatment, the duration of response is inconsistent across cases, suggesting that underlying genetic abnormalities in cancer cells may affect the efficacy of EGFR-TKI treatment. For example, mutations in the TP53 gene have been associated with poor outcomes in EGFR-TKI treatment in various studies, but the underlying mechanisms have not yet been fully elucidated (Non-Patent Documents 8 and 9).

[0005] Alternatively, IL6 is also associated with poor outcomes in NSCLC patients treated with EGFR-TKIs, potentially due to JAK-STAT3 activation inducing tumor cell proliferation, invasion, and metastasis, and suppressing antitumor immune responses (Non-Patent Documents 10-12). However, it is not fully understood how IL6-JAK-STAT3 signaling is regulated in EGFR-mutated NSCLC, or how individual genetic background influences disease progression during EGFR-TKI treatment.

[0006] Chromosomal instability (CIN) increases chromosomal missegregation and is one of the causes of chromosomal aneuploidy. CIN is observed in various types of cancer cells, including lung cancer, and may induce genetic instability in tumors, allowing cancer cells to acquire advantageous traits (Non-Patent Documents 13-15).

[0007] CIN is also known to affect tumor immunity. Specifically, micronuclei formed from chromosomal missegregation burst in the cytoplasm, releasing dsDNA and activating the cGAS-STING pathway (Non-Patent Documents 1 and 17). The cGAS-STING pathway was originally identified as a mechanism for protecting the host from viral infection by activating interferon (IFN) signaling (Non-Patent Document 18). In cancer cells, activation of the cGAS-STING pathway by CIN can lead to activation of interferon signaling, potentially resulting in cell death.

[0008] On the other hand, activation of the cGAS-STING pathway by CIN may cause chronic inflammation in the tumor locale, resulting in the metastasis of cancer cells (Non-Patent Documents 16, 19). In other words, activation of the cGAS-STING pathway may promote metastasis by activating the NF-κB pathway and inducing epithelial-mesenchymal transition (Non-Patent Documents 16, 19).

[0009] The impact of CIN on the antitumor effect of EGFR-TKI treatment in EGFR mutation-positive NSCLC remains unknown. In this study, we performed genomic and transcriptome analyses using tumor tissue to explore the impact of CIN on the tumor microenvironment and the outcome of EGFR-TKI treatment in patients with EGFR mutation-positive NSCLC. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Mitsudomi T, Morita S, Yatabe Y, Negoro S, Okamoto I, Tsurutani J, et al. Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harboring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomized phase 3 trial. Lancet Oncol 2010;11:121-8. [Non-patent document 2] Ramalingam SS, Vansteenkiste J, Planchard D, Cho BC, Gray JE, Ohe Y, et al. Overall survival with osimertinib in untreated, EGFR-mutated advanced NSCLC. N Engl J Med 2020;382:41-50. [Non-patent document 3] Nakagawa K, Garon EB, Seto T, Nishio M, Ponce Aix SP, Paz-Ares L, et al. Ramucirumab plus erlotinib in patients with untreated, EGFR-mutated, advanced non-small-cell lung cancer (RELAY): a randomized, double-blind, placebo-controlled, phase 3 trial. Lancet Oncol 2019;20:1655-69.

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[0011] The standard treatment for non-small cell lung cancer with EGFR-activating gene mutations is the administration of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). While tumors shrink in many cases, all patients eventually acquire resistance to EGFR-TKIs. However, the duration of efficacy of EGFR-TKI treatment varies, and in some cases, resistance develops within a short period of time. When the duration of efficacy of EGFR-TKI treatment is short, combination therapy with other therapeutic agents (e.g., cytotoxic anticancer agents or angiogenesis inhibitors) may be necessary. Therefore, selecting patients for whom the duration of efficacy of EGFR-TKIs is short is desirable. However, it is currently difficult to predict the duration of efficacy of EGFR-TKI treatment "pre-treatment." [Means for solving the problem]

[0012] The present invention has been conceived in view of the above-mentioned problems. More specifically, it relates to a method for selecting patients with non-small cell lung cancer harboring an EGFR activating mutation who have a short progression-free survival period when treated with an epidermal growth factor receptor tyrosine kinase inhibitor.

