WO2023013998A1 - Biomarker for prognosis of lung cancer treatment and use thereof - Google Patents

Abstract

The present invention relates to a biomarker for prognosing the treatment of lung cancer, especially non-small cell lung cancer, and a use thereof. According to the present invention, when it is confirmed that a mutation exists in a specific gene of a subject, prognosis can be made of the treatment of lung cancer, particularly non-small cell lung cancer, of the subject. In addition, the present invention can provide useful information for determining whether adjuvant therapy should be applied following surgical resection in the context of lung cancer treatment.

Description

Biomarkers for predicting the prognosis of lung cancer treatment and their uses

The present invention relates to a biomarker and its use for predicting the prognosis of lung cancer treatment, in particular, the prognosis of non-small cell lung cancer treatment. More specifically, the present invention is intended to predict the prognosis of lung cancer patients who have received or are scheduled to receive adjuvant therapy following surgical resection.

This application claims priority based on Korean Patent Application No. 10-2021-0101280 filed on August 2, 2021 and Korean Patent Application No. 10-2022-0094276 filed on July 28, 2022, All contents disclosed in the specification and drawings of the application are incorporated into this application.

Clonal hematopoiesis (CH) is a condition defined by the expansion of clonally derived hematopoietic stem cells (HSCs) harboring somatic mutations in leukemia-associated genes, which can be detected by next-generation sequencing (NGS) (Genovese G , Kahler AK, Handsaker RE, et al: Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence.N Engl J Med 371:2477-87, 2014;Park SJ, Bejar R: Clonal hematopoiesis in cancer.Exp Hematol 83:105-112, 2020; and Jaiswal S, Ebert BL: Clonal hematopoiesis in human aging and disease. Science 366, 2019). The occurrence of CH is associated with aging and may be found without hematological malignancies. It is also strongly associated with tobacco use, exposure to radiation therapy (RTx) and/or chemotherapy (CTx). In addition, CH is known to increase the incidence of subsequent cardiovascular diseases and hematological malignancies.

On the other hand, lung cancer is the most commonly diagnosed cancer and is a leading cause of cancer-related death worldwide. For lung cancer treatment, surgical excision is preferentially performed when possible, but the 5-year survival rate after surgery remains at 65%. Therefore, it is important to develop predictive factors capable of predicting the prognosis after lung cancer surgery in order to increase the survival rate through additional treatment after surgery and early detection of recurrence. Several prognostic factors such as age, gender, and cancer stage have been identified, but new factors need to be explored in the NGS era.

[Prior art literature]

(Non-Patent Document 1) Jaiswal S, Ebert BL: Clonal hematopoiesis in human aging and disease, Science 366, 2019.

(Non-Patent Document 2) Park SJ, Bejar R: Clonal hematopoiesis in cancer, Exp Hematol 83:105-112, 2020.

(Non-Patent Document 3) Genovese G, Kahler AK, Handsaker RE, et al: Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence, N Engl J Med 371:2477-87, 2014.

The object of the present invention is to solve all of the above problems.

An object of the present invention is to provide a method of predicting the prognosis of lung cancer treatment in a lung cancer patient or a method of providing information for predicting the prognosis of lung cancer treatment.

Another object of the present invention is to provide a composition for predicting the prognosis of lung cancer treatment in lung cancer patients.

Another object of the present invention is to provide a kit for predicting the prognosis of lung cancer treatment in lung cancer patients.

Another object of the present invention is to provide a panel for genetic analysis capable of detecting genetic mutations of clonal hematopoiesis in order to predict the prognosis of lung cancer treatment in lung cancer patients.

Another object of the present invention is to provide a method for treating lung cancer or a method for providing information for lung cancer treatment.

The object of the present invention is not limited to the object mentioned above. The objects of the present invention will become more apparent from the following description, and will be realized by means of the instrumentalities and combinations set out in the claims.

Representative configurations of the present invention for achieving the above object are as follows.

According to one aspect of the present invention, predicting the prognosis of lung cancer treatment of an individual comprising determining whether clonal hematopoiesis exists in an individual through genetic analysis of a biological sample isolated from an individual being treated for lung cancer A method or method of providing information for predicting the prognosis of lung cancer treatment of an individual is provided.

According to another aspect of the present invention, a composition for predicting the prognosis of lung cancer treatment in an individual comprising, as an active ingredient, an agent for confirming the presence of clonal hematopoiesis using a biological sample isolated from an individual being treated for lung cancer. is provided.

According to another aspect of the present invention, a kit for predicting the prognosis of lung cancer treatment of a subject comprising the composition is provided.

According to another aspect of the present invention, a genetic analysis panel for detecting genetic mutations in clonal hematopoiesis comprising the composition is provided.

According to another aspect of the present invention, treatment of lung cancer comprising the step of determining whether clonal hematopoiesis exists in a subject through genetic analysis of a biological sample isolated from the subject prior to administration of a therapeutic agent for lung cancer treatment. A method or method of providing information for treatment of lung cancer is provided.

According to the present invention, when it is confirmed that there is a mutation in a gene related to clonal hematopoiesis (CH) of an individual, it is possible to predict the prognosis according to the treatment of lung cancer, particularly non-small cell lung cancer, of the individual. In addition, the present invention can provide useful information for determining the application of adjuvant therapy following surgical resection in relation to lung cancer treatment, information useful for determining whether to administer a therapeutic agent for lung cancer treatment, and the like, and furthermore, lung cancer In clinical trials of drug candidates for treatment, useful information can be provided to evaluate the efficacy and safety of drug candidates for patients.

1 shows a Consolidated Standards of Reporting Trials (CONSORT) diagram used in an embodiment of the present invention.

Figures 2a and 2b show the overall survival and recurrence-free survival of patients, respectively, according to the presence of clonal hematopoietic (CH) mutations in the entire cohort. Figure 2c shows the overall survival of patients according to the presence of CH mutations after propensity score matching (PSM).

Figure 3 shows the cumulative mortality according to the presence of CH. Specifically, FIGS. 3A, 3B, and 3C show cumulative lung cancer mortality according to the presence of CH, cumulative non-lung cancer mortality according to the presence of CH, and cumulative mortality of unknown cause according to the presence of CH, respectively.

Figures 4a and 4b show overall survival according to the presence of CH mutations in patients receiving adjuvant therapy for stage IIB lung cancer before and after PSM, respectively. 4c and 4d respectively show the overall survival rate according to the presence of CH mutation in patients without adjuvant therapy for stage IIB lung cancer before and after PSM (Tx, Treatment).

5A-5C show the characteristics of CH mutations identified in the entire cohort. Figure 5A shows the prevalence of CH by age of patients in the cohort. 5B shows the number of mutations carried per patient. Figure 5c shows the number of mutations in each CH gene.

Figures 6a to 6c show the overall survival of patients according to the presence of CH mutations in stages IIB, IIIA and IIIB, respectively.

The detailed description of the invention that follows will be described with reference to specific drawings in relation to specific embodiments in which the invention may be practiced, but the invention is not limited thereto and, if properly described, what the claims claim and All scopes of equivalence are limited only by the appended claims. It should be understood that the various implementations/embodiments of the present invention are different from each other but are not necessarily mutually exclusive. For example, specific shapes, structures, and characteristics described herein may be changed from one embodiment/embodiment to another, or a combination of embodiments/embodiments, without departing from the spirit and scope of the present invention. can be implemented Technical and scientific terms used herein, unless defined otherwise, have the same meaning as commonly used in the field to which this invention belongs. For purposes of interpreting this specification, the following definitions will apply and terms used in the singular will include the plural as appropriate and vice versa.

Justice

As used herein, the term "about" refers to the typical error range for each value known to one of ordinary skill in the art. Further, unless otherwise specified, all numbers, values and/or expressions expressing ingredients, conditions, compositions, amounts, etc., used herein mean that such numbers are, among other things, essentially the representations of measurements that would occur to obtain such values. Since these are approximations that reflect various uncertainties, they should be understood to be qualified by the term "about".

The term "clonal haematopoiesis" refers to a condition in which, when hematopoietic stem cells undergo somatic mutations to gain an opportunity for selective proliferation, the mutated clone expands and occupies a certain portion of leukocytes.

The term "subject" can be used interchangeably with "patient" and includes a mammal, such as a primate (eg, a human), a companion animal (eg, a dog, cats, etc.), livestock animals (eg, cows, pigs, horses, sheep, goats, etc.) and laboratory animals (eg rats, mice, guinea pigs, etc.). In one embodiment of the invention, the subject is a human.

The term "prognosis" refers to the course of a disease, such as the likelihood of death or progression due to lung cancer, including onset, recurrence, metastatic spread, survival rate, disease-free survival rate, drug resistance or susceptibility of a disease such as lung cancer, and whether it is cured or not. Specifically, the prognosis may refer to a survival prognosis according to lung cancer treatment including surgery, chemotherapy, chemotherapy, chemoradiation, or a combination of these therapies in lung cancer patients. In addition, the prognosis of lung cancer treatment may mean the patient's responsiveness to a therapeutic agent for lung cancer treatment.

The term “prediction” refers to preliminarily determining the possibility of a patient surviving by responding preferentially or unfavorably to a treatment such as chemotherapy or chemoradiation or a treatment for lung cancer. Predicting survival prognosis can help select the most appropriate treatment method for a patient, confirm whether the patient responds favorably to the treatment method, or predict long-term survival of the patient after performing the treatment method.

The term "biological sample" refers to any biological sample obtained from an individual, which is a tissue, tumor tissue, lung tumor tissue, blood, serum, plasma, lymph, saliva, sputum, Samples such as mucus or urine include, but are not limited to.

The term "adjuvant therapy" includes chemotherapy (CTx), chemoradiation therapy (CRTx), molecular targeted therapy, radiofrequency hyperthermia cancer therapy, immunotherapy using biological agents, etc., performed before and after tumor removal surgery to treat cancer locally. Alternatively, any therapy may be included as long as it can be used as an adjuvant to systemic treatment.

The term “overall survival rate” refers to the rate at which a cancer patient survives 5 years after undergoing surgery, even if the cancer has recurred or metastasized.

The term "recurrence-free survival rate" refers to the rate of cancer recurrence-free survival 5 years after surgery.

The term "missense mutation" refers to a genetic mutation in which a single base substitution occurs at a site on a DNA chain, thereby changing the genetic code of mRNA and designating an amino acid different from the original one to affect a protein.

The term "frameshift mutation" refers to a genetic mutation caused by insertion or deletion of a non-divisible number of bases.

The term "nonsense mutation" refers to a genetic mutation in which a codon encoding an original amino acid is changed to a stop codon that does not encode an amino acid by a single base substitution, so that protein synthesis is stopped at the location of the codon.

The term "splice variation" refers to a variation that occurs through the use of alternative splicing sites within transcribed RNA molecules or between individually transcribed RNA molecules.

The term “primer” refers to a nucleic acid sequence that is capable of forming a base pair with a complementary template and serves as a starting point for copying the template strand. The sequence of the primer does not necessarily have to be exactly the same as the sequence of the template, but is sufficiently complementary to allow hybridization with the template. Primers can initiate DNA synthesis in the presence of reagents for polymerization and four different nucleoside triphosphates in an appropriate buffer solution and temperature. PCR conditions and lengths of sense and antisense primers can be modified based on those known in the art.

The term “probe” refers to a substance capable of specifically binding to a target substance to be detected in a sample, and through the binding, a substance capable of specifically confirming the presence of the target substance in the sample. The probe may be prepared in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, or an RNA probe. Selection of suitable probes and hybridization conditions can be modified based on those known in the art.

The term "antisense nucleic acid" refers to a nucleic acid-based molecule that has a complementary sequence to a target gene variant and can form a dimer with the target gene variant, and can be used to detect the target gene variant. An appropriate length of the antisense nucleic acid may be selected to increase detection specificity.

The term "gene panel" refers to a genetic mutation detection tool in which a panel is composed of a plurality of agents capable of detecting mutations in a plurality of target genes.

The term "therapeutic agent for treating lung cancer" or "therapeutic agent for lung cancer" refers to a substance exhibiting an effect of improving, alleviating or treating the symptoms of a patient with lung cancer, and noting morphological, physiological or genetic changes in lung cancer cells. As long as they are clearly indicated, their physical properties, chemical properties, biological origin, etc. are not particularly limited. The therapeutic agent includes all kinds of substances that can be used in the adjuvant therapy, ie, chemotherapy (CTx), chemoradiation therapy (CRTx), molecular target therapy, immunotherapy using biological agents, and the like.

The term "clinical trial" refers to all processes of research on the application of pharmaceuticals to humans, as well as research procedures conducted to confirm the safety and efficacy of pharmaceuticals, as well as bioequivalence tests to prove the bioequivalence of original and generic drugs. , clinical studies or side effects studies on drugs that have already been approved and are on the market.

Diagnosis of clonal hematopoiesis for prognosis of lung cancer treatment

The present invention is based in part on the surprising discovery that the presence or absence of clonal haematopoiesis (CH) in individuals undergoing lung cancer treatment is significantly associated with the prognosis of lung cancer treatment. In addition, it was confirmed that mutations in specific genes among genes involved in clonal hematopoiesis have a greater relevance. Accordingly, the present invention may include determining whether clonal hematopoiesis exists in a subject being treated for lung cancer in order to provide information necessary for prognosis of lung cancer. In addition, when it is confirmed that clonal hematopoiesis exists in the subject according to the present invention, it may indicate that the prognosis of lung cancer treatment is not good compared to the case where clonal hematopoiesis does not exist.

According to one embodiment of the present invention, individuals with clonal hematopoiesis had a poorer overall survival rate compared to individuals without clonal hematopoiesis, and even after excluding variables other than clonal hematopoiesis by applying the propensity score matching (PSM) technique, Patients who still had clonal hematopoiesis showed poorer survival compared to patients who did not (see Example 6.3 and FIGS. 2A-2C). As such, the presence of clonal hematopoiesis can act as a major factor in poor prognosis in the treatment of lung cancer patients.

According to another embodiment of the present invention, mortality due to lung cancer was similar regardless of the presence or absence of clonal hematopoiesis, but non-lung cancer mortality and mortality from unknown cause were compared with patients without clonal hematopoiesis. It was significantly higher in patients with hematopoiesis (see Example 6.3 and FIGS. 3A-3C). In addition, the presence of clonal hematopoiesis was associated with poorer overall survival in patients receiving adjuvant therapy, but the presence of clonal hematopoiesis did not significantly affect overall survival in patients not receiving adjuvant therapy. It was confirmed (see Example 6.3 and FIGS. 4a to 4d). These results indicate that the death rate is increased due to causes other than exacerbation of lung cancer, such as cardiopulmonary disease, sepsis, stroke, etc., and the existence of clonal hematopoiesis itself amplifies adverse outcomes related to adjuvant therapy, further worsening the prognosis of lung cancer treatment. It is to support that it can be done.

Therefore, confirming the presence or absence of clonal hematopoiesis according to the present invention can predict the prognosis of lung cancer treatment and at the same time help select an appropriate follow-up therapy following lung cancer surgery, thereby increasing the survival rate of individuals after lung cancer treatment. . In addition, it is possible to more efficiently and accurately evaluate the efficacy of a therapeutic agent by selecting an individual to evaluate the efficacy of a therapeutic agent used for lung cancer treatment, such as adjuvant therapy, by checking whether clonal hematopoiesis exists in the individual prior to administration of the therapeutic agent, The cost of evaluating the efficacy of such therapeutics (eg, clinical trials) can be lowered. For example, in recruiting patient groups (subjects) for a clinical trial of a therapeutic agent, before administering the therapeutic agent, it is confirmed whether or not clonal hematopoiesis exists in the target patient, and patients with clonal hematopoiesis are excluded from the patient group for lung cancer. Efficacy evaluation of the therapeutic agent can be performed more efficiently and accurately. Furthermore, by checking whether clonal hematopoiesis exists in an individual before administering the therapeutic agent and selecting an individual highly responsive to the therapeutic agent used for lung cancer treatment, the efficacy of the therapeutic agent in the individual and the lung cancer treatment effect can be increased.

