A method for detecting NSCLC using H3K27Me3 and ctDNA as markers.

Abstract

The present invention relates to a method for analyzing cancer, comprising detecting or measuring the level of trimethylation of histone H3 lysine 27 (H3K27Me3) in cell-free nucleosomes in a body fluid sample obtained from a subject, as well as related uses and kits for using the method.

Description

Technical Field

[0001] (Field of the Invention) The present invention relates to a method for testing body fluids for monitoring and detecting cancer, particularly lung cancer, using biomarkers. The present invention is particularly useful for monitoring lung cancer patients both during and after treatment.

Background Art

[0002] (Background of the Invention) Cancer is a common disease with a high mortality rate. The ecology of this disease is understood to involve progression from a pre-cancer (initiation) state to stage I, II, III, and ultimately stage IV cancer. For most cancer diseases, the mortality rate depends on whether the disease is detected at an early, localized stage where effective treatment options are available, or at a later stage where the disease may have spread within or beyond the affected organ and treatment is more difficult.

[0003] Symptoms of late-stage cancer are diverse and include bleeding due to coughing and unexplained weight loss, as well as many other possible symptoms depending on the type of cancer. However, many cancers diagnosed based on such symptoms are already in a late stage and are difficult to treat. Most cancers are asymptomatic in the early stage or present non-specific symptoms that are not helpful for diagnosis. Therefore, cancer should ideally be detected early using cancer tests.

[0004] The cancer with the highest mortality rate in developed countries is lung cancer. The 5-year survival rate for lung cancer is over 50% for cases detected when the disease is still confined within the lungs, but only 5% when the disease has spread to other organs. Unfortunately, most lung cancer cases are diagnosed when they have already metastasized (57%), and only 16% are diagnosed at an early stage.

[0005] Despite recent advances, only a small number of blood tests are routinely used for cancer screening. There is a need to develop more non-invasive blood tests for individual cancers for general cancer diagnosis, either to include or exclude cancer as a potential diagnosis in symptomatic patients or as an adjunct to other cancer detection methods.

[0006] Similarly, when a patient undergoes treatment, such as surgery or radiation therapy aimed at a cure, it is necessary to monitor the patient for a period of time for the possibility of recurrence. Currently, this may take the form of regular CT scans, PET scans, or X-ray examinations. These scans are costly, and the risk of radiation damage increases over time. PET scans and CT scans using imaging dyes are also invasive, and patients may have adverse reactions to the radioactive imaging tracers or dyes used in these scans. Such scans are generally performed only at least every three months to reduce radiation exposure and to allow radiologists to track the disease over a period of time. A simple blood test that would allow physicians to monitor a patient's response to treatment, whether past or ongoing, would be extremely useful. This would allow for more frequent monitoring and would be cheaper and faster than performing scans. Using such a blood test, information could be provided, for example, whether further treatment is needed if the cancer has recurred, or whether the treatment plan should be changed if the patient is not responding to current treatment.

[0007] This invention solves the aforementioned needs.

[0008] (Description of the invention) According to a first aspect of the present invention, a method for analyzing a target cancer, (i) To detect or measure the level of trimethylation of histone H3 at lysine 27 (H3K27Me3) in cell-free nucleosomes in body fluid samples obtained from subjects; and (ii) Analyze circulating tumor DNA (ctDNA) obtained from the subjects for tumor-associated mutations. A method is provided that includes:

[0009] According to one aspect of the present invention, a method for analyzing a target cancer, To detect or measure the level of trimethylation of lysine 27 of histone H3 in cell-free nucleosomes (H3K27Me3) in body fluid samples obtained from subjects; and Analyzing cancer using levels detected or measured in bodily fluid samples. A method is provided that includes:

[0010] In one embodiment, the presence of ctDNA combined with a higher level of H3K27Me3 compared to a control indicates the presence and / or progression of cancer, that the subject requires further investigation such as a lung biopsy, and / or that the subject requires treatment or a change in treatment; or the absence of ctDNA combined with a higher level of H3K27Me3 compared to a control indicates the presence and / or progression of cancer, that the subject requires further investigation such as a lung biopsy, and / or that the subject requires treatment or a change in treatment.

[0011] In one embodiment, the cancer is lung cancer.

[0012] In one embodiment, the lung cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC).

[0013] In one embodiment, lung cancer is NSCLC.

[0014] In one embodiment, NSCLC is adenocarcinoma, squamous cell carcinoma, large cell carcinoma, adenosquamous carcinoma, or sarcomatoid carcinoma.

[0015] In one embodiment, NSCLC is an adenocarcinoma.

[0016] In one embodiment, the method of the present invention monitors cancer progression, monitors treatment, assesses the need for treatment, assesses the need for treatment modification, assesses the need for investigation in a subject who may have or is suspected of having cancer, and / or aids in the diagnosis of a subject who may have or is suspected of having cancer.

[0017] In one embodiment, the method is for managing a treatment plan in a subject who is suspected of having cancer, has cancer, or has previously received a cancer diagnosis.

[0018] In one embodiment, the method is for assessing recurrence after cancer treatment.

[0019] In one embodiment, the subject has minimal residual disease (MRD).

[0020] In one embodiment, the method is for assessing the success of cancer treatment aimed at cure.

[0021] In one embodiment, the method is for assessing the treatment of a subject or the need for treatment modification of a subject.

[0022] In one embodiment of the method of the present invention, the level of H3K27Me3 is compared to a previous level obtained from a control or a subject.

[0023] [[ID=**28**]]In one embodiment of the method of the present invention, monitoring changes in cancer stage, where an increase compared to an initial sample or control indicates cancer progression from an early stage to a late stage of the disease, is included.

[0024] In one embodiment, the subject is investigated later for cancer, and the investigation includes pulmonary function tests (PFT), imaging, biopsy, and / or surgery.

[0025] In one embodiment, the investigation is a biopsy.

[0026] Note: There was a possible formatting issue in the original text for line break preservation. I've tried to maintain the line breaks as closely as possible while ensuring the translation is accurate. If there are any further formatting or accuracy concerns, please let me know. Also, the text in line 28 seems to be a bit jumbled in terms of grammar and flow in the original, but I've translated it as best as I could while keeping the overall meaning intact.In one embodiment, the imaging is an x-ray, a chest computed tomography (CT) scan, or a positron emission tomography (PET) scan.

[0027] In one embodiment, the level of H3K27Me3 indicating the presence and / or progression of cancer is about 22.5 ng / ml or more.

[0028] In one embodiment, the level of H3K27Me3 is measured as one of a measurement panel including DNA analysis.

[0029] In one embodiment, the cancer is stage I, II, IIIA and B, or IV.

[0030] In one embodiment, the cancer is described as TX, T0, Tis, T1 (including T1mi, T1a, T1b, T1c), T2 (including T2a, T2b), T3, or T4; the cancer is described as NX, N0, N1, N2, or N3, and / or the cancer is described as M0 or M1 (including M1a, M1b, M1c).

[0031] In a second aspect of the present invention, a method for analyzing cancer in a subject, (i) detecting or measuring the level of trimethylation of lysine 27 of histone H3 of cell-free nucleosomes in a body fluid sample obtained from the subject; and (ii) analyzing the ctDNA obtained from the subject for tumor-related somatic mutations such as tumor-related mutations : is provided.

[0032] In a third aspect of the present invention, the use of H3K27Me3 as a biomarker in a body fluid sample for monitoring the progression of cancer, monitoring treatment, assessing the need for treatment, assessing the need for a change in treatment, assessing the need for an investigation in a subject having or suspected of having cancer, or assisting in the diagnosis of a subject having or suspected of having cancer is provided.

[0033] In a fourth aspect of the present invention, a kit is provided comprising H3K27Me3 and a reagent for detecting one or more biomarkers, optionally including ctDNA. [Overview of the project]

[0034] (Summary of the invention) This invention relates to a general-purpose biomarker for quantifying minimal residual disease (MRD) in circulating H3K27 nucleosome-plasma samples for monitoring lung cancer patients undergoing treatment.