[0013] More specifically, the selection method according to the present invention comprises: 1. A method for selecting patients for non-small cell lung cancer with EGFR activating mutations, in which the duration of efficacy of an epidermal growth factor receptor tyrosine kinase inhibitor is shorter than a predetermined standard duration, comprising: calculating a CIN score for tumor tissue taken from said patient; A step of classifying the patients into a CIN-High group or a CIN-Low group by comparing the CIN score with a median CIN score; The method is characterized in that it includes a step of determining that, if the CIN score is in the CIN-High group, the effective period of the epidermal growth factor receptor tyrosine kinase inhibitor for non-small cell lung cancer having the EGFR activating mutation is shorter than the specified standard period. [Effects of the Invention]

[0014] By examining the degree of CIN in tumor tissue in advance using the selection method of the present invention, it is possible to select patients for whom EGFR-TKI will be effective for a short period of time. [Brief explanation of the drawings]

[0015] [Figure 1-A] Diagram of sampling before (pre-treatment sample 32) and after (post-treatment sample 35) EGFR-TKI treatment. [Figure 1-B] Pie chart showing acquired resistance mechanisms and their rates in EGFR-mutated NSCLC treated with EGFR-TKIs. EGFR T790M is a secondary EGFR gene resistance mutation. MET amp is MET gene amplification. HER2 amp is HER2 gene amplification. [Figure 1-C] Progression-free survival curves for EGFR-TKI treatment in patients divided into two groups, CIN-High and CIN-Low, based on median values. [Figure 1-D] Scatter plot of CIN z-scores for individual samples before treatment (post-treatment samples for EGFR T790M). Clinical characteristics are shown, including gender, smoking history, type of EGFR mutation, secondary T790M mutation in the EGFR gene, and TP53 mutation. Data represent mean and SEM values. [Figure 1-E] Figure showing the results of univariate analysis of progression-free survival after EGFR-TKI treatment. Hazard ratios for each variable are shown. Bar graphs show 95% confidence intervals. [Figure 2]CIN analysis using Gene Set Enrichment Analysis (GSEA). Comparison of CIN using tumor tissue before and after EGFR-TKI treatment. NES and p-values are shown. NES, normalized enrichment score. [Figure 3] The top 30 gene signature hallmarks associated with CIN in tumors after EGFR-TKI treatment. The CIN score and gene signature for each tumor sample are shown in a heat map. Background factors such as EGFR gene mutation type, smoking history, and genes responsible for resistance are also shown. The relationship between CIN and each gene signature was evaluated using Pearson analysis, and the r value is shown. [Figure 4] This heat map shows the relationship between various inflammatory cells in tumors after EGFR-TKI treatment and CIN, ranked in order of their correlation with CIN. Inflammatory cells were determined by deconvoluting transcriptome data. The relationship between CIN and various cells was evaluated using Pearson analysis, and the r value is shown. CIN, chromosomal instability; PMN, polymorphonuclear leukocyte; NK cell, natural killer cell; Treg, regulatory T cell; CAF, cancer-associated fibroblast; DC, dendritic cell; MDSC, myeloid-derived suppressor cell. [Figure 5]This heat map shows the gene signatures of cGAS-STING signaling in tumors after EGFR-TKI treatment, as well as the downstream TBK1-type 1 interferon (IFN) signaling and IL6-NFkB signaling, arranged in descending order of their correlation with CIN. Background factors such as EGFR gene mutation type, smoking history, and genes responsible for resistance are also shown. The relationship between CIN and each gene signature was evaluated using Pearson analysis, and the r value is shown. STING, stimulator of interferon genes; cGAS, cyclic GMP-AMP synthase [Figure 6-A] This graph shows the progression-free survival curves of patients treated with EGFR-TKI, divided into low and high cGAS mRNA groups. Each group was divided into two groups based on the median mRNA expression level. Comparisons between the two groups were performed using the log-rank test, and p values are shown. STING, Stimulator of interferon genes; cGAS, cyclic GMP-AMP synthase [Figure 6-B] This graph shows the progression-free survival curves of patients treated with EGFR-TKI, divided into low and high STING mRNA groups. Each group was divided into two groups based on the median mRNA expression level. Comparisons between the two groups were performed using the log-rank test, and p values are shown. STING, Stimulator of interferon genes; cGAS, cyclic GMP-AMP synthase DETAILED DESCRIPTION OF THE INVENTION

[0016] The following describes the method for selecting patients with non-small cell lung cancer harboring an EGFR activating mutation for whom the efficacy period of the epidermal growth factor receptor tyrosine kinase inhibitor is short, with reference to figures and examples. The following description exemplifies one embodiment of the present invention and one example, and the present invention is not limited to the following description. The following description can be modified without departing from the spirit of the present invention. Furthermore, embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included within the technical scope of the present invention. Furthermore, all of the literature described in this specification is incorporated herein by reference. In this specification, when a numerical range is described as "A to B," this means "greater than or equal to A and less than or equal to B."