Methods for predicting the prognosis of lung cancer treatment

According to one aspect of the present invention, there is provided a method for predicting the prognosis of lung cancer treatment of an individual comprising the step of determining whether clonal hematopoiesis exists in an individual through genetic analysis of a biological sample isolated from the individual. In addition, a method for providing information for predicting the prognosis of lung cancer treatment of an individual is provided, which includes determining whether clonal hematopoiesis exists in the individual through genetic analysis of a biological sample isolated from the individual.

Genes causing somatic mutations associated with clonal hematopoiesis include APC, ASXL1, ASXL2, ATM, BCL11B, BCOR, BCORL1, BIRC3, BRAF, BRCC3, CARD11, CASP8, CBL, CD58, CD79B, CNOT3, CREBBP, and CUX1 , DDX3X, DNMT3A, EP300, ETV6, EZH2, FAM46C, FBXW7, FLT3, FOXP1, GNAS, GNB1, GPS2, HIST1H1C, IDH2, IKZF1, IKZF2, JAK1, JAK2, JAK3, JARID2, KDM6A, KIT, KLHL6, KMT2D, KRAS , LUC7L2, MAP3K1, MPL, MYD88, NF1, NFE2L2, NOTCH1, NOTCH2, NRAS, PDS5B, PDSS2, PHF6, PHIP, PIK3CA, PIK3R1, PPM1D, PRDM1, PRPF40B, PTEN, PTPN11, RAD21, RIT1, RPS15, SETD2, SETDB1 , SF1, SF3A1, SF3B1, SMC1A, SMC3, SRSF2, STAG1, STAG2, STAT3, SUZ12, TBL1XR1, TET1, TET2, TNFAIP3, TNFRSF14, TP53, U2AF1, VHL, WT1, ZRSR2 and CHEK2. According to one embodiment of the present invention, the step of determining the presence or absence of clonal hematopoiesis based on whether one or more mutations exist in the one or more genes through genetic analysis using a biological sample isolated from the individual can include

In another embodiment, the one or more genes are DNMT3A, ASXL1, TET2, PPM1D, ATM, BCL11B, CARD11, CBL, CD79B, CHEK2, CUX1, ETV6, FOXP1, JAK2, KMT2D, MAP3K1, MPL, NF1, NOTCH1, NOTCH2, It may include one or more selected from the group consisting of PHIP, PRPF40B, RAD21, SETD2, SF3B1, TET1, TNFAIP3, TP53, U2AF1 and ZRSR2.

In another embodiment, the one or more genes are selected from the group consisting of ASXL1, CBL, CHEK2, CUX1, DNMT3A, FOXP1, JAK2, KMT2D, MPL, NOTCH1, PPM1D, PRPF40B, SF3B1, TET2, TNFAIP3, TP53, U2AF1 and ZRSR2. One or more selected ones may be included.

In another embodiment, the one or more genes may include one or more selected from the group consisting of DNMT3A, ASXL1, TET2, PPM1D, SF3B1, ATM, and TNFAIP3.

In another embodiment, the one or more genes may include any one gene selected from the group consisting of the above-mentioned genes, and in this case, the one or more genes consist of the rest of the genes other than the selected one gene. It may further include one or more selected from the group. As an example, the one or more genes may include DNMT3A. At this time, the one or more genes are ASXL1, TET2, PPM1D, ATM, BCL11B, CARD11, CBL, CD79B, CHEK2, CUX1, ETV6, FOXP1, JAK2, KMT2D, MAP3K1, MPL, NF1, NOTCH1, NOTCH2, PHIP, PRPF40B, It may further include one or more selected from the group consisting of RAD21, SETD2, SF3B1, TET1, TNFAIP3, TP53, U2AF1 and ZRSR2. In addition, the at least one gene is selected from the group consisting of ASXL1, CBL, CHEK2, CUX1, FOXP1, JAK2, KMT2D, MPL, NOTCH1, PPM1D, PRPF40B, SF3B1, TET2, TNFAIP3, TP53, U2AF1 and ZRSR2 Add one or more can be included with In addition, the one or more genes may further include one or more selected from the group consisting of ASXL1, TET2, PPM1D, SF3B1, ATM, and TNFAIP3.

According to another embodiment of the present invention, the presence or absence of clonal hematopoiesis may be determined based on the presence or absence of mutations in the one or more genes and the variant allele frequency (VAF). For example, through genetic analysis using a biological sample isolated from an individual, one or more mutations exist in one or more genes among the genes of the gene group, and the frequency of the variant allele is higher than a certain level, for example, about 1.8% or higher, about 1.8% or higher. If it is 1.9% or more, or about 2% or more, clonal hematopoiesis can be diagnosed as present.

The "individual" may mean a patient being treated for lung cancer. Specifically, the subject may mean a patient with lung cancer. More specifically, the subject may mean a patient before or after undergoing tumor removal surgery. Additionally, the subject may be a patient prior to receiving adjuvant therapy.

The "lung cancer" refers to a tumor originating in the lung, and may include non-small cell cancer and small cell cancer such as squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Specifically, the lung cancer may be non-small cell lung cancer. More specifically, the subject's non-small cell lung cancer may be stage I, stage II or a later stage. More specifically, the non-small cell lung cancer stage of the subject may be stage IA, stage IB, stage IIA, stage IIB, stage IIIA or stage IIIB, and more specifically stage IIB.

In one embodiment, the prognosis of lung cancer treatment may refer to a survival prognosis according to lung cancer treatment including surgery, chemotherapy, anticancer immunotherapy, chemoradiation, or a combination of these therapies or the same in an individual. For example, the prognosis of lung cancer treatment may mean overall survival rate or recurrence-free survival rate according to adjuvant therapy performed before or after tumor removal surgery or lung cancer treatment including the same. In addition, prognosis of lung cancer treatment may include overall survival rate or recurrence-free survival rate after tumor removal surgery or after application of adjuvant therapy following tumor removal surgery.

In another embodiment, the prognosis of lung cancer treatment may include responsiveness to adjuvant therapy such as chemotherapy or radiation therapy after surgery in an individual receiving treatment for lung cancer, survival prognosis, or both thereof. Anticancer chemotherapy may refer to treatment using an anticancer agent. Prognosis may mean, for example, responsiveness to anticancer drugs and/or survival prognosis in lung cancer patients administered with anticancer drugs including platinum-based drugs, taxane-based drugs, vinca alkaloid-based drugs, and anti-metabolites. In the case of lung cancer patients, surgery and adjuvant therapy are performed, and even among patients with similar clinical characteristics or similar stages, the response to each adjuvant therapy varies, and survival prognosis may also show significant differences. When the genetic mutation marker associated with clonal hematopoiesis is used, responsiveness to adjuvant therapy and/or survival prognosis thereof can be predicted, and accordingly, the direction of further treatment can be determined. As a result, the survival rate after lung cancer onset can be remarkably increased.

In one embodiment of the present invention, as a result of confirming patient mortality according to the presence or absence of CH in patients who underwent non-small cell lung cancer surgery, a significant difference according to the presence of CH in patient mortality was not lung cancer mortality but non-lung cancer mortality. and mortality of unknown cause, and these significant differences suggest that in patients with CH, several adverse outcomes associated with CH (e.g., cardiopulmonary disease, sepsis, stroke, etc.) may be amplified by CTx or RTx, eventually affecting survival. support that there is Therefore, the presence or absence of CH in a subject undergoing lung cancer treatment enables the selection of a more appropriate adjuvant therapy. For example, in the case of a patient diagnosed with CH present among individuals undergoing lung cancer treatment, anti-inflammatory drugs (eg, cannabis) alone or in combination with conventional adjuvant anticancer therapy or selected adjuvant anticancer therapy are used to improve the prognosis of lung cancer treatment. Kinumab) can be used.

In one embodiment of the present invention, the step of determining whether clonal hematopoiesis exists is before undergoing tumor removal surgery, after tumor removal surgery, before applying adjuvant therapy after tumor removal surgery, or following tumor removal surgery. This can be done after application of adjuvant therapy. For example, by confirming the presence of clonal hematopoiesis before undergoing tumor removal surgery, it is possible to determine whether chemotherapy or radiation therapy is more favorable for the patient's prognosis instead of surgical resection. As another example, after undergoing tumor removal surgery, whether or not the application of additional adjuvant therapy is favorable for the patient's prognosis can be determined according to the presence or absence of clonal hematopoiesis. As another example, after tumor removal surgery and adjuvant therapy are applied, it may be determined whether to additionally perform a complementary therapy according to the presence or absence of clonal hematopoiesis and/or the type of mutated gene.

Gene mutations associated with the clonal hematopoiesis include missense mutations, frameshift mutations, nonsense mutations, splice mutations, nucleotide insertions, deletions or substitutions, combinations thereof, and the like. can be in the form

For example, the mutation of the DNMT3A gene may be one or more selected from the mutations listed in Table 1 below, but is not limited thereto.

The mutation of the TET2 gene may be one or more selected from the mutations listed in Table 2 below, but is not limited thereto.

Mutations of the ASXL1 gene may be one or more selected from among the mutations listed in Table 3 below, but are not limited thereto.

The mutation of the PPM1D gene may be one or more selected from the mutations listed in Table 4 below, but is not limited thereto.

ATM, BCL11B, CARD11, CBL, CD79B, CHEK2, CUX1, ETV6, FOXP1, JAK2, KMT2D, MAP3K1, MPL, NF1, NOTCH1, NOTCH2, PHIP, PRPF40B, RAD21, SETD2, SF3B1, TET1, TNFAIP3, TP53, U2AF1 And mutations of the ZRSR2 gene may be mutations described in Table 5, respectively, but are not limited thereto.

For example, the mutation at position 5073770 of the JAK2 gene may be a missense mutation in which base G at position 1849 is substituted with T.

In one embodiment of the present invention, the method of the present invention, when it is confirmed that the mutation exists through genetic analysis of a biological sample isolated from the individual, compared to the case where the mutation does not exist, the treatment of lung cancer of the individual It may further include determining that the prognosis is indicative of poor prognosis.

In one embodiment of the present invention, as a result of follow-up of adjuvant therapy treatment progress of lung cancer patients after surgery, in the case of lung cancer patients with the CH-related gene mutation, compared to lung cancer patients without CH-related genetic mutation, after surgery 5 The annual survival rate was statistically significantly lower.

The genetic analysis can be performed using Next Generation Sequencing (NGS) and PCR-based techniques such as real-time quantitative PCR, blocker PCR, digital droplet PCR (ddPCR), clamping PCR, ICE-COLD PCR, castPCR, ARMS PCR, BEAMing, etc., but are not limited thereto. The practitioner can detect and identify the genetic mutation associated with clonal hematopoiesis using known genetic analysis techniques without limitation. Preferably, the genetic analysis may include next-generation genome sequencing analysis. For example, using next-generation genome sequencing analysis, whole genome sequencing, whole exome sequencing, RNA sequencing, etc. information can be analyzed.

Compositions, kits and genetic panels

According to another aspect of the present invention, a composition for predicting the prognosis of lung cancer treatment in a subject comprising an agent for diagnosing clonal hematopoiesis as an active ingredient using a biological sample isolated from a subject being treated for lung cancer is provided. do. The composition can be used in a method for diagnosing clonal hematopoiesis or predicting the prognosis of lung cancer treatment for predicting the prognosis of lung cancer treatment according to the present invention.

In one embodiment, the agent is DNMT3A, ASXL1, TET2, PPM1D, ATM, BCL11B, CARD11, CBL, CD79B, CHEK2, CUX1, ETV6, FOXP1, JAK2, KMT2D, MAP3K1, MPL, NF1, NOTCH1, NOTCH2, PHIP, It may include an agent for detecting whether one or more mutations exist in one or more genes selected from the group consisting of PRPF40B, RAD21, SETD2, SF3B1, TET1, TNFAIP3, TP53, U2AF1 and ZRSR2.

In another embodiment, the agent is one selected from the group consisting of ASXL1, CBL, CHEK2, CUX1, DNMT3A, FOXP1, JAK2, KMT2D, MPL, NOTCH1, PPM1D, PRPF40B, SF3B1, TET2, TNFAIP3, TP53, U2AF1 and ZRSR2 It may include an agent for detecting the presence of one or more mutations in one or more genes.

In another embodiment, the agent may include an agent for detecting the presence of one or more mutations in one or more genes selected from the group consisting of DNMT3A, ASXL1, TET2, PPM1D, SF3B1, ATM, and TNFAIP3.

In another embodiment, the agent may include an agent for detecting whether a mutation exists in one gene selected from the gene group, wherein the agent consists of the remaining genes except for the selected one gene. It may further include an agent for detecting whether there is a mutation in one or more genes selected from the group. As an example, the agent may include an agent for detecting whether there is a mutation in DNMT3A, wherein the agent is ASXL1, TET2, PPM1D, ATM, BCL11B, CARD11, CBL, CD79B, CHEK2, CUX1, ETV6, Detecting whether there is a mutation in one or more genes selected from the group consisting of FOXP1, JAK2, KMT2D, MAP3K1, MPL, NF1, NOTCH1, NOTCH2, PHIP, PRPF40B, RAD21, SETD2, SF3B1, TET1, TNFAIP3, TP53, U2AF1 and ZRSR2 or from the group consisting of ASXL1, CBL, CHEK2, CUX1, DNMT3A, FOXP1, JAK2, KMT2D, MPL, NOTCH1, PPM1D, PRPF40B, SF3B1, TET2, TNFAIP3, TP53, U2AF1 and ZRSR2 Detecting whether there is a mutation in one or more genes selected from the group consisting of ASXL1, TET2, PPM1D, SF3B1, ATM, and TNFAIP3 It may further include an agent for detecting whether there is a mutation in one or more genes selected from the group consisting of ASXL1, TET2, PPM1D, SF3B1, ATM, and TNFAIP3.

The agent may include, for example, an agent capable of detecting a mutant gene, an mRNA derived therefrom, or a protein encoded by the mutant gene.

An agent capable of detecting the expression of the gene or mRNA may be a nucleotide sequence that complementarily binds to the mutant gene or mRNA, for example, sense and antisense primers, probes, or antisense nucleic acids, but is not limited thereto. The agent may specifically be an agent for detecting a gene mutation in clonal hematopoiesis, for example, a primer, a probe, or an antisense nucleic acid.

As examples of agents that can be used to confirm the presence of mutations in clonal hematopoiesis-related genes, probe sequence information is provided in Tables 6 to 9 below.