[0035] Treatment options for lung cancer include radiotherapy and / or combination therapy approaches including chemotherapy, immunotherapy, and targeted therapies based on tumor molecular profiles. Following first-line treatment aimed at cure, clinical surveillance is accompanied by serial CT imaging. However, such surveillance is often inconclusive, as it can only detect macroscopic disease recurrence. Next-generation sequencing (NGS) is used to aid in identifying and monitoring treatment plans. Nucleosomes, complexes of DNA and histone proteins, are released into the bloodstream during cell death. Trimethylation of lysine 27 of histone H3 (H3K27Me3), catalyzed by enhancer of zest homolog 2 (EZH2), is a key epigenetic process in tumorigenesis. We investigated whether H3K27Me3-nucleosome concentration could serve as a biomarker for minimal residual disease (MRD).

[0036] High levels of H3K27Me3 may enable physicians to detect MRD in lung cancer patients after treatment aimed at cure. This can be achieved by monitoring patient examinations at defined treatment-recovery intervals in parallel with imaging to incorporate analysis of the changing molecular landscape during treatment. In these circumstances, H3K27Me3-nucleosome quantification, which completes the molecular search for cfDNA, is particularly promising in advanced NSCLC where re-bioplasty is impractical, expensive, and potentially harmful. H3K27Me3-nucleosome quantification may also be useful in patient identification for specific therapies, such as EZH2 inhibitors.

[0037] This invention also relates to the circulating H3K27 methylated nucleosome concentration in lung cancer, which improves the contribution of ctDNA molecular profiling results at the time of diagnosis.

[0038] In patients with advanced lung adenocarcinoma, certain subsets can be cured by combination regimens including radiotherapy and / or chemotherapy, immunotherapy, or targeted therapy based on somatic molecular profiling. As described above, nucleosomes are fundamental elements of chromatin, consisting of 147 bp of DNA wrapped around a histone octamer. Cell-free DNA (cfDNA) and nucleosomes are released into the bloodstream upon cell death. In addition to somatic mutations in genes, epigenetic modifications are known to play a crucial role in tumorigenesis in various cancers. Lysine 27 trimethylated histone H3 (H3K27Me3) is a well-known transcriptional repression marker and has been shown to be involved in tumorigenesis, cell cycle progression, and regulation of proliferation abnormalities. However, circulating nucleosome concentration as a biomarker of the contribution of circulating tumor DNA (ctDNA) molecular profiling in patient management at the time of diagnosis has not been investigated to date.

[0039] The absence of detection of high levels of H3K27Me3 and somatic mutations strongly supports the presence of corresponding non-mutant ctDNA in plasma. This significantly improves the confidence in negative molecular outcomes of cfDNA in lung cancer. This may allow for the application of personalized treatment regimens through rapid medical decisions and potentially reduce invasive tissue re-biopsies. [Brief explanation of the drawing]

[0040] (Brief explanation of the drawing) [Figure 1] Figure 1 shows an example of current patient pathways in the advanced stage of NSCLC, either post-treatment or during treatment. These pathways are based on the presence or absence of somatic mutations in circulating tumor DNA (ctDNA). The number of samples (n=) is shown for the entire cohort (W; n=304). Percentages represent the portion of the entire cohort involved. [Figure 2] Figure 2 shows an example of a patient pathway in the advanced stage of NSCLC after or during treatment according to the present invention. This pathway is based on the presence or absence of somatic mutations in circulating tumor DNA (ctDNA) and H3K27Me3-nucleosome levels below or above 22.5 ng / mL. The number of samples (n=) is shown for the entire cohort (W; n=304). Percentages represent the portion of the entire cohort involved. [Figure 3] Figure 3 shows an example of a patient pathway at the time of diagnosis according to the present invention. This pathway is based on the presence or absence of somatic mutations in circulating tumor DNA (ctDNA) and H3K27Me3-nucleosome levels below or above 22.5 ng / mL. The number of samples (n=) is shown for the entire cohort (W; n=318). The percentage represents the portion of the entire cohort involved. [Figure 4]Figure 4 shows the quantification of circulating H3K27Me3- nucleosomes in NSCLC and healthy control samples in a global cohort at the time of diagnosis. (A) Box plot analysis of circulating H3K27Me3- nucleosome levels in NSCLC samples compared with healthy control samples. (B) Box plot analysis of circulating H3K27Me3- nucleosome levels in NSCLC samples (ctDNA+) when mutant cfDNA is detected in the blood, compared with NSCLC- derived samples without detected mutations (ctDNA-). The boxes represent the 25th to 75th percentiles, including the median. The whiskers represent the 2.5th to 97.5th percentiles. *** and **** represent p-values ​​<0.001 and <0.0001, respectively, calculated by the Mann-Whitney test (group of interest (k)=2) and the Kruskal-Wallis test (k>2). (C) Receiver operational characteristic (ROC) curve analysis of circulating H3K27Me3- nucleosomes. The dotted line represents the theoretical random probability. [Figure 5] Figure 5 shows the quantification of circulating H3K27Me3- nucleosomes in NSCLC and healthy control samples in a global cohort of advanced-stage disease. (A) Box plot analysis of circulating H3K27Me3- nucleosome levels in NSCLC samples compared with healthy control samples. (B) Box plot analysis of circulating H3K27Me3- nucleosome levels in NSCLC samples (ctDNA+) when mutant cfDNA is detected in the blood, compared with NSCLC- derived samples without detected mutations (ctDNA-). The boxes represent the 25th to 75th percentiles, including the median. The whiskers represent the 2.5th to 97.5th percentiles. *** and **** represent p-values ​​<0.001 and <0.0001, respectively, calculated by the Mann-Whitney test (group of interest (k)=2) and the Kruskal-Wallis test (k>2). (C) Receiver operational characteristic (ROC) curve analysis of circulating H3K27Me3- nucleosomes. The dotted line represents the theoretical random probability. (D) Group size of the target group, area under the curve (AUC), and corresponding p-value. [Modes for carrying out the invention]

[0041] (Detailed description of the invention) Despite recent advances, the diagnosis and treatment of cancer, particularly lung cancer, remain challenging. Currently, there are no circulating protein biomarkers available for routine use in the management of lung cancer, for example, in monitoring cancer recurrence or response to treatment. Similarly, there are currently no circulating protein biomarkers available for routine use to aid in the diagnosis of lung cancer. The availability of immunoassays that can be used as liquid biopsies has the potential to revolutionize the management of cancer, particularly lung cancer. This invention aims to provide such a solution. This invention provides an assay that is cost-effective, easy to use, and provides rapid results.

[0042] For a definitive diagnosis of cancer, biopsy-based pathology is recommended. However, in clinical practice, repeating such tissue biopsies in the advanced stages of the disease is often difficult. Furthermore, this invasive sampling procedure can cause unnecessary health complications, especially in patients with comorbidities, and may further delay the administration of treatment, making it unsuitable for patients in the advanced stages of the disease. Given these limitations, there is an urgent need for less invasive and more convenient techniques for monitoring treatment with assays. This invention attempts to address this need by eliminating or reducing the number of biopsies required throughout the course of the disease. The clinical potential of this invention includes testing, monitoring, and detecting residual lesions in response to treatment.

[0043] The embodiments of the present invention increase the identification rate of disease progression from approximately 43% to approximately 58% compared to conventional methods, thereby increasing the identification rate of disease progression by more than 15%.

[0044] According to one aspect of the present invention, a method for analyzing a target cancer, (i) To detect or measure the level of trimethylation of histone H3 at lysine 27 (H3K27Me3) in cell-free nucleosomes in body fluid samples obtained from subjects; and (ii) Analyze circulating tumor DNA (ctDNA) obtained from the subjects for tumor-associated mutations. A method is provided that includes:

[0045] The levels of H3K27Me3 detected or measured in a bodily fluid sample and the analyzed ctDNA are used to analyze cancer. Therefore, in one embodiment, the method includes (iii) a step of analyzing cancer using the levels of H3K27Me3 and the analysis of ctDNA.

[0046] According to one aspect of the present invention, a method for analyzing a target cancer, To detect or measure the level of trimethylation of lysine 27 of histone H3 in cell-free nucleosomes (H3K27Me3) in body fluid samples obtained from subjects; and Analyzing cancer using levels detected or measured in bodily fluid samples. A method is provided that includes:

[0047] The present invention is particularly useful in lung cancer, and in non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC). In certain embodiments, lung cancer is NSCLC. Examples of NSCLC include adenocarcinoma, squamous cell carcinoma, large cell carcinoma, adenosquamous carcinoma, or sarcomatoid carcinoma. The present invention is particularly useful for analyzing adenocarcinoma.