[0017] <Research method> Study design, tumor specimen and clinical data acquisition This retrospective study was based on genomic and transcriptomic analyses of 67 tumor tissue samples obtained from patients with EGFR-mutated NSCLC before EGFR-TKI treatment (32 cases) and after EGFR-TKI resistance (35 cases) (Non-Patent Document 20). The dataset was collected from a previous study (Non-Patent Document 20). The samples were formalin-fixed, paraffin-embedded (FFPE) tissue sections of tumors obtained from patients with EGFR-mutated NSCLC treated at Kindai University Hospital, Kishiwada Municipal Hospital, and Izumi City General Medical Center between 2018 and 2020.

[0018] Most tumor specimens were obtained from the lungs by bronchoscopic biopsy or surgical resection. Clinical data were collected from each patient's medical record. This study was approved by the institutional review boards of Kindai University Hospital, Kishiwada Municipal Hospital, and Izumi City General Medical Center. Written informed consent was obtained from all participants after providing an opportunity to opt out.

[0019] [DNA and RNA extraction] DNA and RNA extraction was performed as previously described (Non-Patent Document 20). Briefly, FFPE specimens were subjected to histological examination, and only specimens containing sufficient tumor cells (≥50% for RNA and ≥30% for DNA) were subjected to nucleic acid extraction. DNA and RNA were purified using FormaPure (Beckman Coulter Inc.) or Allprep DNA / RNA FFPE kits (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. The extracted DNA / RNA was stored at -80°C until analysis.

[0020] [DNA panel sequencing] DNA panel sequencing was performed as previously described (Non-Patent Document 20). Library preparation was performed using the Illumina TruSight Oncology 500 Kit (TSO500, Illumina Inc.) according to the manufacturer's instructions. A DNA input of 110 ng was used for library preparation, but the maximum DNA input was used if the DNA amount was insufficient. qPCR was performed using a QuantStudio 12 K Flex Real-Time PCR System (Thermo Fisher Scientific Inc.) according to the manufacturer's instructions. To fragment DNA strands into 90-250 bp fragments, DNA from each sample was processed using a Covaris E220 ultrasonicator (Covaris, Inc., Woburn, MA, USA) according to a previous protocol (Non-Patent Document 20).

[0021] The normalized libraries were sequenced using NextSeq NextSeq500 (Illumina, Inc.). After aligning the sequence reads to the human sequence (hg19), mutation calling (single-nucleotide mutations and insertions / deletions) was performed using TSO500 analysis software (v1.3.1), and annotation was performed using Variant Effect Predictor (VEP, release_98.3).

[0022] TMB analysis and copy number variation (CNV) analysis were performed using TSO500 analysis software versions 1.3.1 and 2.1, respectively. Samples with low library concentrations (<3 ng / μL) were excluded, along with SNVs with low numbers of mutant alleles (<5). Known germline mutations from the Human Genetic Variation Database were excluded.

[0023] [RNA panel sequencing] RNA panel sequencing was performed as previously described (20). Library preparation was performed using a TSO500 according to the manufacturer's instructions. 170 ng of RNA input was used for library preparation, but the maximum RNA input was used if the amount of RNA was insufficient. Normalized libraries were sequenced using NextSeq, NextSeq500, or NextSeq550 (Illumina, Inc.) to a length of 100 bp from both ends (2 × 100 bp). Sequence reads were aligned to the human sequence (hg19). Fusion and splice variant identification was performed using TST170 analysis software (v2.0.0). Samples with low library concentrations (<3 ng / μL) were excluded from the dataset.

[0024] [Transcriptional profiling] RNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tumors containing more than 50% cancer cells. cDNA libraries for RNA-seq were prepared for directional sequencing using the following kits and reagents: QIAseq FastSelect (Qiagen Inc., Venlo, Netherlands), NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, Inc., Ipswich, MA, USA), and NEBNext Multiplex Oligos for Illumina (New England Biolabs, Inc.). Library preparation was performed using 20 ng of RNA input; however, the maximum RNA input was used if insufficient RNA was available.