Chromosome	Start	Stop	Gene	Probe Sequence	sequence number
2	25564672	25564792	DNMT3A	GCGGGCGGCGGCGGCGGCGAGAGCAGAGGACGAGCCGGGACGCGGCGCCGCGGCACCAGGGCGCGCAGCCGGGCCGGCCCGACCCCACCGGCCATACGGTAATGAGCGCCGCTGCTGGCG	One
2	25537472	25537592	DNMT3A	CAGAGCCAACCAGGCCTGCAGCCTGAGCTCAGACCTCAGCTTTCTCTGGGCCCAGGCACCGGTCCTGAAGTGAGGTGACATTCATTTCACTACATCCTGTGACTGCACTCACTTCCTCCA	2
2	25537290	25537410	DNMT3A	GGGCCTCTGGGGGAGGCTGGGATTTCCCAGGGGCTGGTTGGGGTGGGAGGGAGGATGCGGGAAGCTTTCAAAGGAGAGAGAGGCTGTGAATGAAGAGAAGGGAGGAGGCGGAGGCAGCTG	3
2	25537170	25537290	DNMT3A	GAGGTCCCAGAAGTGGCACCCATTGGCCGCAGCGACACGGAGGGGAGACTGCTGCCACTCCAAGGCTCTCAGAGGCTGCTGGGGTAGGAGGTGGCCCTGAAGGGAGCCTGCCTCGGTTTC	4
2	25537050	25537170	DNMT3A	TCTGCTCTCTGGGGTGCTGGTGAGGCCAGAAGGAGGAGCAACACCCCAGGCCTCACAGGCCAGGTGTGGCCCTGGGCTGAGGGCCTGACCCAGGGAGGGTGGGGTGCTCCGCCCCACTGA	5
2	25536966	25537086	DNMT3A	CTGACCCAGGGAGGGTGGGGTGCTCCGCCCCACTGATCCTCTTCTCTCCCCCACAGGTGGAGCCATCGAAGCCCCCACCCACAGGCTGACAGAGGCACCGTTCACCAGAGGGCTCAACAC	6
2	25536846	25536966	DNMT3A	CGGGATCTATGTTTAAGTTTTAACTCTCGCCTCCAAAGACCACGATAATTCCTTTCCCCAAAGCCCAGCAGCCCCCCAGCCCCGCGCAGCCCCAGCCTGCCTCCCGGCGCCCAGATGCCCG	7
2	25536726	25536846	DNMT3A	CCATGCCCTCCAGCGGCCCCGGGGACACCAGCAGCTCTGCTGCGGAGCGGGAGGAGGACCGAAAGGTGAGCAGCGGGCTCTGCTGGGGACCCTCTGGCCTGGGAGAGGGAGAGCCCTGTC	8
2	25523000	25523120	DNMT3A	GCCTGCAGGACGGAGAGGAGCAGGAGGAGCCGCGTGGCAAGGAGGAGCGCCAAGAGCCCAGCACCACGGCACGGAAGGTGGGGCGGCCTGGGAGGAAGCGCAAGCACCCCCCGGTGAGTG	9
2	25504380	25504500	DNMT3A	CCTCCCAGGGCCCTGCCAGGTTGTTTTGCATATGTGCATTTTAATTTCAAAAAAGTCTTCCTTCCAAGCGTGTATGATGAAATGAGTAAATTGATTAATTGGCGTAACTTATTTTGCATGG	10
2	25504282	25504402	DNMT3A	TGGCGTAACTTATTTTGCATGGATCCAACCTAATGTTCATGCAGGATAGAGAACATTTCCAGAATACAAATTTCCAAACTTATTAAGAAGCTTTTATTCATTTATTATTCGACAAATATG	11
2	25504745	25504865	DNMT3A	TTGCTGTATTTGTAGCCCCTGGCCTGGGCACTCAAGGGCAGCAGATACCCTGTTTGCCTCCCTGAGTGCAGAGGTCCTGAGCCCACCCTAGTTGGGCTGACTCAACTGGAAATTTGGTTG	12
2	25504650	25504770	DNMT3A	GCTGACTCAACTGGAAATTTGGTTGTGACAGTGGCGTGGGGAGAGGGCTGGGTGATTGTATTCTGTGTACTGCCCAGCCCAGGCCTCTTCATCTGGGGACTTTTTGGCCTAACCCTGGAA	13
2	25504555	25504675	DNMT3A	GGGACTTTTTGGCCTAACCCTGGAAGCCTGGAAAGTTGCCCACTTTTCTCTTTCAGGTTAAGCCAGCAATTTCAAGGGCCAACCGAGCTGTAAACATGTTAGTAATGAGGACAACTAGCAT	14
2	25505503	25505623	DNMT3A	ACTGATCTAACCCTCCTCTTGTGTATCTTTCTTACATTTGCAGGTGGAAAGCGGTGACACGCCAAAGGACCCTGCGGTGATTCTCCAAGTCCCCATCCATGGCCCAGGACTCAGGCGCCTC	15
2	25505383	25505503	DNMT3A	AGAGCTATTACCCAATGGGGACTTGGAGAAGCGGAGTGAGCCCCAGCCAGAGGAGGGGAGCCCTGCTGGGGGGCAGAAGGGCGGGGCCCCAGCAGAGGGAGAGGGTGCAGCTGAGACCCT	16
2	25505263	25505383	DNMT3A	GCCTGAAGCCTCAAGAGCAGTGGAAAATGGCTGCTGCACCCCCAAGGAGGGCCGAGGAGCCCCTGCAGAAGCGGGTGAGTCCTCAGCACCAGGGGCAGCCTCTTCTGGGCCCACCAGCAT	17
2	25505144	25505263	DNMT3A	ACCCTGAGAGTCAGGGACTTGGCTCTCCAGCAGGTCCCAGGAAGGATGGTCTGGGTCGTGGCTAAAGGTCTGCTTGCCAAGGCTATGCCTGGAGGCTACTGGCTGGATGCAGCCTGCG	18
2	25498330	25498450	DNMT3A	ACCAACGAACTGGTCCCTTTGTTCTTCCCTCTCCACAGGCAAAGAACAGAAGGAGACCAACATCGAATCCATGAAAATGGAGGCAAGTGTTCCACCCCCAGGGGCTCTGTGGATCCTGAT	19
2	25497882	25498002	DNMT3A	CGTCAGTGCCCACCCTAATGCCCTAATGTCTGTCTCTCTGTCCTAGGGCTCCCGGGGCCGGCTGCGGGGTGGCTTGGGCTGGGAGTCCAGCCTCCGTCAGCGGCCCATGCCGAGGCTCAC	20
2	25497762	25497882	DNMT3A	CTTCCAGGCGGGGGACCCCTACTACATCAGCAAGCGCAAGCGGGACGAGTGGGCTGGCACGCTGGAAAAGGGAGGGTGAGGCGCCTTCTGGCCTGGGCCCCAGCCCGTCCTGCAGAACTGGA	21
2	25475123	25475243	DNMT3A	CTAGACAAAGAAAATGTTCCCTCCCTCCCCCCCGCCGCCCCCCTCCCCTCCCAGTGGCCCCCTCCGCCCCAGCCCCATCGCCCCCTTCCCCTCCCCCAAGACGGGCAGCTACTTCCAGA	22
2	25475003	25475123	DNMT3A	GCTTCAGGGCCGCGGCTCACACCTGAGCGCGACTGCAGAGGGGCTGCACCTGGCCTTATGGGTAGGTTCCTGGACGGAGCCCCGGGGAGACCGGGGGCGGGGAGGCGGCAGGGCCGGTGG	23
2	25472499	25472619	DNMT3A	ATCAAGGATTTCATTCTCCTTTGTAGGGATCCTGGAGCGGGTTGTGAGAAGGAATGGGCGCGTGGATCGTAGCCTGAAAGACGAGTGTGATACGGTGAAAGGATGGAGGCTGTGCAATGG	24
2	25471013	25471133	DNMT3A	TTGTTTCCCCAGGCTGAGAAGAAAGCCAAGGTCATTGCAGGAATGAATGCTGTGGAAGAAAACCAGGGGCCCGGGGAGTCTCAGAAGGTGGAGGAGGCCAGCCCTCCTGCTGTGCAGCAG	25
2	25470893	25471013	DNMT3A	CCCACTGACCCCGCATCCCCCACTGTGGCTACCACGCCTGAGCCCGTGGGGTCCGATGCTGGGGACAAGAATGCCACCAAAGCAGGCGATGACGAGCCAGAGTACGAGGTGAGGCGGCTTC	26
2	25470538	25470658	DNMT3A	GTGACCACTGTGTAATGATTTCTGCTCCTTGGGGCTCCAGGACGGCCGGGGCTTTGGCATTGGGGAGCTGGTGTGGGGGAAACTGCGGGGCTTCTCCTGGTGGCCAGGCCGCATTGTGTC	27
2	25470418	25470538	DNMT3A	TTGGTGGATGACGGGCCGGAGCCGAGCAGCTGAAGGCACCCGCTGGGTCATGTGGTTCGGAGACGGCAAATTCTCAGTGGTAAGTTGTGGGGTTTGGCAGTAGCCTGGGGTGGGGGAAGG	28
2	25469913	25470033	DNMT3A	CCCCAGGTGTGTGTTGAGAAGCTGATGCCGCTGAGCTCGTTTTGCAGTGCGTTCCACCAGGCCACGTACAACAAGCAGCCCATGTACCGCAAAGCCATCTACGAGGTCCTGCAGGTGAGT	29
2	25469567	25469687	DNMT3A	AGCCTGTCCTGACAACCCCAACCCTGGCGTGTCACCCTCCAGGTGGCCAGCAGCCGCGCGGGGAAGCTGTTCCCGGTGTGCCACGACAGCGATGAGAGTGACACTGCCAAGGCCGTGGAG	30
2	25469447	25469567	DNMT3A	GTGCAGAACAAGCCCATGATTGAATGGGCCCTGGGGGGCTTCCAGCCTTCTGGCCCTAAGGGCCTGGAGCCACCAGAAGGTAAATGAGGGCACCCAGCTTTCTGGGACCCCTGCCCGCCA	31
2	25469103	25469223	DNMT3A	CCTGTCAGCCTGTAACTGACCTTGGCACCTGCTTTCCTCCTCCAGAAGAGAAGAATCCCTACAAAGAAGTGTACACGGACATGTGGGTGGAACCTGAGGCAGCTGCCTACGCACCACCTC	32
2	25468983	25469103	DNMT3A	CACCAGCCAAAAAGCCCCGGAAGAGCACAGCGGAGAAGCCCAAGGTCAAGGAGATTATTGATGAGCGCACAAGAGGTAGTTGGCCTGCTTCTGGAGAGGGTGGCACCAGGAGGCCTGCAT	33
2	25468851	25468971	DNMT3A	CGCGGCCTCCTTCTGACGCCAGCTCTCCTCCCCTTGCAGAGCGGCTGGTGTACGAGGTGCGGCAGAAGTGCCGGAACATTGAGGGTAAGTTTGTTGCCTGGGAACTCTGGCACTCCTAGTG	34
2	25468101	25468221	DNMT3A	CCCCTCCCTCTGCTTTTCCAGACATCTGCATCTCCTGTGGGAGCCTCAATGTTACCCTGGAACACCCCCTCTTCGTTGGAGGAATGTGCCAAAACTGCAAGGTAGGAGCACACCCACCCAG	35
2	25467405	25467525	DNMT3A	CTAGAACTGCTTTCTGGAGTGTGCGTACCAGTACGACGACGACGGCTACCAGTCCTACTGCACCATCTGCTGTGGGGGCCGTGAGGTGCTCATGTGCGGAAACAACAACTGCTGCAGGTG	36
2	25467115	25467235	DNMT3A	CTCCTCTGCTCACTGGGTCTCCTTCCAGGTGCTTTTGCGTGGAGTGTGTGGACCTCTTGGTGGGGCCGGGGGCTGCCCAGGCAGCCATTAAGGAAGACCCCTGGAACTGCTACATGTGCG	37
2	25466995	25467115	DNMT3A	GGCACAAGGGTACCTACGGGCTGCTGCGGCGGCGAGAGGACTGGCCCTCCCGGCTCCAGATGTTCTTCGCTAATAACCACGACCAGGAATTTGTGAGTGCTGGGCCTGGGGCGCGGTCTC	38
2	25466749	25466869	DNMT3A	GTTGGCTCACTGCTTAGGACCCTCCAAAGGTTTACCCACCTGTCCCAGCTGAGAAGAGGAAGCCCATCCGGGTGCTGTCTCTCTTTGATGGAATCGCTACAGGTGAGGGGTGCAGGCCC	39
2	25464503	25464623	DNMT3A	CCAGGGAGATGGCTCCAAGTAACGGTGCTGTCTGCTGGCTGGTGCAGGGCTCCTGGTGCTGAAGGACTTGGGCATTCAGGTGGACCGCTACATTGCCTCGGAGGTGTGTGAGGACTCCAT	40
2	25464383	25464503	DNMT3A	CACGGTGGGCATGGTGCGGCACCAGGGGAAGATCATGTACGTCGGGGACGTCCGCAGCGTCACACAGAAGCATGTATGTCCATGCTGTGGGGCGCAGCCCGTCTTCCCCTCCCTGCACAC	41
2	25463493	25463613	DNMT3A	CTTTATCCTCCCAGATCCAGGAGTGGGGCCCATTCGATCTGGTGATTGGGGGCAGTCCCTGCAATGACCTCTCCATCGTCAACCCTGCTCGCAAGGGCCTCTACGGTAGGTACCATCCTG	42
2	25463245	25463365	DNMT3A	CTATGCAGACAGCCCCAGCTGATGGCTTTCTCTTCCGACCTCTCAGAGGGCACTGGCCGGCTCTTCTTTGAGTTCTACCGCCTCCTGCATGATGCGCGGCCCAAGGAGGGAGATGATCGC	43
2	25463125	25463245	DNMT3A	CCCTTCTTCTGGCTCTTTGAGAATGTGGTGGCCATGGGCGTTAGTGACAAGAGGGACATCTCGCGATTTCTCGAGGTATAGCCAGCAACCTTGGTTTGGCCAGCTCACTAATGGCTTCTA	44
2	25461981	25462101	DNMT3A	CGTCTCCTGTTTTGTAGTCCAACCCTGTGATGATTGATGCCAAGAAGGTCAGCTGCACACAGGGCCCGCTACTTCTGGGGTAACCTTCCCGGTATGAACAGGTTGGTGAAAGCTCCTG	45
2	25459779	25459899	DNMT3A	TTACAGTCTCTCTTCTGCCTCCTAGGCCGTTGGCATCCACTGTGAATGATAAGCTGGAGCTGCAGGAGTGTCTGGAGCATGGCAGGATAGCCAAGGTCAGCTCCAGCGTCTAGAACCTCT	46
2	25458574	25458694	DNMT3A	TTCAGCAAAGTGAGGACCATTACTACGAGGTCAAACTCCATAAAGCAGGGCAAAGACCAGCATTTTCCTGTCTTCATGAATGAGAAAGAGGACATCTTATGGTGCACTGAAATGGAAAGG	47
2	25457218	25457338	DNMT3A	CCTGGTCTGGCCAGCACTCACCCTGCCCTCTCTGCCTTTTCTCCCCCAGGGTATTTGGTTTCCCAGTCCACTATACTGACGTCTCCAACATGAGCCGCTTGGCGAGGCAGAGACTGCTGG	48
2	25457098	25457218	DNMT3A	GCCGGTCATGGAGCGTGCCAGTCATCCGCCACCTCTTCGCTCCGCTGAAGGAGTATTTTGCGTGTGTGTAAGGGACATGGGGGCAAACTGAGGTAGCGACACAAAGTTAAACAAACAAAC	49