[0048] The present inventors have provided methods for analyzing target cancers, particularly for monitoring cancer progression, monitoring treatment, assessing the need for treatment, assessing the need for changes in treatment, assessing the need for investigation in subjects who have or are suspected of having cancer, and / or for assisting in the diagnosis of subjects who have or are suspected of having cancer, for managing treatment plans in subjects who are suspected of having cancer, have cancer, or have been previously diagnosed with cancer, for assessing cancer recurrence after treatment, for assessing the success of cancer treatment aimed at cure, and for assessing the need for treatment or changes in treatment of subjects.

[0049] Minimal residual disease (MRD) is the term given to a small number of leukemia cells (bone marrow-derived cancer cells) that remain in the body during treatment or after treatment when the patient is in remission (i.e., the patient has no symptoms or signs of the disease). However, MRD is a major cause of relapse in cancer and leukemia. Therefore, the method of the present invention is useful for monitoring patients suspected of relapse, particularly patients in remission from cancer.

[0050] The selected biomarkers include the histone post-translational modification (PTM): H3K27Me3 (i.e., trimethylated histone H3 at the lysine residue at position 27). Since the biomarkers described herein refer to histone PTMs present in nucleosomes, these biomarkers may also be called “nucleosome biomarkers.” Therefore, according to one aspect of the present invention, the use of the nucleosome biomarker H3K27Me3 in a body fluid sample for diagnosing and / or monitoring cancer, in particular lung cancer, is provided. In a specific embodiment, the presence of this biomarker in, for example, blood, plasma, or serum is an indicator of lung cancer, but other fluids or tissues may be assayed.

[0051] Nucleosomes are the basic units of chromatin structure and consist of protein complexes of eight highly conserved core histones (each composed of a pair of histones H2A, H2B, H3, and H4). Approximately 146 base pairs of DNA are wrapped around this complex. Another histone, H1 or H5, acts as a linker and participates in chromatin condensation. The DNA wraps around a sequence of nucleosomes in a structure often described as "beads on a string," which forms the basic structure of open chromatin, or euchromatin. In compressed chromatin, or heterochromatin, these strings form coils and supercoils, resulting in a closed, complex structure (Herranz and Esteller (2007)).

[0052] In one embodiment, the nucleosome is a cell-free nucleosome. When the term “nucleosome” is detected in a bodily fluid sample, it may refer to a “cell-free nucleosome.” Throughout this document, the term “cell-free nucleosome” will be understood to include any cell-free chromatin fragment containing one or more nucleosomes.

[0053] In one embodiment, the biomarker comprises a histone isoform. Many histone isoforms are known in the art. Nucleotide sequences of numerous histone isoforms are publicly available, for example, in the National Human Genome Research Institute (NHGRI) histone database (Marino-Ramirez et al., "The Histone Database: an integrated resource for histones and histone fold-containing proteins," Database Vol. 2011), the GenBank (NIH gene sequence) database, the EMBL nucleotide sequence database, and the DNA Databank of Japan (DDBJ). In a preferred embodiment, the biomarker comprises a histone isoform of histone H3, for example, a histone isoform selected from H3.1, H3.2, and H3t.

[0054] In one embodiment, an assay cutoff level is used, and a patient is considered positive if the assay results are above (or below, if applicable) the assay cutoff level for all or a minimum number of assays (e.g., one out of two, two out of two, two out of three, etc.). In one embodiment of the present invention, a decision tree model or algorithm is used to analyze the results. For example, this assay cutoff level may be about 10 ng / ml or higher, about 20 ng / ml or higher, or about 30 ng / ml or higher. In one embodiment, this assay cutoff level is about 22.5 ng / ml or higher. In yet another embodiment, this assay cutoff level is greater than about 22.5 ng / ml.

[0055] A significant proportion of tumor DNA circulates after necrosis and / or apoptosis of primary tumor cells, as well as after the death of circulating tumor cells. This is often called circulating tumor DNA (ctDNA). In cancer patients, this ctDNA forms part of all circulating or cell-free DNA, known as cfDNA. With advances in the sensitivity of mutation detection tools, it has become possible to detect some somatic mutations in small plasma samples, which can be used as an aid in cancer patient management.

[0056] Several techniques, including PCR assays and NGS, are currently available for detecting plasma DNA. Notably, the sensitivity limits of all methods used for detection in plasma can lead to false-negative results, especially with low cfDNA input. Clinical NGS assays, in particular, offer significant opportunities for precision medicine, as they can screen for pathogenic mutations at thousands of genomic loci. Liquid biopsy is particularly promising as it provides a minimally invasive approach to investigating ctDNA. However, like many groundbreaking NGS applications, liquid biopsy requires the detection of low mutant allele frequencies, and therefore, NGS assays can be prone to false negatives. False negatives pose a safety risk and can lead to incorrect treatment decisions or delays in reconfirming results.

[0057] The use of H3K27Me3 according to the present invention offers the advantage of reducing such false negatives. The benefits of the present invention are shown in Figures 1 and 2. Figure 1 shows the current patient pathway in the advanced stage of NSCLC post- or during treatment. In this pathway in a cohort of 304 patients, the absence of somatic mutations in cfDNA in 173 patients (i.e., 56.9% of patients) suggests a therapeutic response to treatment. The presence of somatic mutations in ctDNA in 131 patients (i.e., 43.1%) suggests disease progression. The present invention enables an improvement in the workflow as shown in Figure 2. When the levels of H3K27Me3-nucleosomes are also taken into account, the identification of disease progression increases from 43.1% to 58.2% of patients. That is, the present invention can increase the identification of disease progression in this patient cohort. This improvement in the identification of disease progression in liquid biopsy will yield significant benefits, for example, enabling earlier intervention in treatment management.

[0058] Therefore, in one advantageous embodiment, measuring or detecting H3K27Me3 according to the present invention is used in combination with ctDNA measurement. In particular, the present invention is used in combination with molecular profiling of ctDNA, especially mutation detection assays. Such mutations may be somatic mutations. ctDNA can be analyzed by any convenient method such as PCR or NGS.

[0059] Examples of ctDNA analysis techniques that can be used in this invention are described below: [Table 1]

[0060] Examples of tumor-related genes that can be evaluated as part of the present invention include one or more genes selected from the list consisting of AKT1, ARID1A, BAP1, ARID2, CDK4, CDKN2A, EGFR, FBXW7, FOXL2, IDH2, JAK2, KIT1, APC, ARID2, BRAF, CARD11, CDKN2A, CCND1, DNMT3A, ERBB2, ERBB4, FBXW7, FGFR3, JAK1, KRAS, NRAS, MCL1, MET, NOTCH1, PI3KCA, POLE, PTEN, SF3B1, SMAD4, TP53, ALK, NRG1, EBLN2, LRP1B, EGFR, ELOB, LTBP4, MCM3AP, RET, ESR1, BCL2, GNAQ, RB1, and PDGFR.

[0061] Embodiments of the present invention utilize a combination of H3K27Me3 and ctDNA to improve the accuracy or certainty of lung cancer diagnosis in clinical settings and enhance the ability to detect lung cancer. For example, as shown in Figure 3, of the data included herein concerning 27.7% of patients who were positive for both H3K27Me3 and ctDNA at the time of diagnosis, 25.5% of these patients were positive for H3K27Me3 only, indicating that H3K27Me3 is useful as a biomarker at the time of diagnosis.

[0062] In some embodiments, the mere presence or absence of H3K27Me3, without quantitative determination of the amount of this marker, can be useful and can be correlated with the diagnosis of lung cancer or an increased risk of developing lung cancer.

[0063] In another embodiment, the measurement of H3K27Me3 may involve the quantification of the marker in order to correlate the detection of the marker with a probable diagnosis of lung cancer. Therefore, if the amount of the marker detected in a subject being tested is different from (i.e., higher than) the control amount, the subject being tested is likely to have lung cancer.

[0064] In one embodiment of the present invention, the method includes managing the treatment of a subject based on the state of the cancer. As previously stated, such management includes actions taken after determining the state of the lung cancer. For example, if the results of the method of the present invention indicate the presence or progression of the disease, the physician may direct further investigation. Alternatively, if the state indicates that surgery is appropriate, surgery may be scheduled for the patient. In other examples, the patient may receive chemotherapy or radiation therapy instead of or in addition to surgery. Similarly, if the results are negative, further action may not be necessary. Furthermore, if the results indicate that the treatment was successful, further management may not be necessary.