[0025] The amount of prepared libraries was measured using a 4200 TapeStation. Prepared libraries were pooled into one tube and sequenced at 75 bp from both ends (2 × 75 bp) on a single flow cell using a NovaSeq6000 (Illumina, Inc.) at 1.5 nM. Sequencing reads were aligned to the human genome reference (GRCh38) using STAR software (2.5.3a). The transcripts per million (TPM) and expected number of each gene were estimated using RNA-seq and expectation maximization (RSEM) software (1.3.0).

[0026] Non-cancer cell samples and samples with low total expected counts were excluded and normalized using the trichotomous mean of the M-value normalization method. For data analysis, samples with less than 50% within a species or less than 50M total reads were excluded.

[0027] [Molecular pathway analysis] GSEA was performed using GSEA version 4.2.2 (Non-Patent Document 21) with default settings. Hallmarks h.all.v2023.2.symbol.gmt was selected as the gene set database. The gene set for evaluating STING activation was used according to a previous report (Non-Patent Document 22). Sorting was performed according to normalized enrichment score (NES), and categories were selected in detail.

[0028] [RNA-Seq data analysis and CIN score calculation method] RNA-Seq data were analyzed using the following web tools: CIBERSORTx ( https: / / cibersortx.stanford.edu / ), GenePattern for ssGSEA ( https: / / www.genepattern.org / #gsc.tab=0 ), and Carcinoma EcoTyper ( https: / / ecotyper.stanford.edu / carcinoma / ).

[0029] Next, ssGSEA was performed using the gene set of h.all.v2023.2.symbol.gmt as the gene set database. The CIN score was calculated using the average normalized gene expression data of the 70 genes that make up the signature, as previously reported (Non-Patent Document 23). Note that the "CIN score" may also be referred to as the "degree of CIN."

[0030] [Tumor microenvironment (TME) analysis] The Carcinoma EcoTyper web tool (24) was used to determine the infiltration of multiple immune cells to estimate the tumor microenvironment of EGFR-mutant NSCLC. Data were uploaded to the CIBERSORTx web portal, and the algorithm was run with 1000 permutations using the LM22 signature.

[0031] [Statistical analysis] Statistical analysis was performed using SPSS software (version 22.0; IBM). Progression-free survival (PFS) was defined as the time from the start of EGFR-TKI therapy to tumor progression or death from any cause. Kaplan-Meier curves for PFS or overall survival were constructed and used to calculate the median and 95% confidence interval (CI) for each treatment group. In this specification, progression-free survival (PFS) is also referred to as the "response period."

[0032] Two-sided p-values were determined using the log-rank test, and hazard ratios (HRs) (and 95% CIs) were estimated using the Cox proportional hazards model. Pearson's correlation coefficients (r) were calculated to examine the relationship between CIN score and other genomic characteristics of the tumor. Genomic expression differences were analyzed based on these differences.

[0033] <result> [The impact of chromosomal instability on the outcome of EGFR-mutated NSCLC treated with EGFR-TKIs] In patients with EGFR-mutated NSCLC who responded to EGFR-TKI treatment, a total of 67 tumor tissue samples were collected before EGFR-TKI treatment and after the acquisition of EGFR-TKI resistance (32 samples before treatment, 35 samples after treatment; Figure 1-A). The characteristics of the patients from whom the pre-treatment samples were collected are shown in Table 1.

[0034] [Table 1]

[0035] Although post-treatment samples had acquired secondary mutations that cause EGFR-TKI resistance, including EGFR T790M, HER2 amplification, and MET amplification, nearly half of the samples did not show genetic abnormalities that cause EGFR-TKI resistance (Figure 1-B).

[0036] Next, patients were divided into CIN-High and CIN-Low groups based on the median CIN score of tumor tissue before treatment, and the relationship between CIN and EGFR-TKI treatment outcomes was evaluated by comparing progression-free survival (PFS) between the two groups. The maximum CIN score in this study was 26,598, the minimum was 16,130, and the median CIN score was 21,292. Therefore, if the calculated CIN score was greater than the median CIN score, the patient was classified as the CIN-High group, and if it was less than the median CIN score, the patient was classified as the CIN-Low group.