Chromosome	Start	Stop	Gene	Probe_Sequence	sequence number
4	106155077	106155197	TET2	GGATGGCCCCGAAGCAAGCCTGATGGAACAGGATAGAACCAACCATGTTGAGGGCAACAGACTAAGTCCATTCCTGATACCATCACCTCCCATTTGCCAGACAGAACCTTCTGGCTACAAA	50
4	106156157	106156277	TET2	TTCAGGTTCCAGCAGCAATTTGCAAGCTCCTGGTGGCAGCTCTGAACGGTATTTAAAACAAAATGAAATGAATGGTGCTTACTTCAAGCAAAGCTCAGTGTTCACTAAGGATTCCTTTTC	51
4	106156277	106156397	TET2	TGCCACTACCACACCACCACCACCATCACAATTGCTTCTTTCTCCCCCTCCTCCTCTTCCACAGGTTCCTCAGCTTCCTTCAGAAGGAAAAAGCACTCTGAATGGTGGAGTTTTAGAAGA	52
4	106156397	106156517	TET2	ACACCACCACTACCCCAACCAAAGTAACAACACTTTTAAGGGAAGTGAAAATAGAGGGTAAACCTGAGGCACCACCTTCCCAGAGTCCTAATCCATCTACACATGTATGCAGCCCTTC	53
4	106156517	106156637	TET2	TCCGATGCTTTCTGAAAGGCCTCAGAATAATTGTGTGAACAGGAATGACATACAGACTGCAGGGACAATGACTGTTCCATTGTGTTCTGAGAAAACAAGACCAATGTCAGAACACCTCAA	54
4	106156637	106156757	TET2	GCATAACCCACCAATTTTTGGTAGCAGTGGAGAGCTACAGGACAACTGCCAGCAGTTGATGAGAAACAAAGAGCAAGAGATTCTGAAGGGTCGAGACAAGGAGCAAACACGAGATCTTGT	55
4	106156757	106156877	TET2	GCCCCCAACACAGCACTATCTGAAACCAGGATGGATTGAATTGAAGGCCCCTCGTTTTCACCAAGCGGAATCCCATCTAAAACGTAATGAGGCATCACTGCCATCAATTCTTCAGTATCA	56
4	106156877	106156997	TET2	ACCCAATCTCTCCAATCAAATGACCTCCAAACAATACACTGGAAATTCCAACATGCCTGGGGGGCTCCCAAGGCAAGCTTACACCCAGAAAACAACACAGCTGGAGCACAAGTCACAAAT	57
4	106156997	106157117	TET2	GTACCAAGTTGAAATGAATCAAGGGCAGTCCCAAGGTACAGTGGACCAACATCTCCAGTTCCAAAAACCCTCACACCAGGTGCACTTCTCCAAAACAGACCATTTACCAAAAGCTCATGT	58
4	106157117	106157237	TET2	GCAGTCACTGTGTGGCACTAGATTTCATTTTCAACAAAGAGCAGATTCCCAAACTGAAAAACTTATGTCCCCAAGTGTTGAAACAGCACTTGAATCAACAGGCTTCAGAGACTGAGCCATT	59
4	106157237	106157357	TET2	TTCAAACTCACACCTTTTGCAACATAAGCCTCATAAACAGGCAGCACAAACACAACCATCCCAGAGTTCACATCTCCCTCAAAACCAGCAACAGCAGCAAAAATTACAAATAAAGAATAA	60
4	106155197	106155317	TET2	GCTCCAGAATGGAAGCCCACTGCCTGAGAGAGCTCATCCAGAAGTAAATGGAGACACCAAGTGGCACTCTTTCAAAAGTTATTATGGAATACCCTGTATGAAGGGAAGCCAGAATAGTCG	61
4	106157357	106157477	TET2	AGAGGAAATACTCCAGACTTTTCCTCACCCCCAAAGCAACAATGATCAGCAAAGAGAAGGATCATTCTTTGGCCAGACTAAAGTGGAAGAATGTTTTCATGGTGAAAATCAGTATTCAAA	62
4	106157477	106157597	TET2	ATCAAGCGAGTTCGAGACTCATAATGTCCAAATGGGACTGGAGGAAGTACAGAATATAAATCGTAGAAATTCCCCTTATAGTCAGACCATGAAATCAAGTGCATGCAAAATACAGGTTTC	63
4	106157597	106157717	TET2	TTGTTCAAACAATACACACCTAGTTTCAGAGAATAAAGAACAGACTACACATCCTGAACTTTTTGCAGGAAACAAGACCCAAAACTTGCATCACATGCAATATTTTCCAAATAATGTGAT	64
4	106157717	106157837	TET2	CCCAAAGCAAGATCTTCTTCACAGGTGCTTTCAAGAACAGGAGCAGAAGTCACAACAAGCTTCAGTTCTACAGGGATATAAAAATAGAAACCAAGATATGTCTGGTCAACAAGCTGCGCA	65
4	106157837	106157957	TET2	ACTTGCTCAGCAAAGGTACTTGATACATAACCATGCAAATGTTTTTCCTGTGCCTGACCAGGGAGGAAGTCACACTCAGACCCCTCCCCAGAAGGACACTCAAAAGCATGCTGCTCTAAG	66
4	106157957	106158077	TET2	GTGGCATCTCTTACAGAAGCAAGAACAGCAGCAAACACAGCAACCCCAAACTGAGTCTTGCCATAGTCAGATGCACAGGCCAATTAAGGTGGAACCTGGATGCAAGCCACATGCCTGTAT	67
4	106158077	106158197	TET2	GCACACAGCACCACCAGAAAACAAAACATGGAAAAAGGTAACTAAGCAAGAGAATCCACCTGCAAGCTGTGATAATGTGCAGCAAAAGAGCATCATTGAGACCATGGAGCAGCATCTGAA	68
4	106158197	106158317	TET2	GCAGTTTCACGCCAAGTCGTTATTTGACCATAAGGCTCTTACTCTCAAATCACAGAAGCAAGTAAAAGTTGAAATGTCAGGGCCAGTCACAGTTTTGACTAGACAAACCACTGCTGCAGA	69
4	106158317	106158437	TET2	ACTTGATAGCCACACCCCAGCTTTAGAGCAGCAAACAACTTCTTCAGAAAAGACACCAACCAAAAGAACAGCTGCTTCTGTTCTCAATAATTTTATAGAGTCACCTTCCAAATTACTAGA	70
4	106158437	106158557	TET2	TACTCCTATAAAAAATTTATTGGATACACCTGTCAAGACTCAATATGATTTCCCATCTTGCAGATGTGTAGGTAAGTGCCAGAAATGTACTGAGACACATGGCGTTTATCCAGAATTAGC	71
4	106155317	106155437	TET2	TGTGAGTCCTGACTTTACACAAGAAAGTAGAGGGTATTCCAAGTGTTTGCAAAATGGAGGAATAAAACGCACAGTTAGTGAACCTTCTCTCTCTGGGCTCCTTCAGATCAAGAAATTGAA	72
4	106158557	106158677	TET2	AAATTTATCTTCAGATATGGGATTTTCCTTCTTTTTTTAAATCTTGAGTCTGGCAGCAATTTGTAAAGGCTCATAAAAATCTGAAGCTTACATTTTTTGTCAAGTTACCGATGCTTGTGT	73
4	106158677	106158797	TET2	CTTGTGAAAGAGAACTTCACTTACATGCAGTTTTTCCAAAAGAATTAAATAATCGTGCATGTTTATTTTTCCCTCTCTTCAGATCCTGTAAAATTTGAATGTATCTGTTTTAGATCAATT	74
4	106158797	106158917	TET2	CGCCTATTTAGCTCTTTGTATATTATCTCCTGGAGAGACAGCTAGGCAGCAAAAAAACAATCTATTAAAATGAGAAAATAACGACCATAGGCAGTCTAATGTACGAACTTTAAATATTTT	75
4	106158917	106159037	TET2	TTAATTCAAGGTAAAATATATTAGTTTCACAAGATTTCTGGCTAATAGGGAAATTATTATCTTCAGTCTTCATGAGTTGGGGGAAATGATAATGCTGACACTCTTAGTGCTCCTAAAGTT	76
4	106159037	106159156	TET2	TCCTTTTCTCCATTTATACATTTGGAATGTTGTGATTTATATTCATTTTGATTCCCTTTTCTCTAAAATTTCATCTTTTTGATTAAAAAAATATGATACAGGCATACCTCAGAGATATTG	77
4	106155437	106155557	TET2	ACAAGACCAAAAGGCTAATGGAGAAAGACGTAACTTCGGGGTAAGCCAAGAAAGAAATCCAGGTGAAAGCAGTCAACCAAATGTCTCCGATTTGAGTGATAAGAAAGAAATCTGTGAGTTC	78
4	106155557	106155677	TET2	TGTAGCCCAAGAAAATGCAGTTAAAGATTTCACCAGTTTTTCAACACATAACTGCAGTGGGCCTGAAAATCCAGAGCTTCAGATTCTGAATGAGCAGGAGGGGAAAAGTGCTAATTACCA	79
4	106155677	106155797	TET2	TGACAAGAACATTGTATTACTTAAAAACAAGGCAGTGCTAATGCCTAATGGTGCTACAGTTTCTGCCTCTTCCGTGGAACACACACATGGTGAACTCCTGGAAAAAACACTGTCTCAATA	80
4	106155797	106155917	TET2	TTATCCAGATTGTGTTTCCATTGCGGTGCAGAAAACCACATCTCACATAAATGCCATTAACAGTCAGGCTACTAATGAGTTGTCCTGTGAGATCACTCACCCATCGCATACCTCAGGGCA	81
4	106155917	106156037	TET2	GATCAATTCCGCACAGACCTCTAACTCTGAGCTGCCTCCAAAGCCAGCTGCAGTGGTGAGTGAGGCCTGTGATGCTGATGATGCTGATAATGCCAGTAAACTAGCTGCAATGCTAAATAC	82
4	106156037	106156157	TET2	CTGTTCCTTTCAGAAACCAGAACAACTACAACAACAAAAATCAGTTTTTGAGATATGCCCATCTCCTGCAGAAAATAACATCCAGGGAACCACAAAGCTAGCGTCTGGTGAAGAATTCTG	83
4	106161458	106161578	TET2	AATAAAACGAGGTATGCCTGTATTTTTAAAAAAAGCTTTTTGTTAAAATTCAGGATATGTAATAGGTCTGTAGGAATAGTGAAATATTTTTGCTGATGGATGTAGATATATACGTGGATA	84
4	106162538	106162658	TET2	AGGAGCAGGTCCTAATGTGGCAGCTATTAGAGAAATCATGGAAGAAAGGTAATTAACGCAAAGGCACAGGGCAGATTAACGTTTATCCTTTTGTATATGTCAATTTTTCCAGCCTTCA	85
4	106162658	106162778	TET2	CACACAAAGCAGTAAACAATTGTAAATTGAGTAATTATTAGTAGGCTTAGCTATTCTAGGGTTGCCAACACTACACACTGTGCTATTCACCAGAGAGTCACAATATTTGACAGGACTAAT	86
4	106162778	106162898	TET2	AGTCTGCTAGCTGGCACAGGCTGCCCACTTTGCGATGGATGCCAGAAAACCCAGGCATGAACAGGAATCGGCCAGCCAGGCTGCCAGCCACAAGGTACTGGCACAGGCTCCAACGAGAGG	87
4	106162898	106163018	TET2	TCCCACTCTGGCTTTCCCACCTGATAATAAAGTGTCAAAGCAGAAAGACTGGTAAAGTGTGGTATAAGAAAAGAACCACTGAATTAAATTCACCTAGTGTTGCAAATGAGTACTTATCTC	88
4	106163018	106163138	TET2	TAAGTTTTCTTTTACCATAAAAAGAGAGCAAGTGTGATATGTTGAATAGAAAGAGAAACATACTATTTACAGCTGCCTTTTTTTTTTTTTTTCGCTATCAATCACAGGTATACAAGTACT	89
4	106163138	106163258	TET2	TGCCTTTACTCCTGCATGTAGAAGACTCTTATGAGCGAGATAATGCAGAGAAGGCCTTTCATATAAATTTATACAGCTCTGAGCTGTTCTTCTTCTAGGGTGCCTTTTCATTAAGAGGTA	90
4	106163258	106163378	TET2	GGCAGTATTATTATTAAAGTACTTAGGATACATTGGGGCAGCTAGGACATATTCAGTATCATTCTTGCTCCATTTCCAAATTATTCATTTCTAAATTAGCATGTAGAAGTTCACTAAATA	91
4	106163378	106163498	TET2	ATCATCTAGTGGCCTGGCAGAAATAGTGAATTTCCCTAAGTGCCTTTTTTTTGTTGTTTTTTTGTTTTGTTTTTTAAACAAGCAGTAGGTGGTGCTTTGGTCATAAGGGAAGATATAGTC	92
4	106163498	106163618	TET2	TATTTCTAGGACTATTCCATATTTTCCATGTGGCTGGATACTAACTATTTGCCAGCCTCCTTTTCTAAATTGTGAGACATTCTTGGAGGAACAGTTCTAACTAAAATCTATTATGACTCC	93
4	106163618	106163738	TET2	CCAAGTTTTAAAATAGCTAAATTTAGTAAGGGAAAAAATAGTTTATGTTTTAGAAGACTGAACTTAGCAAACTAACCTGAATTTTGTGCTTTGTGAAATTTTATATCGAAATGAGCTTTC	94
4	106161578	106161698	TET2	GAGATGAAGATCTTAATTATAGCTATGCAGCATAGATTTAGTCAAAGACATTTGAAAAGACAAATGTTAAATTAGTGTGGCTAATGACCTACCCGTGCCATGTTTTCCCTCTTGCAATGA	95
4	106163738	106163858	TET2	CCATTTTCACCCACATGTAATTTACAAAATAGTTCATTACAATTATCTGTACATTTTGATATTGAGGAAAAACAAGGCTTAAAAACCATTATCCAGTTTGCTTGGCGTAGACCTGTTTAA	96
4	106163858	106163978	TET2	AAAATAATAAACCGTTCATTTCTCAGGATGTGGTCATAGAATAAAGTTATGCTCAAATGTTCAAATATTTTGATTGCCTCTTGAATTCATTTGCTAATTGTATGTGTGTGTGTTTCTGTG	97
4	106161698	106161818	TET2	GATACCCCACACTGTGTAGAAGGATGGAGGGAGGACTCCTACTGTCCCTCTTTGCGTGTGGTTATTAAGTTGCCTCACTGGGCTAAAACACCACACATCTCATAGATAATATTTGGTAAG	98
4	106161818	106161938	TET2	TTGTAATCGTCTTCACTCTTCTCTTATCACCCACCCCTATCTTCCCACTTTTCCATCTTTGTTGGTTTGCAACAGCCCCTTCTTTTTGCCTGACTCTCCAGGATTTTCTCTCATCATAAA	99
4	106161938	106162058	TET2	TTGTTCTAAAGTACATACTAATATGGGTCTGGATTGACTATTCTTATTTGCAAAACAGCAATTAAATGTTATAGGGAAGTAGGAAGAAAAAGGGGTATCCTTGACAATAAACCAAGCAAT	100
4	106162058	106162178	TET2	ATTCTGGGGGTGGGATAGAGCAGGAAATTTTATTTTTAATCTTTTAAAATCCAAGTAATAGGTAGGCTTCCAGTTAGCTTTAAATGTTTTTTTTTTCCAGCTCAAAAAATTGGATTGTAG	101
4	106162178	106162298	TET2	TTGATACTACATATAATACATTCTAATTCCCTCACTGTATTCTTTGTTTAGTTTCATTTATTTGGTTTAAAATAATTTTTTATCCCATATCTGAAATGTAATATATTTTTATCCAACAAC	102
4	106162298	106162418	TET2	CAGCATGTACATATACTTAATTATGTGGCACATTTTCTAATAGATCAGTCCATCAATCTACTCATTTTAAAGAAAAAAAAATTTTAAAGTCACTTTTAGAGCCCTTAATGTGTAGTTGGG	103
4	106162418	106162538	TET2	GGTTAAGCTTTGTGGATGTAGCCTTTATATTTAGTATAATTGAGGTCTAAAATAATAATCTTCTATTATCTCAACAGAGCAAATTATTGAAAAAGATGAAGGTCCTTTTTATACCCATCT	104
4	106163977	106164097	TET2	GGGTTTCTTTAAGGTTTGGACAGAAGGGTAAAGCTATTAGGATTGAAAGAGTCATCTATACTGGTAAAGAAGGCAAAAGTTCTCAGGGATGTCCTATTGCTAAGTGGGTAAGTGTGACTT	105
4	106164710	106164830	TET2	ATGGTGATCCACGCAGGTGGTTCGCAGAAGCAGCAGTGAAGAGAAGCTACTGTGTTTGGTGCGGGAGCGAGCTGGCCACACCTGTGAGGCTGCAGTGATTGTGATTCTCATCCTGGTGTG	106
4	106164830	106164950	TET2	GGAAGGAATCCCGCTGTCTCTGGCTGACAAACTCTACTCGGAGCTTACCGAGACGCTGAGGAAATACGGCACGCTCACCAATCGCCGGTGTGCCTTGAATGAAGAGTAAGTGAAGCCCAG	107
4	106180731	106180851	TET2	ATAATGCTATCCATAGCAATGAATTTGGTCTTTTGATTTTTCAGGAGAACTTGCGCCTGTCAGGGGCTGGATCCAGAAACCTGTGGTGCCTCCTTCTCTTTTGGTTGTTCATGGAGCATG	108
4	106180851	106180971	TET2	TACTACAATGGATGTAAGTTTGCCAGAAGCAAGATCCCAAGGAAGTTTAAGCTGCTTGGGGATGACCCAAAAGAGGTTTGTTTACTTCCTGATGTATAATCGCTTTATTTTTCATAGAGA	109
4	106182900	106183020	TET2	ATTCACTTTATACAGGAAGAGAAACTGGAGTCTCATTTGCAAAACCTGTCCACTCTTATGGCACCAACATATAAGAAACTTGCACCTGATGCATATAATAATCAGGTAAGTTTAAATAAT	110
4	106190715	106190835	TET2	GTAAGAGTAAAACTAACTACTTTCGCATTCACACACACTTTTATTTTTCAGATTGAATATGAACACAGAGCACCAGAGTGCCGTCTGGGTCTGAAGGAAGGCCGTCCATTCTCAGGGGTC	111
4	106190835	106190955	TET2	ACTGCATGTTTGGACTTCTGTGCTCATGCCCACAGAGACTTGCACAACATGCAGAATGGCAGCACATTGGTAAGTTGGGCTGAGGACAGCTTAGCAGCTGTTGAGTCTGTTCTCACACTG	112
4	106193718	106193838	TET2	AGGTATGCACTCTCACTAGAGAAGACAATCGAGAATTTGGAGGAAAACCTGAGGATGAGCAGCTTCACGTTCTGCCTTTATACAAAGTCTCTGACGTGGATGAGTTTGGGAGTTGTGGAAG	113
4	106193838	106193958	TET2	CTCAGGAGGAGAAAAAACGGAGTGGTGCCATTCAGGTACTGAGTCTTTTCGGCGAAAAGTCAGGATGTTAGCAGAGCCAGTCAAGACTTGCCGACAAAGGAAACTAGAAGCCAAGAAAG	114
4	106193958	106194078	TET2	CTGCAGCTGAAAAGCTTTCCTCCCTGGAGAACAGCTCAAATAAAAATGAAAAGGAAAAGTCAGCCCCATCACGTACAAAACAAACTGAAAACGCAAGCCAGGCTAAACAGTTGGCAGGTA	115
4	106196122	106196241	TET2	TTTAAGTATCCTCACTAGCCTTCATAAAATAATCATCAACATCAAAGATACCTGTTTCTGTTCTCTCTTACCCTGTCCACAGAACTTTTGCGACTTTCAGGACCAGTCATGCAGCAGTC	116
4	106196292	106196412	TET2	GAGACCCCAGCAGCAGCAGCCACATCACCCTCAGACAGAGTCTGTCAACTCTTATTCTGCTTCTGGATCCACCAATCCATACATGAGACGGCCCAATCCAGTTAGTTAGTCCTTATCCAAACTC	117
4	106197372	106197492	TET2	CCAGCATAAGAGCATGAATGAGCCAAAACATGGCTTGGCTCTTTGGGAAGCCAAAATGGCTGAAAAAGCCCGTGAGAAAGAGGAAGAGTGTGAAAAGTATGGCCCAGACTATGTGCCTCA	118
4	106197492	106197612	TET2	GAAATCCCATGGCAAAAAAGTGAAACGGGAGCCTGCTGAGCCACATGAAACTTCAGAGCCCACTTACCTGCGTTTCATCAAGTCTCTTGCCGAAAGGACCATGTCCGTGACCACAGACTC	119
4	106197612	106197732	TET2	CACAGTAACTACATCTCCATATGCCTTCACTCGGGTCACAGGGCCTTACAACAGATATATATGATATCACCCCCTTTTGTTGGTTACCTCACTTGAAAAGACCACAACCAACCTGTCAGT	120
4	106196412	106196532	TET2	TTCACACACTTCAGATATCTATGGAAGCACCAGCCCTATGAACTTCTATTCCACCTCATCTCAAGCTGCAGGTTCATATTTGAATTCTTCTAATCCCATGAACCCTTACCCTGGGCTTTT	121
4	106196532	106196652	TET2	GAATCAGAATACCCAATATCCATCATATCAATGCAATGGAAACCTATCAGTGGACAACTGCTCCCCATATCTGGGTCCTATTCTCCCCAGTCTCAGCCGATGGATCTGTATAGGTATCC	122
4	106196652	106196772	TET2	AAGCCAAGACCCTCTGTCTAAGCTCAGTCTACCACCCATCCATACACTTTACCAGCCAAGGTTTGGAAATAGCCAGAGTTTTACATCTAAATACTTAGGTTATGGAAACCAAAATATGCA	123
4	106196772	106196892	TET2	GGGAGATGGTTTCAGCAGTTGTACCATTAGACCAAATGTACATCATGTAGGGAAATTGCCTCCTTATCCCACTCATGAGATGGATGGCCACTTCATGGGAGCCACCTCTAGATTACCACC	124
4	106196892	106197012	TET2	CAATCTGAGCAATCCAAACATGGACTATAAAAATGGTGAACATCATTCACCTTCTCACATAATCCATAACTACAGTGCAGCTCCGGGCATGTTCAACAGCTCTCTTCATGCCCTGCATCT	125
4	106197012	106197132	TET2	CCAAAACAAGGAGAATGACATGCTTTCCCACACAGCTAATGGGTTATCAAAGATGCTTCCAGCTCTTAACCATGATAGAACTGCTTGTGTCCAAGGAGGCTTACACAAATTAAGTGATGC	126
4	106197132	106197252	TET2	TAATGGTCAGGAAAAGCAGCCATTGGCACTAGTCCAGGGTGTGGCTTCTGGTGCAGAGGACAACGATGAGGTCTGGTCAGACAGCGAGCAGAGCTTTCTGGATCCTGACATTGGGGGAGT	127
4	106197252	106197372	TET2	GGCCGTGGCTCCAACTCATGGGTCAATTCTCATTGAGTGTGCAAAGCGTGAGCTGCATGCCACAACCCCTTTAAAGAATCCCAATAGGAATCACCCCACCAGGATCTCCCTCGTCTTTTA	128