[0065] Possible further investigations include X-ray, CT scan, PET scan, or biopsy.

[0066] One aspect of the present invention relates to the detection of cancer in individuals, particularly lung cancer, or the identification of individuals at increased risk of developing such cancer. In some embodiments, the present invention provides diagnostic benefits to these patients, for example, by helping healthcare providers diagnose cancer at a very early stage, which can significantly improve the survival rate of cancer patients.

[0067] In one embodiment of the present invention, a combination of H3K27Me3 and ctDNA (hereinafter referred to as the "biomarker combination") is provided for use as a diagnostic tool for lung cancer. This combination provides a composite scoring in the evaluation and / or diagnosis of cancer. In one embodiment, the level of H3K27Me3 is measured as one of a measurement panel, including, for example, DNA analysis. In a further embodiment, the DNA is analyzed for circulating tumor DNA (ctDNA). In an even further embodiment, the ctDNA is analyzed for tumor-associated mutations, such as tumor-associated somatic mutations.

[0068] Therefore, according to one aspect of the present invention, a method for analyzing a target cancer, (i) To detect or measure the level of trimethylation of histone H3 at lysine 27 (H3K27Me3) in cell-free nucleosomes in body fluid samples obtained from subjects; and (ii) Analyze the ctDNA obtained from the subjects for tumor-associated mutations such as tumor-associated somatic mutations. A method is provided that includes:

[0069] In some embodiments, the method further includes analyzing cancer by using H3K27Me3 levels detected or measured in a bodily fluid sample in combination with ctDNA analysis.

[0070] In some embodiments, the presence of ctDNA combined with higher levels of H3K27Me3 compared to a control indicates the presence and / or progression of cancer, that the subject requires further investigation such as a lung biopsy, and / or that the subject requires treatment or a change in treatment.

[0071] In some embodiments, the absence of ctDNA combined with higher levels of H3K27Me3 compared to a control indicates the presence and / or progression of cancer, indicating that the subject requires further investigation, such as a lung biopsy, and / or that the subject requires treatment or a change in treatment.

[0072] In some embodiments, the absence of ctDNA in combination with lower levels of H3K27Me3 compared to a control indicates the absence and / or regression of cancer (e.g., during treatment), indicating that the subject does not require further investigation and / or that the subject does not require treatment or is responding well to treatment.

[0073] In a further embodiment, a method for monitoring cancer progression in a subject (for example, a subject with cancer such as lung cancer), (i) to detect or measure the level of H3K27Me3 in cell-free nucleosomes in body fluid samples obtained from subjects; and (ii) Analyze the ctDNA obtained from the subjects for tumor-associated mutations such as tumor-associated somatic mutations. : includes, Herein, a method is provided in which the level of H3K27Me3 and / or the presence of tumor-associated mutations in ctDNA indicate disease progression (e.g., cancer).

[0074] In a further embodiment, a method for diagnosing a target cancer (for example, lung cancer), (i) to detect or measure the level of H3K27Me3 in cell-free nucleosomes in body fluid samples obtained from subjects; and (ii) Analyze the ctDNA obtained from the subjects for tumor-associated mutations such as tumor-associated somatic mutations. : includes, Herein, a method is provided in which the level of H3K27Me3 and / or the presence of tumor-associated mutations in ctDNA indicates that a subject has cancer.

[0075] As described herein, if this method indicates that a subject has cancer, the diagnosis can be confirmed by further diagnostic investigations, such as biopsy. In one embodiment, the presence of ctDNA combined with higher levels of H3K27Me3 compared to a control indicates that the subject requires further investigation, such as a lung biopsy.

[0076] The present invention also provides a method in which a biomarker (or combination of biomarkers) is measured again after patient management. In these cases, the method is used to monitor the state of cancer, for example, response to cancer treatment, remission of the disease, or progression of the disease. Because the method is easy to use and, in some embodiments, non-invasive, it can be repeated after each treatment a patient receives. This makes it possible to track the effectiveness of the treatment course. If the results indicate that the treatment is ineffective, the treatment plan can be changed accordingly. This allows for flexibility in treatment options.

[0077] In one embodiment, treatment is selected from chemotherapy, radiotherapy (including stereotactic radioablation (SABR) or stereotactic body radiotherapy (SBRT), and serial high-fractionation accelerated radiotherapy (CHART)), chemoradiotherapy, surgery, targeted cancer drugs, immunotherapy, radiofrequency ablation, photodynamic therapy, and / or symptom control therapy. The treatment is a second-line treatment.

[0078] In one embodiment of the present invention, a positive result may indicate that EZH2-targeted therapy, such as therapy with an EZH2 inhibitor, is appropriate.

[0079] In other specific embodiments, the present invention provides the following:

[0080] A method for treating and / or evaluating lung cancer, Obtaining bodily fluid samples from the subject; (i) Measure the level of H3K27Me3 in the sample; (ii) Identifying subjects with lung cancer based on higher levels of H3K27Me3 compared to controls; and (iii) Administering treatment to the subject Methods that include:

[0081] A method for monitoring the success of lung cancer treatment aimed at cure, comprising detecting the level of H3K27Me3 in a fluid sample from a subject undergoing treatment for lung cancer for comparison with previous levels obtained from the subject.

[0082] A method for monitoring the progression of lung cancer in a subject, (a) Determine the level of H3K27Me3 in the body fluid samples obtained from the subject; (b) optionally compare the determined amount of H3K27Me3 in the sample derived from the subject with the amount of H3K27Me3 in the normal control; and (c) Repeat steps (a) and (b) at intervals of two or more time intervals (where an increase in patient-derived H3K27Me3 levels over time is associated with an increase in lung cancer progression, and a decrease in patient-derived H3K27Me3 levels over time is associated with a decrease in lung cancer progression). Methods that include:

[0083] A method for predicting the clinical outcome of a patient with lung cancer, comprising: a) evaluating the level of H3K27Me3 in the patient's biological sample; b) comparing the level of H3K27Me3 with a reference value; and c) predicting the clinical outcome based on the comparison in step b). Methods that include:

[0084] A method for evaluating the activity of antitumor therapy in lung cancer patients treated with antitumor therapy, (a) Before administering antitumor therapy, measure the level of H3K27Me3 in a body fluid sample, preferably a plasma sample, derived from the cancer patient; and (b) Measuring the level of H3K27Me3 in a biological sample derived from a cancer patient, preferably a plasma sample: Here, the reduced level of methylation compared to the level of H3K27Me3 before administering antitumor therapy to lung cancer patients indicates the activity of the antitumor therapy in lung cancer patients, for example, here, the activity is dose-dependent.

[0085] In one preferred embodiment, these aforementioned embodiments can be carried out using a combination of H3K27Me3 and ctDNA analysis as a biomarker.

[0086] The method of the present invention has other applications. For example, markers can be used to screen for compounds that modulate the expression of markers in vitro or in vivo, and these compounds may then be useful in treating or preventing lung cancer in patients. In another example, markers can be used to monitor the response to lung cancer treatment. In yet another example, markers can be used in genetic studies to determine whether a subject is at risk of developing lung cancer. For example, certain markers may be genetically linked. This can be determined, for example, by analyzing samples from a population of lung cancer patients with a family history of lung cancer. The results can then be compared, for example, with data obtained from lung cancer patients without a family history of lung cancer. Genetically linked markers can be used as a tool to determine whether a subject with a family history of lung cancer is more likely to develop lung cancer.

[0087] As used herein, the term “biomarker” refers to a marker that enables the detection of disease, or assists in the detection of disease in an individual when compared to a healthy individual, including the detection of residual disease, disease recurrence, and the early stages of disease. In specific embodiments, the level of a biomarker measured by an immunoassay in a fluid sample from the individual to be tested is compared to the respective levels in a past fluid sample from the same individual or a fluid sample from another healthy individual.