[0037] The results are shown in Figure 1-C. Referring to Figure 1-C, the vertical axis represents progression-free survival (unitless), and the horizontal axis represents the observation period (years). The observation period begins on the day EFGR-TKI treatment was initiated. The CIN-High group is represented by a dashed line, and the CIN-Low group by a solid line. PFS was worse in the CIN-High group (16 patients) than in the CIN-Low group (16 patients) (HR 3.274; p=0.005; median PFS 16.7 vs. 11.0 months, respectively).

[0038] In large-scale clinical trials of Tagrisso (registered trademark), a known oral treatment and standard treatment for non-small cell lung cancer, the 2-year progression-free survival rate was reported to be 40-45%. Therefore, it is reasonable to consider that progression-free survival (effectiveness period) of 2 years or less after the initiation of epidermal growth factor receptor tyrosine kinase inhibitor treatment is low and that the effective period of the epidermal growth factor receptor tyrosine kinase inhibitor is short. A low progression-free survival rate is considered to be lower than 40%. This is called the predetermined criterion. In other words, a progression-free survival rate lower than 40% can be considered low.

[0039] On the other hand, Figure 1-C shows that if the standard period for determining short progression-free survival (called the "predetermined standard period") is 2 years, the progression-free survival rate was approximately 10% in the CIN-High group and approximately 50% in the CIN-Low group. Therefore, if the CIN score of the target patient belongs to the CIN-High group, the progression-free survival rate is low, and it is worth considering combining other drugs with an epidermal growth factor receptor tyrosine kinase inhibitor.

[0040] Based on the above, the short duration of efficacy of epidermal growth factor receptor tyrosine kinase inhibitors is defined as two years or less, at which point the progression-free survival rate falls below 40%. The CIN score of tumor specimens obtained from the lungs by bronchoscopic biopsy or surgical resection is calculated, and patients with a CIN score higher than the median score (CIN-High group) can be classified (selected) as patients with a short duration of efficacy of epidermal growth factor receptor tyrosine kinase inhibitors. These patients can also be said to have a low two-year progression-free survival rate.

[0041] In this way, patients who have been identified as having a low 2-year progression-free survival rate can be used as an indicator that they should be considered for combined treatment with other therapeutic drugs in addition to treatment with epidermal growth factor receptor tyrosine kinase inhibitors.

[0042] Next, we investigated the relationship between the CIN score and several factors that may affect PFS with EGFR-TKI treatment (gender, smoking history, type of EGFR gene mutation, EGFR secondary T790M mutation, and TP53 gene mutation) (Figure 1-D).CIN scores were higher in tumors with TP53 gene mutations (*p<0.05).

[0043] We investigated the relationship between PFS in EGFR-TKI therapy and factors such as clinical background and genetic mutations (CIN, type of EGFR mutation, PS performance status, type of EGFR-TKI, gender, age, smoking history, and TP53 gene mutation). Multivariate analysis revealed that only CIN was independently correlated with PFS (Figure 1-E).

[0044] We also evaluated the changes in CIN before and after EGFR-TKI treatment using gene signature analysis. Increased CIN was observed in tumor tissue after treatment compared with before treatment (Figure 2).

[0045] In Figure 2, the vertical axis represents the enrichment score, and the horizontal axis represents the gene ranking. Before treatment, the further to the left you go, and after treatment, the further to the right you go, the greater the change in gene expression. Many of the relevant genes clearly change after treatment, and have high enrichment scores. The NES value is also negative, indicating that the amount of mutated genes increased after treatment, and that CIN was exacerbated.

[0046] [Enhanced inflammation-related signals in CIN-High tumors] Next, we evaluated the impact of CIN on tumor properties, particularly those related to progression and drug resistance, using gene signature analysis. In EGFR-mutant NSCLC tumors that had acquired resistance to EGFR-TKIs, we found a correlation between CIN and several gene signatures associated with cancer progression or drug resistance (Figure 3). Specifically, activation of PI3K-AKT-mTOR signaling, which suggests cancer cell proliferation, was associated with CIN. Furthermore, several inflammation-related signals (inflammatory response, IFN Alpha responses, and IL6 / JAK / STAT3 signaling) were found to be associated with CIN (each signaling is marked with an asterisk (★)).