Chromosome	Start	Stop	Gene	Probe Sequence	sequence number
20	30946588	30946708	ASXL1	AACAGAAGAAGAAGAAGGAGCGCACGTGGGCCGAGGCCGCGCGCCTGGTGAGGCGGACAGCCGAGGGGGGCTCCGTGGGGCCCGGGGTGGGGGGGGCTCGCCGCGCACCCCCCCACTGGG	129
20	30954168	30954288	ASXL1	ACGTTTATATTTCTTCAAGGTATTAGAAAACTACTCGGATGCTCCAATGACACCAAAACAGATTCTGCAGGTCATAGAGGCAGAAGGACTAAAGGAAATGAGGTTTGTATTGTTCTTGTTG	130
20	30954286	30954406	ASXL1	TGCTTAACATGAGGGTTTCATAGGATATGTGTCTTTCTGTAGTGTAGTGATATGAGTAAGTACACCTGTGTATGTATTTGTGCTTCTGTGTCTGGTAGTGAGATGGAACTATCCATTTC	131
20	30956811	30956931	ASXL1	TTACAGTGGGACTTCCCCTCTCGCATGCCTCAATGCTATGCTACATTCCAATTCAAGAGGAGGAGAGGGGTTGTTTTATAAACTGCCTGGCCGAATCAGCCTTTTCACGCTCAAGGTAAG	132
20	30959880	30960000	ASXL1	TTGTAATTTGGCTCTGGGCAGAATTCTTGGCATAGTACGTGCTTTATGTGTGAACTGAATTATATTTCTTCATCTTAATTTTACAGGTGTGAGCCACTGCACCAGGCCCCTTCATCTTAA	133
20	30960000	30960120	ASXL1	TTTTAATATATCTTTGAATAAACACCATTGTATGAACCTGCTGTAAGCTTGGGAGTGGTCTGTTAGTCTACAGCTTGTGTCTGAGATGTGCTAATTGAATATTTGCTCAGTACCTCATCT	134
20	30960120	30960239	ASXL1	TAACTGCCTTTGGCTTTATGTTGCTTATCCTTCATAGTATCTTGTTCATTGGCCTTTTACATCCATAGGCATCACTTCTCTGATATTCGTTGTGCTCTTTTAATGGATTAATGGTTTGC	135
20	31015870	31015990	ASXL1	CTGCCTTGCTTTAAGAATTTGTAGGGTTTTGTTCACCTGAGTTGTACCTTGCTGTCACAGAAGGATGCCCTGCAGTGGTCTCGCCATCCAGCTACAGTGGAGGGAGAGGAGCCAGAGGAC	136
20	31015990	31016110	ASXL1	ACGGCTGATGTGGAGAGCTGTGGGTCTAATGAAGCCAGCACTGTGAGTGGTGAAAACGATGGTAAGGACCCTTTAATGGATGGGTGAGGGAGCCACAGCAGGCACTAGGGACTAACCTTT	137
20	31016116	31016236	ASXL1	CTCTCTTCCAGTATCTCTTGATGAAACATCTTCGAACGCATCCTGTTCTACAGAATCTCAGAGTCGACCTCTTTCCAATCCCAGGGACAGCTACAGAGCTTCCTCACAGGTAAGGAAGAG	138
20	31017127	31017247	ASXL1	TCTTGGAACGCAGGCGAACAAACAAAAGAAAAAAGACTGGGGTGATGCTGCCTCGAGTGTCCTGACTCCTCTGAAGGTAAACGGGGCCCACGTGGAATCTGCATCAGGTATGTGTAAACT	139
20	31017659	31017779	ASXL1	GCGGCTTGGTGATACTTTTGACCAGTGGAATGCTGTGCCTTCAGGGTTCTCGGGCTGCCACGCCGATGGCGAGAGCGGCAGCCCGTCCAGCAGCAGCAGCGGCTCTCTGGCCCTGGGCAG	140
20	31017779	31017899	ASXL1	CGCTGCTATTCGTGGCCAGGCCGAGGTCACCCAGGACCCTGCCCCGCTCCTGAGAGGCTTCCGGAAGCCAGCCACAGGTGAGTGGCGTGGCACTTATTTCTCTGCCTGTAAAGGGGGCCC	141
20	31019085	31019205	ASXL1	AAAAAGCTGAAATCTATACCTTGCTTCAAAAATCATAGGTCAAATGAAGCGCAACAGAGGGGAAGAAATAGATTTTGAGACACCTGGGTCCATTCTTGTCAACACCAACCTCCGTGCCCT	142
20	31019205	31019325	ASXL1	GATCAACTCTCGGACCTTCCATGCCTTACCATCACACTTCCAGCAGCAGCTCCTCTTCCTCCTGCCTGAAGTAGACAGACAGGTGCACATGGGCAGCCTCCCCTTTGCCTCTCTCTGGGT	143
20	31019373	31019493	ASXL1	TGATCCTTCTAGGTGGGGACGGATGGCCTGTTGCGTCTCAGCAGCAGTGCACTAAATAACGAGTTTTTTACCCATGCGGCTCAGAGCTGGCGGGAGCGCCTGGCTGATGGTATGTAGACT	144
20	31020621	31020740	ASXL1	TCCATAAGACAGACATTAATATCCCGAATGCACTTACTAGAAGAGGTTTATTTCTCCCTAGGTGAATTTACTCATGAGATGCAAGTCAGGATACGACAGGAAATGGAGAAGGAAAAGAA	145
20	31020742	31020862	ASXL1	TGGAACAATGGAAAGAAAAGTTCTTTGAAGACTACTATGGACAGAAGTAAGGCAGTTGGAGCTATGAGTCCTGGTCTGGGGTTTTGAGGGGATAGAGGTAGATGGTCTCAAAATAGCATA	146
20	31021043	31021163	ASXL1	TCCACTCTGGCCTGAAACTGATGGCTGTGATTTGATTTGCAGGCTGGGTTTGACCAAAGAAGAGTCATTGCAGCAGAACGTGGGCCAGGAGGAGGCTGAAATCAAAAGTGGCTTGTGTG	147
20	31021163	31021283	ASXL1	TCCCAGGAGAATCAGTGCGTATACAGCGTGGTCCAGCCACCCGACAGCGAGATGGGCATTTTAAGAAACGCTCTCGGCCAGATCTCCGAACCAGAGCCAGAAGGAATCTGTACAAAAAAC	148
20	31021283	31021403	ASXL1	AGGAGTCAGAACAAGCAGGGGTTGCTAAGGATGCAAAATCTGTGGCCTCAGATGTTCCCCTCTACAAGGATGGGGAGGCTAAGACTGACCCAGCAGGGCTGAGCAGTCCCCATCTGCCAG	149
20	31021403	31021523	ASXL1	GCACATCCTCTGCAGCACCCGACCTGGAGGGTCCCGAATTCCCAGTTGAGTCTGTGGCTTCTCGGATCCAGGCTGAGCCAGACAACTTGGCACGTGCCTCTGCATCTCCAGACAGAATTC	150
20	31021523	31021643	ASXL1	CTAGCCTGCCTCAGGAAACTGTGGATCAGGAACCCAAGGATCAGAAGAGGAAATCCTTTGAGCAGGCGGCCTCTGCATCCTTTCCCGAAAAGAAGCCCCGGCTTGAAGATCGTCAGTCCT	151
20	31021643	31021763	ASXL1	TTCGTAACACAATTGAAAGTGTTCACACCGAAAAGCCACAGCCCACTAAAGAGGAGCCCAAAGTCCCGCCCATCCGGGTAGGAGACTGTTTGATTCCTGGCTGCCCTGGAGCCAGGTTTT	152
20	31022188	31022308	ASXL1	AGATCACCCAGTCAGTTAAAACTATTTTCTAATTCTTTTTTTGCAGATTCAACTTTCACGTATCAAACCACCCTGGGTGGTTAAAGGTCAGCCCACTTACCAGATATGCCCCCGGATCAT	153
20	31023268	31023388	ASXL1	CATACCATCTGTTGAGCCCCAGGTTGGAGAGGAGTGGGAGAAAGCTGCTCCCACCCCTCCTGCATTGCCTGGGGATTTGACAGCTGAGGAGGGTCTAGATCCTCTTGACAGCCTTACTTC	154
20	31023388	31023508	ASXL1	ACTCTGGACTGTGCCATCTCGAGGAGGCAGTGACAGCAATGGCAGTTACTGTCAACAGGTGGACATTGAAAAGCTGAAAATCAACGGAGACTCTGAAGCACTGAGTCCTCACGGTGAGTC	155
20	31023508	31023628	ASXL1	CACGGATACAGCCTCTGACTTTGAAGGTCACCTCACGGAGGACAGCAGTGAGGCTGACACTAGAGAAGCTGCAGTGACAAAGGGATCTTCGGTGGACAAGGATGAGAAACCCAATTGGAA	156
20	31023628	31023748	ASXL1	CCAATCTGCCCCACTGTCCAAGGTGAATGGTGACATGCGTTCTGGTTACAAGGACAGATGGGATGGTTGCTCCTCAGAGCTGGGTGTCTCGAGTATGTGCGGTCCGCCAAAAGATCCCAGA	157
20	31023748	31023868	ASXL1	TTCCCTACTGCTGGCCAGTACTGAGTACCAGCCAAGAGCCGTGTGCCTGTCCATGCCTGGGTCCTCAGTGGAGGCCACTAACCCACTTGTGATGCAGTTGCTGCAGGGTAGCTTGCCCCT	158
20	31023868	31023988	ASXL1	AGAGAAGGTTCTTCCACCAGCCCACGATGACAGCATGTCAGAATCCCCACAAGTACCACTTACAAAAGACCAGAGCCATGGCTCGCTACGCATGGGATCTTTACATGGTCTTGGAAAAAA	159
20	31023988	31024108	ASXL1	CAGTGGCATGGTTGATGGAAGCAGCCCCAGTTCTTTAAGGGCTTTGAAGGAGCCTCTTCTGCCAGATAGCTGTGAAACAGGCACTGGTCTTGCCAGGATTGAGGCCACCCAGGCTCCTGG	160
20	31024108	31024228	ASXL1	AGCACCCCAAAAGAATTGCAAGGCAGTCCCAAGTTTTGACTCCCTCCATCCAGTGACAAATCCCATTACATCCTCTAGGAAACTGGAAGAAATGGATTCCAAAGAGCAGTTCTCTTCCTT	161
20	31024228	31024348	ASXL1	TAGTTGTGAAGATCAGAAGGAAGTCCGTGCTATGTCACAGGACAGTAATTCAAATGCTGCTCCAGGAAAGAGCCCAGGAGATCTTACTACCTCGAGAACACCTCGTTTCTCATCTCCAAA	162
20	31024348	31024468	ASXL1	TGTGATCTCCTTTGGTCCAGAGCAGACAGGTCGGGCCCTGGGTGATCAGAGCAATGTTACAGGCCAAGGGAAGAAGCTTTTTGGCTCTGGGAATGTGGCTGCAACCCTTCAGCGCCCCAG	163
20	31022308	31022428	ASXL1	CCCCACCACGGAGTCCTCCTGCCGGGGTTGGACTGGCGCCAGGACCCTCGCAGACATTAAAGCCCGTGCTCTGCAGGTCCGAGGGGCGAGAGGTCACCACTGCCATAGAGAGGCGGCCAC	164
20	31024468	31024588	ASXL1	GCCTGCGGACCCGATGCCTCTTCCTGCTGAGATCCCTCCAGTTTTTCCCAGTGGGAAGTTGGGACCAAGCACAAACTCCATGTCTGGTGGGGTACAGACTCCAAGGGAAGACTGGGCTCC	165
20	31024588	31024708	ASXL1	AAAGCCACATGCCTTTGTTGGCAGCGTCAAGAATGAGAAGACTTTTGTGGGGGGTCCTCTTAAGGCAAATGCCGAGAACAGGAAAGCTACTGGGCATAGTCCCCTGGAACTGGTGGGTCA	166
20	31024708	31024828	ASXL1	CTTGGAAGGGATGCCCTTTGTCATGGACTTGCCCTTCTGGAAATTACCCCGAGAGCCAGGGAAGGGGCTCAGTGAGCCTCTGGAGCCTTCTTCTCTCCCCTCCCAACTCAGCATCAAGCA	167
20	31024828	31024948	ASXL1	GGCATTTTATGGGAAGCTTTCTAAACTCCAACTGAGTTCCACCAGCTTTAATTATTCCTCTAGCTCTCCCACCTTTCCCAAAGGCCTTGCTGGAAGTGTGGTGCAGCTGAGCCACAAAGC	168
20	31024948	31025068	ASXL1	AAACTTTGGTGCGAGCCACAGTGCATCACTTTCCTTGCAAATGTTCACTGACAGCAGCACGGTGGAAAGCATCTCGCTCCAGTGTGCGTGCAGCCTGAAAGCCATGATCATGTGCCAAGG	169
20	31025068	31025188	ASXL1	CTGCGGTGCGTTCTGTCACGATGACTGTATTGGACCCTCAAAGCTCTGTGTATTGTGCCTTGTGGTGAGATAATAAATTATGGCCATGGGAAACATTGTATATTTAGTGTGTGTATTTTG	170
20	31022428	31022548	ASXL1	CACTGCCATCGGAGGGGGGGGGTGGCCCGGGTGGAGGTGGCGGCGGGGCCACCGATGAGGGAGGTGGCAGAGGCAGCAGCAGTGGTGATGGTGGTGAGGCCTGTGGCCACCCTGAGCCCAG	171
20	31022548	31022668	ASXL1	GGGAGGCCCGAGCACCCCTGGAAAGTGTACGTCAGATCTACAGCGAACACAACTACTGCCGCCTTATCCTCTAAATGGGGAGCATACCCAGGCCGGAACTGCCATGTCCAGAGCTAGGAG	172
20	31022668	31022788	ASXL1	AGAGGACCTGCCTTCTCTGAGAAAGGAGGAAAGCTGCCTACTACAGAGGGCTACAGTTGGACTCACAGATGGGCTAGGAGATGCCTCCCAACTCCCCGTTGCTCCCACTGGGGACCAGCC	173.
20	31022788	31022908	ASXL1	ATGCCAGGCCTTGCCCCTACTGTCCTCCCAAACCTCAGTAGCTGAGAGATTAGTGGAGCAGCCTCAGTTGCATCCGGATGTTAGAACTGAATGTGAGTCTGGCACCACTTCCTGGGAAAG	174
20	31022908	31023028	ASXL1	TGATGATGAGGAGCAAGGACCCACCGTTCCTGCAGACAATGGTCCCATTCTGTCTCTAGTGGGAGATGATACATTAGAGAAAGGAACTGGCCAAGCTCTTGACAGTCATCCCACTATGAA	175
20	31023028	31023148	ASXL1	GGATCCTGTAAATGTGACCCCCAGTTCCACACCTGAATCCTCACCGACTGATTGCCTGCAGAACAGAGCATTTGATGACGAATTAGGGCTTGGTGGCTCATGCCCTCCTATGAGGGAAAG	176
20	31023148	31023268	ASXL1	TGATACTAGACAAGAAAACTTGAAAACCAAGGCTCTCGTTTCTAACAGTTCTTTGCATTGGATACCCATCCCATCGAATGATGAGGTAGTGAAACAGCCCAAACCAGAATCCAGAGAACA	177