[0088] The sample may be any biological fluid (or body fluid) sample taken from a subject, including, but not limited to, cerebrospinal fluid (CSF), whole blood, serum, plasma, menstrual blood, endometrial fluid, urine, saliva, or other body fluids (feces, tears, synovial fluid, sputum), exhaled breath, such as condensed breath, or extracts or purified products thereof, or dilutions thereof. In a preferred embodiment, the body fluid sample is selected from blood, serum, or plasma. Biological samples may also include specimens from living subjects or specimens collected postmortem. The sample can be prepared by conventional methods, for example, by diluting or concentrating as appropriate, and stored. It will be understood that the method and use of the present invention are particularly useful with blood, serum, or plasma samples obtained from patients. In one embodiment, the sample is a blood or plasma sample. In a further embodiment, the sample is a serum sample. In a further embodiment, both serum and plasma samples are used to measure different components of the assay panel.

[0089] In one embodiment, the biomarker is intended for use in diagnosing the stage of cancer. Cancer can be assigned stages 0, 1, 2, 3, and 4. The definitions of the stages vary depending on the type of cancer and are well known in the art. Typically, stage 1 is classified as when the cancer is small and locally confined to the original tissue. Stage 2 is classified as when the cancer is larger and has grown beyond its origin into neighboring tissues or nearby lymph nodes within the organ. Stage 3 is classified as when the cancer has grown beyond the original organ into neighboring tissues but has not spread to other more distant parts of the body. Stage IV is classified as when the cancer has spread to one or more distant parts of the body, such as the liver or lungs. Early-stage cancer typically includes stages 0, 1, and 2. Late-stage cancer typically includes stages 3 and 4.

[0090] More specifically, in NSCLC, cancer staging is performed using a system called TNM, where: T describes the size of the tumor (cancer tissue). • N describes the spread of cancer to the lymph nodes. • M describes whether the cancer has spread to another part of the body, such as the liver (metastasis).

[0091] (T) Regarding T, there are four main stages:

[0092] T1 lung cancer means that the cancer is still inside the lung. T1 is classified into three substages: • T1a tumors are approximately 1 cm wide; • The T1b tumor is 1 cm to 2 cm wide; and • T1c tumors are 2cm to 3cm wide.

[0093] T2 is • The tumor is 3cm to 5cm wide, or • The tumor has spread to the main airway or the inner layer of the chest wall, • The lungs are either collapsing or obstructed due to inflammation. This is used to describe the following three possibilities:

[0094] T3 is • The tumor is 5cm to 7cm wide, or • There are more than one tumor in the lung lobe, or • The tumor has spread to the chest wall, the phrenic nerve (a nerve close to the lungs), or the outer layer of the heart (pericardium). This is used to describe the following three possibilities:

[0095] T4 is • The tumor is wider than 7 cm, or • The tumor has spread to both parts of the lung (each lung is composed of two parts known as lobes), or • The tumor has spread to areas of the body close to the lungs, such as the heart, trachea, esophagus (food pipe), or major blood vessels. It is used to describe various possibilities, including the colon (:).

[0096] (N) For N, there are three main stages:

[0097] N1 is used to describe cancer cells in lymph nodes located inside the lung or in the area (hilum) where the lung connects to the airway.

[0098] N2 is • The cancer cells are located in the central lymph nodes on the same side of the chest as the affected lung, or • Cancer cells are located in the lymph nodes below the trachea. This is used to describe the two possibilities:

[0099] N3 is • The cancer cells are located in the lymph nodes in the central part of the chest opposite the affected lung, or • Cancer cells are located in the lymph nodes above the clavicle, or • Cancer cells are located in the lymph nodes in the upper part of the lungs. This is used to describe the following three possibilities:

[0100] (M) Regarding M, there are two main stages: • M0 - The cancer has not spread to other parts of the body outside the lung; and • M1 - The cancer has spread to other parts of the body outside the lungs.

[0101] Small cell lung cancer is less common than non-small cell lung cancer. The cancer cells in this type of cancer are smaller in size than the cells that cause non-small cell lung cancer.

[0102] Small cell lung cancer is, • Localized disease – In this case, the cancer may be in only one lung and possibly in a nearby lymph node; or • Advanced disease – In this case, the cancer has spread to the other lung, even further to lymph nodes, or to other parts of the body. There are only two possible stages:

[0103] (Measurement method) In one embodiment, the level or concentration of the detected H3K27Me3 nucleosome biomarker is compared to a control. The control subjects may be selected based on various criteria, and it will be apparent to those skilled in the art that such control subjects may include, for example, subjects known to be disease-free, or subjects with different diseases (for example, for differential diagnosis investigations). "Control" may include healthy subjects, disease-free subjects, and / or subjects without cancer. Comparison with a control is well known in the field of diagnosis.

[0104] Once a "normal range" is established, it can be used as the baseline for all subsequent tests, so it will be understood that it is not always necessary to measure healthy / unaffected controls for comparison. The normal range can be established by taking samples from multiple control subjects without cancer and examining the levels of biomarkers. The results (i.e., biomarker levels) of subjects suspected of having cancer can then be examined to determine whether they fall within or outside their respective normal ranges. The use of a "normal range" is standard practice for disease detection.

[0105] Even if a subject is determined not to have cancer, the present invention can still be used to monitor disease progression. For example, if this use involves blood, serum, or plasma samples from a subject determined not to have cancer, the biomarker level can be repeated at a later time to determine whether the biomarker level has changed.

[0106] References to “subject” or “patient” are used interchangeably herein. In one embodiment, the patient is a human patient. In one embodiment, the patient is a (non-human) animal. Uses, panels, and methods described herein are preferably performed in vitro.

[0107] In one embodiment, the detection or measurement of a biomarker includes immunoassay, immunochemistry, mass spectrometry, chromatography, chromatin immunoprecipitation, or biosensor methods.

[0108] In one embodiment, detection or measurement includes an immunoassay. In a preferred embodiment of the present invention, a two-site immunoassay method for nucleosome moieties is provided. In particular, such a method is preferred for measuring nucleosomes or epigenetic features incorporated into nucleosomes in situ, utilizing two anti-nucleosome conjugates or anti-nucleosome conjugates combined with an anti-histone modification or anti-histone variant or anti-DNA modification or anti-addition protein detection conjugate. In another embodiment of the present invention, a two-site immunoassay is provided, utilizing a labeled anti-nucleosome detection conjugate combined with an immobilized anti-histone modification or anti-histone variant or anti-DNA modification or anti-addition protein conjugate.

[0109] The detection or measurement of the biomarker level can be carried out using one or more reagents, such as a suitable binder. In one embodiment, the one or more binders include a desired H3K27Me3 biomarker, or a ligand or binder specific to a structural / shape mimic of the biomarker or a component thereof.

[0110] It will be apparent to those skilled in the art that the terms “antibody,” “binder,” or “ligand” in any aspect of the present invention are not limiting and are intended to include any binder capable of binding to a particular molecule or entity, and that any suitable binder can be used in the methods of the present invention.

[0111] Methods for detecting biomarkers are known in the art. In one embodiment, the reagent comprises one or more ligands or binders. In one embodiment, the ligand or binder of the present invention comprises naturally occurring or chemically synthesized compounds capable of specific binding to a desired target. The ligand or binder may include peptides, antibodies or fragments thereof, or synthetic ligands such as plastic antibodies, or aptamers or oligonucleotides capable of specific binding to a desired target. The antibody may be a monoclonal antibody or a fragment thereof. If an antibody fragment is used, it will be understood that it retains the ability to bind to a biomarker so that the biomarker can be detected (according to the present invention). The ligand / binder may be labeled with a detectable marker, e.g., a luminescent, fluorescent, enzyme, or radioactive marker; or, further, the ligand according to the present invention may be labeled with an affinity tag, e.g., biotin, avidin, streptavidin, or His (e.g., hexa-His) tag. Alternatively, ligand binding may be determined using label-free techniques, e.g., ForteBio's label-free techniques.

[0112] A diagnostic or monitoring kit (or panel) for carrying out the method of the present invention is provided. Such a kit preferably includes one or more ligands for the detection and / or quantification of biomarkers according to the present invention, and / or biosensors and / or arrays described herein, optionally together with instructions for use of the kit.

[0113] A further aspect of the present invention is a kit for detecting the presence of a disease state, comprising a biosensor capable of detecting and / or quantifying one or more of the biomarkers defined herein. As used herein, the term “biosensor” means anything capable of detecting the presence of a biomarker. Examples of biosensors are described herein. A biosensor may comprise a ligand binder or ligand described herein, capable of specific binding to a biomarker. Such a biosensor is useful in the detection and / or quantification of the biomarkers of the present invention.