[0047] [Chromosomal instability-dependent infiltration of tumor-promoting inflammatory cells in EGFR-mutated NSCLC] The increased inflammatory signaling associated with CIN was a mixture of "anti-tumor" inflammatory signals such as IFNα and "tumor-promoting" inflammatory signals such as IL6. Therefore, we deconvolved tumor transcriptome data to estimate the various inflammatory cell infiltrates in the tumor microenvironment.

[0048] Although CD8+ T cell infiltration was observed in CIN-High tumors, these cells were presumed to be exhausted and their function was impaired (Figure 4). Macrophages (tumor-associated macrophages), both M1-like and M2-like, infiltrated relatively more frequently in CIN-High tumors. Tumor-promoting Treg (regulatory T cells) and fibroblast cells were also presumed to infiltrate more frequently in CIN-High tumors (Figure 4, each cell type is marked with an inverted triangle (▼)).

[0049] Furthermore, endothelial cells and epithelial cells, suggesting enhanced angiogenesis, were relatively more abundant in CIN-High tumors (Figure 4). In contrast, CIN-Low tumors showed relatively few infiltrating tumor-promoting inflammatory cells, with the exception of M2-like macrophages (tumor-associated macrophages) (Figure 4). However, antitumor NK cells and B cells, suggestive of tertiary lymphoid structures, were relatively more abundant in CIN-Low tumors (Figure 4).

[0050] Next, we evaluated the relationship between CIN and intracellular signaling pathways in tumors after EGFR-TKI treatment using gene signature analysis. Consistent with previous reports, we observed increased cGAS mRNA expression and activation of the STING pathway in CIN-High tumors (Figure 5). Furthermore, activation of TBK1-type 1 IFN signaling and NFkB-IL6 signaling downstream of STING was also suggested, suggesting that these signaling pathways contribute to inflammation in the tumor microenvironment (Figure 5). Expression of cGAS mRNA and STING mRNA tended to increase or decrease, respectively, depending on the severity of CIN (Figure 5). Specifically, the correlation coefficient r between cGAS mRNA and CIN was 0.782, and the correlation coefficient r between STING mRNA and CIN was -0.666.

[0051] Next, we investigated the relationship between these expression levels and progression-free survival (PFS) after EGFR-TKI treatment. Specifically, we divided patients into cGAS-Low and cGAS-High groups based on the median cGAS mRNA expression level using pretreatment tumor tissue, and compared PFS between the two groups. The results showed that PFS tended to be shorter in the cGAS-High group compared with the cGAS-Low group (HR 2.616; p = 0.018; Figure 6-A).

[0052] On the other hand, for STING mRNA, the STING-High group tended to have shorter PFS than the STING-Low group (HR 0.542; p = 0.088; Figure 6-B). In Figures 6-A and 6-B, the vertical axis represents progression-free survival (PFS), and the horizontal axis represents the observation period (years). The observation period begins on the day EGFR-TKI treatment was initiated.

[0053] These results demonstrate that CIN negatively impacts the outcome of EGFR-TKI treatment in EGFR-mutated lung cancer. It is suggested that CIN induces tumor cells to activate their own cGAS-STING signaling, inducing an inflammatory response. This may lead to a deterioration of the tumor immune environment, accompanied by exhaustion of CD8+ cells and infiltration of tumor-promoting monocytes, Tregs (regulatory T cells), and fibroblasts in the tumor microenvironment. [Industrial Applicability]

[0054] The present invention can be suitably used as a guideline for treating non-small cell lung cancer with EGFR activating mutations.

Claims

1. 1. A method for selecting patients for non-small cell lung cancer having an EGFR activating mutation, in which the duration of efficacy of an epidermal growth factor receptor tyrosine kinase inhibitor is shorter than a predetermined standard duration, comprising: calculating a CIN score for tumor tissue taken from said patient; A step of classifying the patients into a CIN-High group or a CIN-Low group by comparing the CIN score with a median CIN score; determining that the effective period of the epidermal growth factor receptor tyrosine kinase inhibitor for non-small cell lung cancer having an EGFR activating mutation is shorter than the predetermined standard period if the CIN score is in the CIN-High group.

2. the median CIN score is 21292; 2. The method of claim 1, wherein the predetermined reference period is two years after the start of treatment with the epidermal growth factor receptor tyrosine kinase inhibitor.

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