Chromosome	Start	Stop	Gene	Probe_Sequence		sequence number
17	58677676	58677796	PPM1D	TCGAAGATAAACAATAGTTGGCCGGCGAGCGCCTAGTGTGTCTCCCGCCGCCGGATTCGGCGGGCTGCGTGGGACCGGCGGGATCCCGGCCAGCCGGCCATGGCGGGGCTGTACTCGCTG	178
17	58677796	58677915	PPM1D		GGAGTGAGCGTCTTCTCCGACCAGGGCGGGAGGAAGTACATGGAGGACGTTACTCAAATCGTTGTGGAGCCCGAACCGACGGCTGAAGAAAAGCCCTCGCCGCGGCGGTCGCTGTCTCA	179
17	58677926	58678046	PPM1D	CGCGGCCGTCGCCGGCCGCCCTTCCCGGCGGCGAAGTCTCGGGGAAAGGCCCAGCGGTGGCAGCCCGAGAGGCTCGCGACCCTCTCCCGGACGCCGGGGCCTCGCCGGCACCTAGCCGCT	180
17	58678046	58678166	PPM1D	GCTGCCGCCGCCGTTCCTCCGTGGCCTTTTTCGCCGTGTGCGACGGGCACGGCGGGCGGGAGGCGGCACAGTTTGCCCGGGAGCACTTGTGGGGTTTCATCAAGAAGCAGAAGGGTTTCA	181
17	58678166	58678286	PPM1D		CCTCGTCCGAGCCGGCTAAGGTTTGCGCTGCCATCCGCAAAGGCTTTCTCGCTTGTCACCTTGCCATGTGGAAGAAACTGGGTAAGTTCCCTGGCTTGTTTGGCGCCCGCCCCTTTTTCA	182
17	58700875	58700995	PPM1D	TTACAGCGGAATGGCCAAAGACTATGACGGGTCTTCCTAGCACATCAGGGACAACTGCCAGTGTGGTCATCATTCGGGGCATGAAGATGTATGTAGCTCACGTAGGTGACTCAGGGGTGG	183
17	58700995	58701115	PPM1D	TTCTTGGAATTCAGGATGACCCGAAGGATGACTTTGTCAGAGCTGTGGAGGTGACACAGGACCATAAGCCAGAACTTCCCAAGGAAAGAGAACGAATCGAAGGACTTGGTGGGAGGTAAC	184
17	58711155	58711275	PPM1D	TATCTTAGTTGTTGTATTTTAATCATTTAGATTATTATGTGAACTCTTTATTTTTAGTGTAATGAACAAGTCTGGGGTGAATCGTGTAGTTTGGAAACGACCTCGACTCACTCACAATG	185
17	58711275	58711395	PPM1D	GACCTGTTAGAAGGAGCACAGTTATTGACCAGATTCCTTTTCTGGCAGTAGCAAGAGCACTTGGTAAGTAGGACTTAATTTGGTGAAAATTATATTGAATTTTCTACAATATATGGTGGCA	186
17	58725228	58725348	PPM1D	GCTTTTTCTGCTCCCTTCCCCCAGGTGATTTGTGGAGCTATGATTTCTTCAGTGGTGAATTTGTGGTGTCACCTGAACCAGACACAAGTGTCCACACTCTTGACCCTCAGAAGCACAAGT	187
17	58725348	58725468	PPM1D	ATATTATATTGGGGAGTGATGGACTTTGGAATATGATTCCACCACAAGATGCCATCTCAATGTGCCAGGACCAAGAGGAGAAAAAATACCTGATGGTGAGATGTGATTGAATAACTTGAT	188
17	58733901	58734021	PPM1D	AGATGTAGTGGCAGCTAAATCTGAGTTACTTTCCTTCTCCTTGTTCTTTTGAATACAGGTGGAGCATGGACAATCTTGTGCCAAAATGCTTGTGAATCGAGCATTGGGCCGCTGGAGGCA	189
17	58734021	58734141	PPM1D	GCGTATGCTCCGAGCAGATAACACTAGTGCCATAGTAATCTGCATCTCTCCAGAAGTGGACAATCAGGGAAACTTTACCAATGAAGATGAGTTATACCTGAACCTGACTGACAGCCCTTC	190
17	58734141	58734261	PPM1D	CTATAATAGTCAAGAAACCTGTGTGATGACTCCTTCCCCATGTTCTACACCACCAGTCAAGGTATATAGTTCCATAGTTTTTAAGTTATGTTTTAATAGACACCAGTTCTTTGGTTAGCG	191
17	58734240	58734360	PPM1D	ACACCAGTTCTTTGGTTAGCGTCACCTGGAAACAATTTTTAAATTCTTACAGGATTTTGGATTTGAACTCGATTCAAGAAAGTGATGTAACTTATTATCAGAGAGCCATCTTTACATCAA	192
17	58734360	58734479	PPM1D	TACTAATCTGAATGCCAGCCATGTGTACAGCACTAATAAAAAGGTGATTGTGGGCCCTTAAGAATATTATTGTTTGCCATCTAATGCTATGAACATTATTTTTAAAGCATTTAGAAGTT	193
17	58740334	58740454	PPM1D	TTTTACCTTCTTATTTTTCAGTCACTGGAGGAGGATCCATGGCCAAGGGTGAATTCTAAGGACCATATACCTGCCCTGGTTCGTAGCAATGCCTTCTCAGAGAATTTTTTAGAGGTTTCA	194
17	58740454	58740574	PPM1D	GCTGAGATAGCTCGAGAGAATGTCCAAGGTGTAGTCATACCCTCAAAAGATCCAGAACCACTTGAAGAAAATTGCGCTAAAGCCCTGACTTTAAGGATACATGATTCTTTGAATAATAGC	195
17	58740574	58740694	PPM1D		CTTCCAATTGGCCTTGTGCCTACTAATTCAACAAACACTGTCATGGACCAAAAAAATTTGAAGATGTCAACTCCTGGCCAAATGAAAGCCCAAGAAATTGAAAGAACCCCTCCAACAAAC	196
17	58740694	58740814	PPM1D	TTTAAAAGGACATTAGAAGAGTCCAATTCTGGCCCCCTGATGAAGAAGCATAGACGAAATGGCTTAAGTCGAAGTAGTGGTGCTCAGCCTGCAAGTCTCCCCACAACCTCACAGCGAAAG	197
17	58740814	58740934	PPM1D	AACTCTGTTAAACTCACCATGCGACGCAGACTTAGGGGCCAGAAGAAAATTGGAAATCCTTTACTTCATCAACACAGGAAAACTGTTTGTGTTTGCTGAAATGCATCTGGGAAATGAGGT	198

This is an example of probe sequence information for chromosomal sequences in which somatic sequence mutations are detected among the entire sequences of the NGS panel for DNMT3A, TET2, ASXL1, and PPM1D genes, and the mutation detection agent is not limited thereto.

The composition can be used for genetic analysis of a biological sample isolated from an individual, and the genetic analysis includes Next Generation Sequencing (NGS) and PCR-based techniques such as real-time quantitative PCR, blocker PCR, digital droplet PCR (ddPCR), clamping PCR, ICE-COLD PCR, castPCR, ARMS PCR, BEAMing, and the like.

In addition, the agent capable of detecting the protein is a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a fragment (scFv) of these antibodies, or an aptamer ( aptamer), but is not limited thereto.

According to another aspect of the present invention, a kit for predicting the prognosis of lung cancer treatment of a subject comprising the composition is provided.

Specifically, the kit consists of one or more other component compositions, solutions or devices suitable for the assay method. For example, the kit may be a reverse transcription polymerase chain reaction (RT-PCR) kit, a DNA chip kit, an enzyme-linked immunosorbent assay (ELISA) kit, a protein chip kit, or a rapid kit.

In one embodiment, the composition included in the kit may be in the form of a panel, but is not limited thereto.

According to another aspect of the present invention, a panel for genetic analysis comprising the composition is provided. The genetic analysis panel may be based on next-generation sequencing (NGS), which may be used to search for genetic mutations associated with clonal hematopoiesis or to predict prognosis in association with lung cancer treatment. Through the gene analysis panel, the practitioner can perform analysis on the region of the gene to be sequenced and furthermore, the region to search for mutations in the gene. In addition, the operator can perform simultaneous analysis on a plurality of target genes in one analysis through the gene analysis panel. The gene analysis panel may include probes having complementary nucleotide sequences for each target gene, and each probe is specific for a target gene region in a biological sample isolated from an individual by hybridization. can be antagonistically combined. For example, panels for genetic analysis for detecting genetic variants associated with clonal hematopoiesis include APC, ASXL1, ASXL2, ATM, BCL11B, BCOR, BCORL1, BIRC3, BRAF, BRCC3, CARD11, CASP8, CBL, CD58, CD79B KIT , KLHL6, KMT2D, KRAS, LUC7L2, MAP3K1, MPL, MYD88, NF1, NFE2L2, NOTCH1, NOTCH2, NRAS, PDS5B, PDSS2, PHF6, PHIP, PIK3CA, PIK3R1, PPM1D, PRDM1, PRPF40B, PTEN, PTPN11, RAD21, RIT1 , RPS15, SETD2, SETDB1, SF1, SF3A1, SF3B1, SMC1A, SMC3, SRSF2, STAG1, STAG2, STAT3, SUZ12, TBL1XR1, TET1, TET2, TNFAIP3, TNFRSF14, TP53, U2AF1, VHL, WT1, ZRSR2 and CHEK2 genes. It may contain probes for one or more genes selected from the group consisting of: The above probes can be used to search for nucleotide sequence mutations of the gene. The gene to which the probe is attached can be amplified through PCR to prepare a library for sequencing, and the presence or absence of nucleotide sequence mutation in the gene can be finally detected through next-generation sequencing analysis.

For detailed descriptions of the same elements such as "clonal hematopoiesis", "lung cancer", "subject", "biological sample", "prognosis", "prediction", and "mutation", refer to the above.

Diagnosis of clonal hematopoiesis for the treatment of lung cancer

According to another aspect of the present invention, treatment of lung cancer comprising the step of determining whether clonal hematopoiesis exists in a subject through genetic analysis of a biological sample isolated from the subject prior to administration of a therapeutic agent for lung cancer treatment. A method or method of providing information for treatment of lung cancer is provided. As described above, the presence or absence of clonal hematopoiesis in lung cancer patients is closely related to the prognosis of lung cancer treatment. can be determined, thereby increasing the survival rate of lung cancer patients.

For the configuration of the genes in which somatic mutations related to the clonal hematopoiesis occur, refer to the contents described in "Method for Predicting Prognosis of Lung Cancer Treatment" of the present specification.

In one embodiment, the step of determining whether clonal hematopoiesis exists is DNMT3A, ASXL1, TET2, PPM1D, ATM, BCL11B, CARD11, CBL, CD79B, CHEK2, CUX1, ETV6, FOXP1, JAK2, KMT2D, MAP3K1 , MPL, NF1, NOTCH1, NOTCH2, PHIP, PRPF40B, RAD21, SETD2, SF3B1, TET1, TNFAIP3, TP53, U2AF1 and ZRSR2 may include determining whether one or more mutations exist in one or more genes selected from the group consisting of and, based on this, determining the presence or absence of clonal hematopoiesis may be further included.

In another embodiment, the one or more genes are selected from the group consisting of ASXL1, CBL, CHEK2, CUX1, DNMT3A, FOXP1, JAK2, KMT2D, MPL, NOTCH1, PPM1D, PRPF40B, SF3B1, TET2, TNFAIP3, TP53, U2AF1 and ZRSR2 may include one or more.

In another embodiment, the one or more genes may include one or more selected from the group consisting of DNMT3A, ASXL1, TET2, PPM1D, SF3B1, ATM, and TNFAIP3.

In another embodiment, the one or more genes may include any one gene selected from the group consisting of the above-mentioned genes, and in this case, the one or more genes consist of the rest of the genes other than the selected one gene. It may further include one or more selected from the group.

In another embodiment, the presence or absence of clonal hematopoiesis may be determined based on the presence or absence of a mutation in the one or more genes and the variant allele frequency (VAF). For example, through genetic analysis using a biological sample isolated from an individual, one or more mutations exist in one or more genes among the genes of the gene group, and the frequency of the variant allele is higher than a certain level, for example, about 1.8% or higher, about 1.8% or higher. If it is 1.9% or more, or about 2% or more, it can be classified as an individual with clonal hematopoiesis.

In another embodiment, when it is determined that clonal hematopoiesis does not exist in the subject, the step of administering a lung cancer treatment may be further included.