[0114] Preferably, the biosensor for detecting one or more biomarkers of the present invention combines the recognition of a biomolecule with appropriate means for converting the detection of the presence of a biomarker in a sample or the quantification of a biomarker in a sample into a signal. The biosensor can be adapted for "alternative site" diagnostic testing in, for example, hospital wards, outpatients' departments, operating rooms, homes, field, and workplaces. Examples of biosensors for detecting one or more biomarkers of the present invention include acoustic sensors, plasmon resonance sensors, holographic sensors, biolayer interference (BLI) sensors, and microengineered sensors. Imprint recognition elements, thin-film transistor technology, magnetoacoustic resonator devices, and other novel acoustic-electric systems can be utilized in the biosensor for detecting one or more biomarkers of the present invention.

[0115] Biomarkers for detecting the presence of disease are essential targets for the discovery of novel targets and drug molecules that can delay or halt the progression of the disorder. Since the results for biomarkers or biomarker panels indicate disorder and drug response, biomarkers are useful for identifying novel therapeutic compounds in in vitro and / or in vivo assays. The biomarkers and biomarker panels of the present invention can be used in methods for screening compounds that modulate the activity of biomarkers.

[0116] Accordingly, further embodiments of the present invention provide the use of a binder or ligand described herein, which may be a peptide, antibody or fragment thereof, or an aptamer or oligonucleotide directed to a biomarker according to the present invention; or the use of a biosensor, array or kit according to the present invention for identifying a substance that can promote and / or inhibit the generation of a biomarker.

[0117] The term "biomarker" refers to a differential biological or bio-derived indicator of a process, event, or state. Biomarkers can be used in diagnostic methods, such as clinical screening and prognosis assessment, as well as in monitoring treatment outcomes, identifying individuals most likely to respond to specific therapeutic interventions, and screening and developing drugs. Biomarkers and their use are beneficial in identifying new drug therapies and discovering new targets for drug treatment.

[0118] As used herein, the terms “detect” or “diagnose” encompass the identification, confirmation, and / or characterization of a disease state. The detection, monitoring, and diagnostic methods according to the present invention are useful for confirming the presence of a disease, for monitoring the development of a disease by assessing its onset and progression, or for assessing the improvement or mitigation of a disease. The detection, monitoring, and diagnostic methods are also useful for evaluating clinical screening, prognosis, treatment selection, and evaluation of treatment benefits, i.e., for drug screening and development.

[0119] Identification and / or quantification can be carried out by any method suitable for identifying the presence and / or amount of a specific protein in a biological sample derived from the subject, or in a purified or extracted product or dilution thereof. In the method of the present invention, quantification can be carried out by measuring the concentration of the target in the sample(s). Biological samples that can be examined in the method of the present invention include the biological samples defined herein. Samples can be prepared by conventional methods, and for example, they can be diluted or concentrated as appropriate and stored.

[0120] Identification and / or quantification of biomarkers can be carried out by detecting the biomarker or a fragment thereof, for example, a fragment having a C-terminal cleavage or a fragment having an N-terminal cleavage. The fragments are preferably longer than 4 amino acids, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. It should be noted that peptides with sequences identical to or related to histone tail sequences are particularly useful histone protein fragments.

[0121] For example, detection and / or quantification can be carried out using immunological methods, such as immunoassays. Immunosays include any method utilizing one or more antibodies or other specific binders directed to bind to the biomarkers defined herein. Immunosays include two-site immunoassays or immunoassays utilizing enzyme detection methods (e.g., ELISA), fluorescently labeled immunoassays, time-resolved fluorescently labeled immunoassays, chemiluminescent immunoassays, immunoturbidimetric assays, particulate-labeled immunoassays, and immunoradiometric assays, as well as single-site immunoassays, reagent-limited immunoassays, competitive immunoassays including labeled antigens and labeled antibodies, and single-antibody immunoassays using various labeling types, including radioactive, enzyme, fluorescent, time-resolved fluorescence, and particulate labeling.

[0122] In another example, detection and / or quantification can be performed by one or more methods selected from the group consisting of SELDI(-TOF), MALDI(-TOF), 1-D gel-based analysis, 2-D gel-based analysis, mass spectrometry (MS), reverse-phase (RP)LC, size permeation (gel filtration), ion exchange, affinity, HPLC, UPLC, and other LC or LC-MS-based techniques. Suitable LC-MS techniques include ICAT® (Applied Biosystems, CA, USA) or iTRAQ® (Applied Biosystems, CA, USA). Liquid chromatography (e.g., high-pressure liquid chromatography (HPLC) or low-pressure liquid chromatography (LPLC)), thin-layer chromatography, and NMR (nuclear magnetic resonance) spectroscopy may also be used.

[0123] The method of the present invention, involving the identification and / or quantification of one or more biomarkers, can be performed using a tabletop instrument or incorporated into a disposable diagnostic or monitoring platform that can be used in a non-laboratory environment, such as a physician's office or at the patient's bedside. A suitable biosensor for performing the method of the present invention is a "credit" card equipped with an optical or acoustic reader. The biosensor can be configured to electronically transmit the collected data to a physician for interpretation, and thus can form the basis of e-medicine.

[0124] Identifying biomarkers of disease status enables the integration of diagnostic procedures and treatment regimens. Using the detection of biomarkers of the present invention, subjects can be screened before participating in clinical trials. Biomarkers provide a means of indicating treatment response, poor response, undesirable adverse event profiles, degree of medication compliance, and achievement of appropriate serum drug levels. Biomarkers can be used to provide warnings of adverse drug responses. Biomarkers are useful in the development of personalized medicine because evaluation of responses allows for fine-tuning of dosages, minimizing the number of prescribed medications, reducing delays in achieving effective treatment, and avoiding adverse drug reactions. Therefore, monitoring the biomarkers of the present invention allows for precise tailoring of subject care to the needs determined by the subject's disability and pharmacogenome profile; thus, biomarkers can be used to adjust optimal doses, predict positive treatment responses, and identify subjects at high risk of severe adverse events.

[0125] Biomarker-based testing provides a primary assessment of "new" subjects and offers an objective measure for accurate and rapid diagnosis that cannot be achieved using current scales.

[0126] Furthermore, methods, biosensors, and kits for monitoring biomarkers are crucial as targeted monitoring tools that enable physicians to determine whether relapses are due to worsening of the disability. If pharmacological treatment is deemed inadequate, treatment can be restarted or increased; and, where appropriate, treatment can be modified. Because biomarkers are sensitive to the state of the disability, they provide indicators of the impact of drug treatment.

[0127] (Kit and panel testing) Using the kit combinations described herein, kits or panel tests can be prepared, in particular, for use in the diagnosis of lung cancer and / or monitoring of patients with lung cancer or suspected lung cancer.

[0128] Therefore, according to a further aspect of the present invention, a kit is provided comprising a reagent for detecting the level of H3K27Me3. According to a further aspect of the present invention, a kit is provided comprising a reagent for detecting H3K27Me3 and ctDNA. For the detection of H3K27Me3 (particularly when associated with circulating nucleosomes), the reagent may comprise a binder that specifically binds to H3K27Me3 and optionally one or more further binders that specifically bind to nucleosomes or their further epigenetic features. For the detection of cfDNA, the reagent may comprise a reagent for DNA extraction and / or sequencing. The kits described herein may be for use in the diagnosis of lung cancer.

[0129] A further aspect of the present invention provides the use of a kit defined herein for identifying patients who require treatment for lung cancer.

[0130] A further aspect of the present invention provides the use of a kit as defined herein for monitoring a patient for progression of lung cancer (e.g., further tumor growth or progression to different stages of cancer). Embodiments of this aspect include use for detecting disease progression in follow-up, active surveillance, and postoperative monitoring or in treatment for other recurrences.

[0131] A further aspect of the present invention provides the use of a kit as defined herein for evaluating the effectiveness of lung cancer treatment in a patient.

[0132] A further aspect of the present invention provides the use of a kit as defined herein for selecting a treatment for a patient with lung cancer.

[0133] It will be understood that the embodiments described herein are applicable to all aspects of the present invention, that is, the embodiments described for use are equally applicable to the claimed methods and the like.

[0134] The present invention will now be described with reference to the following non-limiting embodiments. [Examples]

[0135] (Examples) (Methods and materials) (cfDNA collection) The total EDTA blood sample was centrifuged at 1600 g for 10 minutes. The supernatant was then centrifuged at 6000 g for 10 minutes, and the resulting plasma was stored at -80°C until cfDNA extraction and molecular analysis. cfDNA was extracted using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Valencia, CA, USA, catalog number 55114) with a Qiagen vacuum manifold, according to the manufacturer's instructions. The cfDNA sample was quantified using the Qubit® dsDNA HS assay kit (Invitrogen®, catalog number 32854) with a Qubit® 4-fluorometer (Invitrogen®, catalog number Q33238, Carlsbad CA, 92008, United States).

[0136] (Library preparation for DNA sequencing) For custom validated NGS library preparation, 10–100 ng of cfDNA was used with a capture-based custom technique provided by SOPHiA GENECTICS (Lausanne, Switzerland) and performed according to the manufacturer's instructions. The custom panel covered 78 genes involved in cancer (e.g., EGFR, TP53, or KRAS). The libraries were sequenced using a NextSeq 550 (Illumina technology, San Diego, CA 92122) with 2 × 150 pair-end-run sequencing. Subsequent variant calling files were subjected to inter-sample background filtering to remove potential artifacts less than 3 standard deviations of mean background noise for each location. The filter criteria for variant calling were set to absolute value of mutant allele read count ≥ 40, minimum total depth > 1000x, and MAF threshold ≥ 0.5%. Bioinformatics was performed using the SophiaDDM platform.

[0137] (H3K27Me3 immunoassay:) Nucleosome structure was measured using a sandwich immunoassay based on magnetic beads and chemiluminescence technology. 50 μL of K2-EDTA plasma (the same as that used for DNA sequencing analysis) was incubated with acridinium ester-labeled anti-nucleosome antibody. Subsequently, magnetic particle beads coated with the corresponding monoclonal anti-histone H3K27Me3 capture antibody were added. Finally, after a washing step, a chemiluminescent substrate was added, and the light emitted by the acridinium ester was measured using a luminometer system. Results are expressed in relative light units (RLU), and concentrations were estimated using four-parameter logistic regression on a reference standard curve. All samples were analyzed in pairs.

[0138] (result) (Circulating H3K27Me3 nucleosomes in NSCLC at the time of diagnosis) The results for the NSCLC cohort at the time of diagnosis are shown in Figure 4. In the entire NSCLC cohort (n=318), 46.9% of patients (n=149) showed H3K27Me3- nucleosome levels below the cutoff, and 53.1% (n=169) showed levels above the cutoff (Figure 4A). In the entire NSCLC cohort, 41.2% of the samples evaluated by the NGS panel showed at least one mutation or copy number variation (CNV) (hereinafter referred to as ctDNA-positive samples (ctDNA+; n=131)), and 58.8% of patients showed no genetic alteration in their blood samples. These are hereafter defined as ctDNA-negative (ctDNA-; n=187). H3K27Me3-nucleosome levels were statistically significantly higher in the ctDNA+ group compared to the ctDNA- group (median = 33.9 ng / mL vs. 18.5 ng / mL; p-value < 0.001), and also statistically significantly higher compared to the healthy control group (median = 33.9 ng / mL vs. 8.0 ng / mL; p-value < 0.0001) (Figure 4B). Furthermore, H3K27Me3-nucleosome levels in the ctDNA- group remained higher than in the healthy control group (median = 18.5 ng / mL vs. 8.0 ng / mL; p-value < 0.0001).

[0139] High clinical outcomes were obtained through ROC curve analysis, with an area under the curve (AUC) of approximately 0.89.

[0140] In summary, circulating H3K27Me3- nucleosome levels were significantly increased in NSCLC samples at the time of diagnosis, and even more so when somatic mutations were detected (ctDNA+).

[0141] Based on these observations, the mutagenic tumor burden, defined by the ctDNA status and H3K27Me3-nucleosome concentration of the samples, was compared (Figure 3). The following was observed: i) 33.3% of the samples were double-negative for ctDNA and H3K27me3, indicating the absence of oncogenetic material containing released ctDNA or epigenetic biomarkers within the tissue tumor, or decreased expression of H3K27Me3; ii) 13.5% of the samples had low levels of H3K27Me3 despite the detection of somatic mutations, indicating a decrease in the tumor epigenetic remodeling process in these NSCLC cancers; iii) 25.5% of the samples were negative for somatic mutations but had high levels of H3K27me3 nucleosomes, indicating the presence of a tumor epigenetic process; iv) 27.7% of the samples were found to be positive for both ctDNA and H3K27me3-nucleosomes, indicating the maintenance of both the epigenetic and mutagenic burden processes.

[0142] (Circulating H3K27Me3 nucleosomes in advanced NSCLC) The results for the advanced NSCLC cohort are shown in Figure 5. Circulating H3K27Me3 nucleosome concentrations were measured in NSCLC samples collected during treatment (n=304) and compared to healthy samples (n=201) under normal use conditions. A very significant increase was observed in NSCLC compared to healthy samples (median = 16.89 ng / mL vs. 7.99 ng / mL, p-value < 0.0001, respectively) (Figure 5A). In this NSCLC population during treatment, H3K27Me3 nucleosome levels were lower than those detected in NSCLC samples at diagnosis (median 進行期 = 16.89 ng / mL vs. median 診断時 (=24.01 ng / mL).

[0143] Molecular profiles of NSCLC samples collected during treatment were analyzed by NGS. At least one of the 78 screened mutations was detected in 43.1% of the samples (n=131), confirming the presence of ctDNA in the plasma samples. Next, the inventors compared circulating H3K27Me3- nucleosome levels in mutation-positive (ctDNA+) and mutation-negative (ctDNA-) ctDNA samples collected during treatment. Circulating H3K27Me3- nucleosome levels were lower in the ctDNA-negative group compared to the ctDNA-positive group (median values). ctDNA- = 13.35 ng / mL vs. median ctDNA+ =26.05 ng / mL, p_value < 0.0001) (Figure 5B). As previously described, a cutoff value of 22.5 ng / mL was determined for H3K27Me3- nucleosomes. Based on this cutoff value, NSCLC samples collected during treatment can be classified into H3K27Me3-negative (H3K27Me3- below 22.5 ng / mL) and H3K27Me3-positive (H3K27Me3+ greater than 22.5 ng / mL). The majority of ctDNA- samples were also H3K27Me3-, represented by the upper limit of the box plot close to the cutoff (Figure 5B), while the majority of ctDNA+ samples were also H3K27Me3+, indicated by the median greater than 22.5 ng / mL.

[0144] A wider range of circulating H3K27Me3- nucleosomes was observed in the ctDNA+ group compared to the ctDNA- group and the healthy control group (minimum 3.11 ng / mL and maximum over 1200 ng / mL in ctDNA+ samples; minimum 1.15 ng / mL and maximum 456.10 ng / mL in ctDNA- samples; minimum 0.47 ng / mL and maximum 20.51 ng / mL in healthy control samples).

[0145] Clinical outcomes were evaluated in the ctDNA- and ctDNA+ subgroups compared to the entire cohort and healthy control samples (Figures 5C-D). Identification of NSCLC samples was superior in the ctDNA+ subgroup, with area under the curve being approximately 0.87, compared to 0.74 for the ctDNA- subgroup and 0.79 for the entire cohort (p-value < 0.0001).

[0146] In summary, circulating H3K27Me3- nucleosome levels were significantly increased in advanced NSCLC samples and even more so when somatic mutations were detected (ctDNA+). After subgrouping NSCLC samples into ctDNA- and ctDNA+, the samples were classified with respect to circulating H3K27Me3- nucleosome levels and finally organized into four classes described in a decision tree (Figure 2). 41.8% of the samples were double-negative for both ctDNA and circulating H3K27Me3- nucleosome levels. 15.1% of the samples that were negative for somatic mutations had high concentrations of circulating H3K27Me3- nucleosome levels. 19.4% of the samples had weak levels of circulating H3K27Me3- nucleosomes even when somatic mutations were detected. 23.68% of the samples were found to be positive for both ctDNA and circulating H3K27Me3- nucleosome levels.