In another embodiment, when it is determined that clonal hematopoiesis exists in the subject, a step of determining whether to administer a lung cancer treatment may be further included. For example, when it is determined that clonal hematopoiesis exists in the subject, it may be determined not to administer the lung cancer treatment or to administer the lung cancer treatment.

In another embodiment, the step of determining whether to administer the lung cancer treatment is a single CH mutation in a significant blood cell count abnormality, high VAF (> 10%), multiple CH with the presence or absence of clonal hematopoiesis (CH) Mutations, TP53 and/or PPM1D variants, DNMT3A variants, IDH1/2 hotspot mutations, and the like can be comprehensively considered. In addition to this, the stage of lung cancer, the patient's age, medical history, and the type and number of current accompanying diseases may be additionally considered.

In consideration of the above-described various variables, when it is determined that clonal hematopoiesis exists in the subject, a step of administering a lung cancer treatment may be further included. For example, when it is determined that clonal hematopoiesis is present in a patient prior to administration of lung cancer treatment, a lung cancer treatment that has relatively less effect on clonal hematopoiesis or has a lower probability of amplifying adverse outcomes associated with clonal hematopoiesis. administration can be selected.

Meanwhile, according to another embodiment of the present invention, the therapeutic agent for treating lung cancer includes a drug candidate for clinical trials. For example, the lung cancer treatment method according to the present invention can be applied to clinical trials for lung cancer therapeutics.

Therefore, according to another aspect of the present invention, prior to administering a drug candidate for lung cancer treatment to a subject, genetic analysis of a biological sample isolated from the subject confirms whether clonal hematopoiesis exists in the subject A method for treating lung cancer or a method for clinical testing of a drug candidate for treating lung cancer is provided.

In one embodiment, the method may further include administering a drug candidate when it is determined that the subject does not have clonal hematopoiesis.

In another embodiment, the method may further include administering a drug candidate when it is determined that the subject has clonal hematopoiesis.

For example, when the lung cancer treatment method of the present invention is applied to a clinical trial for a lung cancer treatment, clonal hematopoiesis occurs in the individual through genetic analysis of a biological sample isolated from an individual who has participated or is scheduled to participate in a clinical trial for a lung cancer treatment. It may include a step of confirming whether or not it is present, and by selecting a population in which clonal hematopoiesis exists through this confirmation step, it is possible to increase the probability of success in a clinical trial for a lung cancer treatment.

As described above, since it has been proven that the presence of clonal hematopoiesis in lung cancer patients leads to poor prognosis for lung cancer treatment, confirming the presence or absence of clonal hematopoiesis according to the present invention is the success of clinical trials for lung cancer therapeutics. Can be used to increase odds. Clinical trials of pharmaceuticals take an average of 10 years or more from phase 1 clinical trial to approval, so the safety and efficacy of medicines are evaluated over a long period of time. At this time, in addition to the effect of the drug being tested, if there are other hidden factors that negatively affect the prognosis of treatment, there is a possibility that the safety and effectiveness of the drug may be underestimated more than it actually is, or the variation depending on patients may be large. As a result, there is a risk that the probability of success of the clinical trial will be lowered. Therefore, it is also very important to select a uniform patient group in clinical trials of pharmaceuticals. The present invention can increase the probability of success of a clinical trial by selecting a clinical trial patient group according to the presence or absence of clonal hematopoiesis, which negatively affects the prognosis of lung cancer treatment.

In one embodiment, the presence of clonal hematopoiesis in the subject may indicate that the safety or effectiveness of a lung cancer treatment is underestimated compared to the case where clonal hematopoiesis is not present.

In another embodiment, the subject may be a subject who has or is currently conducting a clinical trial for a lung cancer treatment. For example, evaluation of safety, efficacy, etc. for a lung cancer therapeutic agent confirms the presence or absence of clonal hematopoiesis in an individual to exclude a population in which clonal hematopoiesis exists, or to exclude a population in which clonal hematopoiesis exists and clonal hematopoiesis. Hematopoiesis can be differentiated and progressed in non-existent populations.

In addition, the subject may be an individual scheduled to conduct a clinical trial for a lung cancer treatment or a lung cancer patient registered as a clinical trial candidate. For example, by confirming the presence or absence of clonal hematopoiesis in the process of selecting patients (individuals) for clinical trials of lung cancer therapeutics, individuals with clonal hematopoiesis are excluded from clinical trials or populations with clonal hematopoiesis are excluded. A clinical trial can be conducted by separately selecting a population in which hyperclonal hematopoiesis does not exist.

On the other hand, in the clinical trial method further comprising the step of administering a drug candidate when it is determined that clonal hematopoiesis exists in the subject, the drug candidate has a relatively greater effect on clonal hematopoiesis. Substances that are less likely to amplify adverse outcomes associated with small or clonal hematopoiesis can be chosen and administered. At this time, for the drug candidate administered to the subject determined to have clonal hematopoiesis, treatment efficacy and safety for lung cancer as well as effects on clonal hematopoiesis may be evaluated. For detailed descriptions of the same elements such as "clonal hematopoiesis", "lung cancer", "subject", "biological sample", and "mutation", refer to the above.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only for exemplifying the present invention, and the scope of the present invention is not limited only to these.

Example

The following example describes the clinical impact of preoperative clonal hematopoiesis on the survival outcome of lung cancer patients, particularly non-small cell lung cancer (NSCLC) patients who received adjuvant therapy following surgical resection in a large single-center serial surgery cohort. -scale single center consecutive surgical cohort). A propensity score matching (PSM) technique was used to rule out the possibility of selection bias according to adjuvant therapy and CH status.

Example 1. Patient group

All clinical records of patients (1211 patients) who underwent NSCLC surgery between January 2011 and December 2017 were reviewed in the lung cancer database of Asan Medical Center. This example was performed on patients receiving adjuvant therapy for stage IIB or III NSCLC (FIG. 1). Specifically, 369 patients out of 1211 patients who underwent NSCLC surgery were excluded, and the exclusion criteria were as follows: i) patients with past or present malignancies other than lung cancer; ii) patients receiving neoadjuvant therapy before surgery; iii) patients who underwent sublobar resection (wedge resection or segmentectomy); iv) patients with incomplete resection; and v) patients who died within 30 days of surgery.

After exclusion according to the above criteria, 563 patients who received adjuvant chemotherapy (CTx) or chemoradiation therapy (CRTx) for stage IIB or III NSCLC were identified. Of these, 424 patients with blood samples collected prior to surgery and stored at the Asan Medical Center's Asan Bio-Resource Center, a Korea Biobank Network, were identified. Nine of these samples were excluded due to sample degradation, and 415 patients with blood samples were enrolled into the final cohort.

In order to confirm the clinical effect of CH according to adjuvant therapy in stage IIB patients, blood samples from patients without adjuvant therapy for stage IIB NSCLC were further analyzed (FIG. 1).

This study was approved by the Clinical Research Review Committee of Asan Medical Center (2020-0906). Written informed consent was obtained from all participants.

Example 2. Sample processing and sequencing

Targeted NGS was performed with a custom panel comprising the following 89 genes using blood-derived DNA collected from patients enrolled in this study (Table 10).

APC	ASXL1	ASXL2	ATM	BCL11B	BCOR	BCORL1	BIRC3
BRAF	BRCC3	CARD11	CASP8	CBL	CD58	CD79B	CNOT3
CREBBP	CUX1	DDX3X	DNMT3A	EP300	ETV6	EZH2	FAM46C
FBXW7	FLT3	FOXP1	GNAS	GNB1	GPS2	HIST1H1C	IDH2
IKZF1	IKZF2	JAK1	JAK2	JAK3	JARID2	KDM6A	KIT
KLHL6	KRAS	LUC7L2	MAP3K1	KMT2D	MPL	MYD88	NF1
NFE2L2	NOTCH1	NOTCH2	NRAS	PDS5B	PDSS2	PHF6	PHIP
PIK3CA	PIK3R1	PPM1D	PRDM1	PRPF40B	PTEN	PTPN11	RAD21
RIT1	RPS15	SETD2	SETDB1	SF1	SF3A1	SF3B1	SMC1A
SMC3	SRSF2	STAG1	STAG2	STAT3	SUZ12	TBL1XR1	TET1
TET2	TNFAIP3	TNFRSF14	TP53	U2AF1	VHL	WT1	ZRSR2
CHEK2

Sequencing libraries were prepared according to the SureSelect XT HS Target Enrichment System (Agilent, Santa Clara, CA) protocol. Libraries were sequenced on an Illumina NovaSeq6000 platform (Illumina, San Diego, CA) using 150 bp paired-ends according to the manufacturer's protocol. The average coverage depth of Analysis ready BAM was over 800 times.

Example 3. Preoperative and postoperative management of patients

Patient work-up for diagnosis, staging, and surgical resection was performed according to well-established and widely used protocols (Yun JK, Bok JS, Lee GD, et al: Long-term outcomes of upfront surgery in patients with resectable pathological N2 non-small-cell lung cancer. Eur J Cardiothorac Surg 58:59-69, 2020).

If computed tomography (CT) or positron emission tomography (PET) suggests clinical N2 (cN2) disease, bronchoscopy ultrasonography (EBUS), mediastinoscopy, or endoscopic ultrasonography (EUS) for the suspicious nodule is recommended. A mediastinal LN biopsy was performed using Treatment plans for biopsy-proven N2 disease were determined by a multidisciplinary team including medical oncologists, radiologists, and thoracic surgeons. Patients in the study sample were retrospectively staged according to the American Joint Committee on Cancer (AJCC) 8th edition (Detterbeck FC, Boffa DJ, Kim AW, et al: The Eighth Edition Lung Cancer Stage Classification. Chest 151 :193-203, 2017]).

Based on the judgment of the multidisciplinary team, adjuvant CTx was recommended for all stage II and III patients, except when the patient was >75 years of age or in poor physical condition. Systemic CTx with platinum-based therapy was recommended for a total of 4 cycles of treatment for 4 to 6 weeks postoperatively. As the use of targeted therapies for patients with activating mutations in the epidermal growth factor receptor (EGFR) has become common since 2008, tyrosine kinase inhibitors have been primarily used when relapsed after first-line adjuvant CTx. For adjuvant radiation therapy (RTx) for stage III, 1.8 Gy daily up to a total dose of 50.4 Gy for patients undergoing complete resection or 55 to 60 Gy for patients with a positive resection margin. dose was administered. Among patients who underwent total resection, adjuvant RTx was omitted for a significant number of patients with single N2 nodal metastases.

Follow-up information for all patients was obtained from clinic follow-up records every 6 months for the first 2 years after surgery and annually thereafter. A chest CT scan was performed at every clinical visit or whenever disease recurrence was suspected. Treatment and chemotherapy for relapse cases were determined at the discretion of the attending physician.

Example 4. Genetic mutation detection and survival outcome measurement

The primary endpoint was survival according to the presence of CH in patients receiving adjuvant therapy for stage IIB or stage III NSCLC, and stage IIB patients according to the presence of CH and adjuvant therapy. survival outcomes were included as secondary end points (FIG. 1).

89 genes frequently detected in CH were selected and examined (see Table 10 for a list of genes), and a variant allele frequency (VAF) of 2% or more was defined as CH positive.

The following criteria were used to classify potential driver (PD) mutations among the mutations: 1) reported more than 1 time in carcinomas in the “hematopoietic and lymphoid” category in COSMIC, or more than 10 times in carcinomas in all categories any somatic mutation that occurs with; and 2) any somatic mutations with loss-of-function effects (frameshift, stop gain, mutations at splice donor/acceptor sites). Mutations that did not meet the criteria for PD mutations were classified as non-PD mutations. The resulting PD mutation list is shown in Table 11 below.

Gene	Position	Variant classification	Protein change	CDS
ASXL1	31022441	FRAME_SHIFT	p.642Gly_643Glyfs	c.1927_1928insG
ASXL1	31022286	STOP_GAINED	p.590Tyr_591GlninsTer???	c.1772_1773insA
ASXL1	31021211	STOP_GAINED	p.Arg404*	c.1210C>T
ASXL1	31022441	FRAME_SHIFT	p.642Gly_643Glyfs	c.1927_1928insG
ASXL1	31023478	FRAME_SHIFT	p.987Asp_994Hisfs	c.2964_2971delCTCTGAAGCACTGAGTC
ASXL1	31024343	FRAME_SHIFT	p.1276Pro_1277Asnfs	c.3829_3830delC
ASXL1	31022402	FRAME_SHIFT	p.629His_637Thrfs	c.1888_1896delCACCACTGCCATAGAGAGGCGGC
ASXL1	31022927	FRAME_SHIFT	p.804Pro_805Thrfs	c.2413_2414insC
ASXL1	31022700	FRAME_SHIFT	p.728Ser_729Cysfs	c.2186_2187delG
ASXL1	31022463	FRAME_SHIFT	p.649Gly_650Glyfs	c.1949_1950delGT
ASXL1	31022441	FRAME_SHIFT	p.642Gly_643Glyfs	c.1927_1928insG
ASXL1	31022288	STOP_GAINED	p.Tyr591*	c.1773C>A
ASXL1	31022936	FRAME_SHIFT	p.807Pro_808Alafs	c.2422_2423delC
ASXL1	31022441	FRAME_SHIFT	p.642Gly_643Glyfs	c.1927_1928insG
CBL	119148973	NON_SYNONYMOUS_CODING	p.His398Arg	c.1193A>G
CHEK2	29083949	FRAME_SHIFT	p.565Arg_566Glufs	c.1696_1697delC
CUX1	101459346	STOP_GAINED	p.Trp12*	c.36G>A
DNMT3A	25463600	SPLICE_SITE_ACCEPTOR	-	-
DNMT3A	25462077	NON_SYNONYMOUS_CODING	p.Pro777Arg	c.2330C>G
DNMT3A	25468888	SPLICE_SITE_DONOR	-	-
DNMT3A	25468201	SPLICE_SITE_ACCEPTOR	-	-
DNMT3A	25462018	NON_SYNONYMOUS_CODING	p.Asn797Asp	c.2389A>G
DNMT3A	25463286	NON_SYNONYMOUS_CODING	p.Arg736His	c.2207G>A
DNMT3A	25467168	FRAME_SHIFT	p.568Pro_569Glyfs	c.1706_1707delC
DNMT3A	25469079	FRAME_SHIFT	p.459Ser_460Thrfs	c.1378_1379insTA
DNMT3A	25469986	FRAME_SHIFT	p.351Ser_352Alafs	c.1055_1056delG
DNMT3A	25467449	NON_SYNONYMOUS_CODING	p.Gly543Cys	c.1627G>T
DNMT3A	25467071	FRAME_SHIFT	p.600Trp_601Profs	c.1803_1804delG
DNMT3A	25469504	FRAME_SHIFT	p.416Ser_421Leufs	c.1251_1256delCGGGAATCCCGGT
DNMT3A	25457243	NON_SYNONYMOUS_CODING	p.Arg882Cys	c.2644C>T
DNMT3A	25469527	FRAME_SHIFT	p.410Leu_414Glnfs	c.1233_1237delTCGGGGGG
DNMT3A	25462068	NON_SYNONYMOUS_CODING	p.Ile780Thr	c.2339T>C
DNMT3A	25457243	NON_SYNONYMOUS_CODING	p.Arg882Cys	c.2644C>T
DNMT3A	25458595	FRAME_SHIFT	p.858Leu_859Trpfs	c.2577_2578insA
DNMT3A	25470028	SPLICE_SITE_ACCEPTOR	-	-
DNMT3A	25467470	FRAME_SHIFT	p.534Ser_535Tyrfs	c.1605_1606delC
DNMT3A	25457243	NON_SYNONYMOUS_CODING	p.Arg882Cys	c.2644C>T
DNMT3A	25458595	NON_SYNONYMOUS_CODING	p.Trp860Arg	c.2578T>C
DNMT3A	25463172	NON_SYNONYMOUS_CODING	p.Glu774Val	c.2321A>T
DNMT3A	25467208	SPLICE_SITE_ACCEPTOR	-	-
DNMT3A	25470472	FRAME_SHIFT	p.333Gly_334Lysfs	c.1001_1002delG
DNMT3A	25467190	NON_SYNONYMOUS_CODING	p.Cys562Tyr	c.1685G>A
DNMT3A	25467023	SPLICE_SITE_DONOR	-	-
DNMT3A	25457159	FRAME_SHIFT	p.908Phe_909Alafs	c.2727_2728insTT
FOXP1	71026147	NON_SYNONYMOUS_CODING	p.Tyr492Cys	c.1475A>G
FOXP1	71064710	FRAME_SHIFT	p.320Gln_321Serfs	c.963_964insAA
JAK2	5073770	NON_SYNONYMOUS_CODING	p. Val617Phe	c.1849G>T
KMT2D	49434156	STOP_GAINED	p.Leu2466*	c.7397T>A
MPL	43815009	NON_SYNONYMOUS_CODING	p.Trp515Leu	c.1544G>T
NOTCH1	139390743	NON_SYNONYMOUS_CODING	p.Thr2483Met	c.7448C>T
PPM1D	58740438	FRAME_SHIFT	p.447Asn_448Phefs	c.1344_1345delT
PPM1D	58740357	FRAME_SHIFT	p.420Ser_421Leufs	c.1263_1264insA
PPM1D	58740532	FRAME_SHIFT	p.479Lys_480Alafs	c.1438_1439insA
PPM1D	58740505	FRAME_SHIFT	p.470Pro_471Glufs	c.1411_1412insC
PPM1D	58740749	STOP_GAINED	p.Arg552*	c.1654C>T
PPM1D	58740623	FRAME_SHIFT	p.509Gln_510Lysfs	c.1529_1530insA
PPM1D	58740694	FRAME_SHIFT	p.533Phe_534Lysfs	c.1600_1601delT
PRPF40B	50027317	FRAME_SHIFT	p.189Asp_190Aspfs	c.568_569insGATGACCTAGAGGGTGA
PRPF40B	50027317	FRAME_SHIFT	p.189Asp_190Aspfs	c.568_569insGATGACCTAGAGGGTGA
SF3B1	198267359	NON_SYNONYMOUS_CODING	p.Lys666Asn	c.1998G>T
SF3B1	198267360	NON_SYNONYMOUS_CODING	p.Lys666Arg	c.1997A>G
TET2	106157878	FRAME_SHIFT	p.926Val_927Phefs	c.2780_2781delT
TET2	106182979	FRAME_SHIFT	p.1339Leu_1340Alafs	c.4019_4020delT
TET2	106193794	FRAME_SHIFT	p.1418Pro_1420Tyrfs	c.4257_4259delTT
TET2	106196243	STOP_GAINED	p.Gln1526*	c.4576C>T
TET2	106155853	FRAME_SHIFT	p.251Ile_252Asnfs	c.755_756insTT
TET2	106180853	NON_SYNONYMOUS_CODING	p.Tyr1294Cys	c.3881A>G
TET2	106157989	FRAME_SHIFT	p.963Gln_964Thrfs	c.2891_2892insA
TET2	106157969	FRAME_SHIFT	p.956Leu_957Glnfs	c.2871_2872insA
TET2	106155281	FRAME_SHIFT	p.60Tyr_61Glyfs	c.183_184insT
TET2	106157789	FRAME_SHIFT	p.896Gln_897Glyfs	c.2691_2692delG
TET2	106157560	STOP_GAINED	p.Gln821*	c.2461C>T
TNFAIP3	138197265	FRAME_SHIFT	p.255His_256Phefs	c.768_769insT
TNFAIP3	138196885	STOP_GAINED	p.Arg183*	c.547C>T
TNFAIP3	138200309	FRAME_SHIFT	p.575Pro_581Alafs	c.1728_1734delGCATTCTTGCCACA
TP53	7574003	STOP_GAINED	p.Arg342*	c.1024C>T
U2AF1	44524456	NON_SYNONYMOUS_CODING	p.Ser34Tyr	c.101C>A
ZRSR2	15833956	FRAME_SHIFT	p.238Phe_239Tyrfs	c.715_716delT