[0147] (Clause) The following are a series of clauses defining the present invention and its preferred embodiments: Article 1. A method for analyzing the target cancer, To detect or measure the level of trimethylation of lysine 27 of histone H3 in cell-free nucleosomes (H3K27Me3) in a body fluid sample obtained from the subject; and Analyzing the cancer using the levels detected or measured in the bodily fluid sample. The method including: Article 2. The method described in Article 1, wherein the cancer is lung cancer. Clause 3. The method described in Clause 2, wherein the lung cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC). Clause 4. The method described in Clause 3, wherein the lung cancer is NSCLC. Clause 5. The method according to Clause 4, wherein the NSCLC is adenocarcinoma, squamous cell carcinoma, large cell carcinoma, adenosquamous carcinoma, or sarcomatoid carcinoma. Clause 6. The method described in Clause 5, wherein the NSCLC is adenocarcinoma. Clause 7. Methods described in any of Clauses 1-6 for monitoring cancer progression, monitoring treatment, assessing the need for treatment, assessing the need for changes in treatment, assessing the need for investigation in subjects with or suspected of having cancer, and / or assisting in the diagnosis of subjects with or suspected of having cancer. Clause 8. Any method described in any of Clauses 1-7 for managing treatment plans in subjects suspected of having cancer, having cancer, or having previously been diagnosed with cancer. Clause 9. A method described in any of Clauses 1-8 for evaluating recurrence of cancer after treatment. Clause 10. The method described in Clause 9, wherein the subject has minimal residual disease (MRD). Clause 11. A method for evaluating the success of cancer treatment aimed at curative therapy, as described in any of Clauses 1 to 10. Clause 12. A method described in any of Clauses 1 to 11 for assessing the need for the treatment or a change in the treatment. Clause 13. The method described in any of Clauses 1 to 12, wherein the level of H3K27Me3 is compared to a control or a previous level obtained from the subject. Clause 14. A method described in any of Clauses 1 to 13 for monitoring changes in cancer staging, wherein an increase compared to an initial sample or control indicates progression of cancer from early to late stages of the disease. Clause 15. The subject is subsequently investigated for cancer, and such investigation is by any of the methods described in Clauses 1 to 14, including pulmonary function tests (PFT), imaging, biopsy, and / or surgery. Article 16. The method described in Article 15, wherein the investigation is a biopsy. Clause 17. The method according to Clause 15, wherein the imaging is x-ray, a computed tomography (CT) scan of the chest, or a positron emission tomography (PET) scan. Clause 18. The method described in any of Clauses 1 to 17, wherein the level of H3K27Me3 indicating the presence and / or progression of the cancer is approximately 22.5 ng / ml or higher. Clause 19. The method described in any of Clauses 1 to 18, wherein the level of H3K27Me3 is measured as one of a measurement panel including DNA analysis. Clause 20. The method described in Clause 19, wherein the DNA is analyzed for circulating tumor DNA (ctDNA). Clause 21. The method described in Clause 20, wherein the ctDNA is analyzed for tumor-associated mutations, such as tumor-associated somatic mutations. Clause 22. The method described in any one of Clauses 19 to 21, wherein the presence of ctDNA in combination with a higher level of H3K27Me3 compared to a control indicates the presence and / or progression of cancer, that the subject requires further investigation such as a lung biopsy, and / or that the subject requires treatment or a change in treatment. Clause 23. The method described in any one of Clauses 19-21, wherein the absence of ctDNA in combination with a higher level of H3K27Me3 compared to a control indicates the presence and / or progression of cancer, that the subject requires further investigation such as a lung biopsy, and / or that the subject requires treatment or a change in treatment. Clause 24. The method described in any of Clauses 19 to 23, wherein the cancer is stage I, II, III, or IV. Clause 25. The method described in any of Clauses 19 to 24, wherein the cancer is described as TX, T0, Tis, T1 (including T1mi, T1a, T1b, T1c), T2 (including T2a, T2b), T3, or T4; and the cancer is described as NX, N0, N1, N2, or N3, and / or the cancer is described as M0 or M1 (including M1a, M1b, M1c). Article 26. A method for analyzing the target cancer, (i) To detect or measure the level of trimethylation of histone H3 lysine 27 (H3K27Me3) in cell-free nucleosomes in a body fluid sample obtained from the subject; and (ii) Analyze the ctDNA obtained from the subject for tumor-associated mutations such as tumor-associated somatic mutations. The method including: Clause 27. Use of H3K27Me3 as a biomarker in bodily fluid samples to monitor cancer progression, monitor treatment, assess the need for treatment, assess the need for changes in treatment, assess the need for investigation in subjects with or suspected of having cancer, or to assist in the diagnosis of subjects with or suspected of having cancer. Clause 28. A kit comprising reagents for detecting one or more biomarkers, including H3K27Me3 and optionally ctDNA.

Claims

1. A method for analyzing the target cancer, (i) To detect and measure the level of trimethylation of histone H3 lysine 27 (H3K27Me3) in cell-free nucleosomes in a body fluid sample obtained from the subject; and (ii) Analyze the circulating tumor DNA (ctDNA) obtained from the subject for tumor-associated mutations. The method including:

2. (i) The presence of ctDNA in combination with higher levels of H3K27Me3 compared to the control indicates the presence and / or progression of cancer, that the subject requires further investigation, and / or that the subject requires treatment or a change in treatment; or (ii) The absence of ctDNA in combination with higher levels of H3K27Me3 compared to the control indicates the presence and / or progression of cancer, that the subject requires further investigation, and / or that the subject requires treatment or a change in treatment. The method according to claim 1.

3. The method according to claim 1 or claim 2, wherein the cancer is lung cancer.

4. The method according to claim 3, wherein the lung cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC).

5. The method according to claim 4, wherein the lung cancer is NSCLC.

6. The method according to claim 5, wherein the NSCLC is adenocarcinoma, squamous cell carcinoma, large cell carcinoma, adenosquamous carcinoma, or sarcomatoid carcinoma.

7. The method according to claim 6, wherein the NSCLC is an adenocarcinoma.

8. The method according to any one of claims 1 to 7, wherein the tumor-associated mutation is a tumor-associated somatic mutation.

9. A method according to any one of claims 1 to 8 for monitoring cancer progression, monitoring treatment, assessing the need for treatment, assessing the need for changes in treatment, assessing the need for investigation in subjects who have or are suspected of having cancer, assisting in the diagnosis of subjects who have or are suspected of having cancer, and / or managing a treatment plan in subjects who are suspected of having cancer, have cancer, or have been previously diagnosed with cancer.

10. A method according to any one of claims 1 to 9 for evaluating cancer recurrence after treatment.

11. The method according to claim 9, wherein the subject has minimal residual disease (MRD).

12. A method according to any one of claims 1 to 11 for evaluating the success of cancer treatment aimed at curative treatment and / or for evaluating the need for a change in the treatment of the subject.

13. The method according to any one of claims 1 to 12, wherein the level of H3K27Me3 is compared with a control or a previous level obtained from the subject.

14. A method according to any one of claims 1 to 13 for monitoring changes in cancer staging, wherein an increase compared to an initial sample or control indicates the progression of cancer from the early to the late stages of the disease.

15. The method according to any one of claims 1 to 14, wherein the subject is subsequently investigated for cancer, and the investigation includes pulmonary function testing (PFT), imaging, biopsy, and / or surgery.

16. The method according to claim 15, wherein the investigation is a biopsy.

17. The method according to claim 15, wherein the imaging is an X-ray, a computed tomography (CT) scan of the chest, or a positron emission tomography (PET) scan.

18. The method according to any one of claims 1 to 17, wherein the level of H3K27Me3 indicating the presence and / or progression of the aforementioned cancer is approximately 22.5 ng / ml or higher.

19. The method according to any one of claims 1 to 18, wherein the cancer is stage I, II, III, or IV.

20. The method according to any one of claims 1 to 19, wherein the cancer is described as TX, T0, Tis, T1 (including T1mi, T1a, T1b, T1c), T2 (including T2a, T2b), T3, or T4; and the cancer is described as NX, N0, N1, N2, or N3, and / or the cancer is described as M0 or M1 (including M1a, M1b, M1c).

21. The method according to any one of claims 1 to 20, wherein the level of H3K27Me3 is measured as one of the measurement panels.

22. Use of H3K27Me3 as a biomarker in bodily fluid samples to monitor cancer progression, monitor treatment, assess the need for treatment, assess the need for treatment changes, assess the need for investigation in subjects with or suspected of having cancer, or to support the diagnosis of subjects with or suspected of having cancer.

23. A kit containing reagents for detecting H3K27Me3 and ctDNA.

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