Overall survival (OS) was calculated based on the time interval between the date of surgery and the date of death, and was determined by reviewing records in the Korean National Security Death Index Database. Lung cancer mortality included deaths due to overt tumor progression. Non-lung cancer mortality was defined as mortality from known causes not due to lung cancer progression. Recurrence-free survival (RFS) was calculated based on the time between the date of surgery and the date of recurrence.

Example 5. Statistical Analysis

Continuous variables were expressed as mean and standard deviation, and categorical variables as counts and percentages. The normality of individual parameter distributions was evaluated with the Shapiro-Wilk test. Student's t- test or Wilcoxon rank-sum test was used to compare two groups in terms of continuous variables, and chi-square test or Fisher's exact test was applied for categorical variables. After propensity score matching (PSM), McNemar's test and paired-sample t-test were used to analyze propensity score matching pairs. OS and RFS results were defined using Kaplan-Meier curves. Differences in survival rates were analyzed using the log-rank test. Since the two causes of death were mutually exclusive, significant differences in cumulative incidence function values between subgroups were assessed using the Gray test. A Cox proportional hazards model was used for univariate and multivariate analysis to determine the clinical impact of CH on survival outcomes. We developed two types of multivariate Cox models: age, sex, smoking history and number of comorbidities were included as covariates to adjust the sensitivity test for CH (related to Table 13 in Example 6.1 below) and ), forward as the procedure of choice for likelihood ratio test (p-value ≤ 0.10 for model input and p-value ≤ 0.05 for model maintenance) to ascertain the prognostic effect of CH in patients receiving adjuvant therapy. A stepwise selection was used (refers to Table 17 in Example 6.3 below). The proportional hazards assumption for the Cox regression model was tested with Schoenfeld residuals.

PSM was applied to adjust for possible selection bias derived from a retrospective nonrandomized cohort to generate two groups (CH-positive and CH-negative) with similar characteristics. A total of 12 variables (related to Table 14 in Example 6.2 below) were used to balance the clinical characteristics of the two groups. For PSM, pairs of observations with equivalent propensity scores were selected with nearest-neighbor matching and a caliper width of standard deviation 0.25. CH-negative patients were randomly matched with CH-positive patients in a 2:1 ratio. Balance between groups was assessed using standardized mean differences (SMDs). An absolute standardized difference of 0.1 or less was considered to represent an ideal balance, and an absolute standardized difference of 0.2 or less was considered to represent an acceptable balance.

All statistical calculations were performed using R version 4.0.2 ( The R Foundation for Statistical Computing, Vienna, Austria) was used. All reported P values are two-tailed. A P value of less than 0.05 was considered significant.

Example 6. Sample Analysis Results

Example 6.1. Characteristics of CH

The mean age of patients in the final cohort was 60.2 ± 8.3 years. Of a total of 415 patients, CH was found in 86 (20.7%). The prevalence of CH was 10.4%, 14.9%, 23.8%, and 34.5% in patients in their 40s, 50s, 60s, and 70s, respectively, and showed a continuous increase with age (FIG. 5a). As for the number of mutations, a single mutation was the most common in 82.6% of patients, two mutations in 14.0%, and three mutations in 3.5% (Fig. 5b). Mutations in DNMT3A (33.0%) were the most common, followed by mutations in ASXL1 (13.2%), TET2 (11.3%), and PPM1D (7.5%). These four genes accounted for 65.1% of all detected mutations, and other mutations such as SF3B1, ATM, and TNFAIP3 were present (FIG. 5c). Details of CH mutations detected for individual patients are summarized in Tables 1-5 above.

Sensitivity tests based on multiple cutoff values for VAF showed that when the cutoff value for VAF was greater than or equal to 1.8%, P-values for OS were univariate and multivariate (adjusted for age, gender, smoking history and number of comorbidities) Cox analysis. showed the lowest at 0.002 and 0.005, respectively (see Table 12 below showing the sensitivity test results for CH based on VAF values). However, the cutoff value of VAF of 2% or more had similar results compared to that of 1.8% or more (p values = 0.006 and 0.012 for univariate and multivariate analysis, respectively) (see Table 12 below).

The prevalence of CH-PD and CH non-PD in study patients was 16.3% (68/415) and 6.0% (25/415), respectively, and 79.1% (68/86) and 29.1% (25/86) in patients with CH mutations, respectively. /86). Sensitivity tests were also performed in these patients. As a result, CH-PD was still observed as a significant factor even after adjustment by several covariates in multivariate analysis (p = 0.006), whereas CH non-PD was not significant in multivariate analysis (p = 0.399) (CH , CH-PD, and CH non-PD, see Table 13 below showing the sensitivity test results).

Example 6.2. patient characteristics

The mean follow-up period after surgery was 44.6 ± 24.3 months. Baseline demographics and tumor characteristics of patients are shown in Table 14 below (baseline characteristics of patients receiving adjuvant therapy for stage IIB or III according to presence of clonal hematopoiesis (final cohort)). Before PSM, patients with CH (n = 86) were older than those without CH (n = 329) (p < 0.001). Between the two groups, gender (p = 0.367), smoking history (p = 0.785), number of comorbidities (p = 0.988), proportion of EGFR mutations (p = 0.501), histological distribution (p = 0.647), and overall stage (p = 0.647). = 0.548), there was no significant difference between adjuvant therapy types (p = 0.146).

After PSM, all variables including age (p = 0.620) became similar between the two groups and were well balanced (all SMDs < 0.2) (stage IIB or III according to the presence of clonal hematopoiesis after propensity score matching). See Table 15 below showing baseline characteristics (final cohort) of patients who received adjuvant therapy for qi).

Example 6.3. survival rate assay

Overall, 45 patients with CH (n = 86) and 124 patients without CH (n = 329) died by the end of follow-up, and their 5-year OS rates were 45.1% and 61.9%, respectively . Recurrent events occurred in 48 and 161 patients with and without CH, respectively, and their 5-year RFS rates were 39.1% and 44.2%, respectively. Detailed information on the cause of death is summarized in Table 16 (cause of death during the entire observation period) below.

Kaplan-Meier survival curves according to the presence of CH are plotted in FIGS. 2A-2C. Although there was no significant difference in RFS between the two groups (p = 0.251), patients with CH had poorer OS than those without CH (p < 0.001) (FIGS. 2A and 2B). After PSM, patients with CH had significantly poorer survival than those without CH (p = 0.029) (Fig. 2c).

According to cause of death, lung cancer mortality was similar regardless of CH (p = 0.568) (Fig. 3a). However, patients with CH had statistically higher non-lung cancer mortality (p = 0.042) and unexplained mortality (p = 0.018) compared to patients without CH (FIGS. 3B and 3C).

In a multivariate Cox analysis, the presence of CH was a significant prognostic factor for OS in patients with advanced NSCLC receiving adjuvant therapy, along with a DLCO <60%, the rate of EGFR mutations, histologic type, and overall stage (hazard ratio [HR] [95% confidence interval] = 1.62 [1.10 to 2.38], p = 0.014) (see Table 17 below showing the results of univariate and multivariate analyzes for OS in all patients). Age and the number of comorbidities that were significant in the univariate analysis became insignificant after adjusting for several covariates, including the presence of CH.

To determine the clinical impact of CH according to adjuvant therapy in stage IIB, patients were divided into patients who received adjuvant therapy and those who did not. For patients receiving adjuvant therapy for stage IIB NSCLC, the presence of CH was associated with poorer OS before PSM (p < 0.001) and after PSM (p = 0.007) (FIGS. 4A and 4B). For patients not receiving adjuvant therapy, the prognosis of patients with CH was significantly worse than that of patients without CH (p = 0.045), but became similar after PSM (p = 0.452) (Figures 4C and 4D). ). Details of baseline characteristics of stage IIB patients before and after PSM are shown in Table 18 (baseline characteristics of patients who received adjuvant chemotherapy for stage IIB NSCLC before and after PSM, according to presence of clonal hematopoiesis) and Table 19 below. (Baseline characteristics of patients without adjuvant chemotherapy for stage IIB NSCLC with presence of clonal hematopoiesis before and after PSM).

Example 7. Consideration

Through the above examples, the present inventors investigated the prevalence and characteristics of clonal hematopoiesis (CH) in patients with advanced non-small cell lung cancer (NSCLC). In addition, we evaluated the clinical impact of preoperative CH on survival outcomes in all patients and in patients after PSM.

As a result, 21% of the patients had CH before surgery, which increased with age. Among mutations in genes associated with the presence of CH, mutations in DNMT3A were the most common, followed by ASXL1, TET2, and PPM1D. In addition, CH-PD accounted for 3/4 of CH and served as a more important prognostic factor for overall survival (OS) than CH itself. Presence of CH before surgery was significantly associated with increased overall mortality, especially non-lung cancer mortality and unexplained mortality. Multivariate Cox analysis confirmed the presence of CH as an important prognostic factor among patients receiving adjuvant therapy followed by surgery for advanced-stage NSCLC. The prognostic effect of CH was the same after adopting a rigorous risk-adjustment methodology to appropriately adjust baseline covariates between the two groups.

Since the first detection of non-random X chromosome inactivation in healthy elderly women, research on CH and our understanding has grown considerably over the past few years. According to several previous studies, CH is a common phenomenon associated with aging and is closely related to subsequent hematological malignancies, cardiovascular disease and poor prognosis in patients with advanced solid tumors. Cancer patients have higher CH rates than healthy individuals, and CH is associated with shorter patient survival. This is presumably due to a high genetic predisposition to malignancies, prolonged exposure to carcinogenic environments and cancer-related therapies using genotoxic therapies. Particularly in cancer-related therapy, CH is thought to be highly relevant to CTx and RTx, meaning that local and systemic therapies can promote clonal outgrowth of hematopoietic stem cells (HSCs). In this respect, we hypothesize that CH present before surgery amplifies a series of processes that lead to adverse CH-related outcomes through cancer-related treatment, leading to poor survival outcomes.

In a survival assay according to one example, the presence of CH was significantly associated with poor OS (p = 0.001) (FIG. 2A). However, considering the positive correlation between CH and age, we could not conclude the effect of CH on OS from this result alone. To overcome this problem, we performed two types of statistical adjustments. As a result, even after adjusting for age, gender, smoking history, and number of comorbidities associated with multivariate analysis, the effect of CH as a predictor of prognosis was still significant (p = 0.012) (Table 12 above). In addition, the fact that CH, but not age, was included as the final prognostic factor through progressive stage selection in multivariate analysis suggests that CH is more closely related to survival than age (Table 17 above). After PSM, all clinical variables, including age, were similar regardless of CH, and patients with CH still had poorer OS compared to those without CH (p = 0.029) (Fig. 2c). Thus, we could conclude that the presence of CH is an independent prognostic factor for OS in patients receiving adjuvant therapy for advanced-stage NSCLC.

In terms of cause of death, we found that a significant difference according to the presence of CH appeared in non-lung cancer mortality (p = 0.042) and unknown cause mortality (p = 0.018), but not in lung cancer mortality (p = 0.568). . Judging from the good compliance with postoperative monitoring in patients who completed adjuvant therapy, it is speculated that unexplained deaths are due to acute events, such as cardiopulmonary disease, sepsis, and stroke, rather than relatively slow cancer progression. Thus, these findings support that various adverse outcomes associated with CH in patients with CH are amplified by CTx or RTx, eventually affecting survival.

Most cytotoxic chemotherapeutic agents, including platinum-based compounds, such as cisplatin and the like, target the organ of DNA replication. Conventional chemotherapy is designed to kill rapidly dividing cells, causing severe DNA damage and killing the cells. However, mutations in cancer-related genes such as TP53, PPM1D, and CHEK2 impair the cell death process that should be normally activated by DNA damage, so that hematopoietic stem cells (HSCs) with damaged DNA continue to survive despite the action of cytotoxic drugs. make it possible Therefore, cancer-related therapies are thought to influence the evolutionary trajectory of emerging CH clones. A recent study evaluated the clonal kinetics of CH in response to cancer treatment through sequential sampling, cancer treatment was preferentially selected for clones with mutations in DNA damage response (DDR) genes, and these clones were selected for cytotoxic or radiotherapy. reported that competitive fitness was lower than non-DDR gene mutations in the absence of this.

A recent study focusing on CH mutations classified CH based on its association with PD, CH-PD or CH non-PD in hematological malignancies and then reported its impact. According to the report by Coombs et al., although CH itself did not show an independent prognostic effect, CH-PD was associated with a poorer prognosis regardless of age, gender, and smoking. Consistent with some of these results, in our multivariate analysis, the prognostic effect of CH-PD (p = 0.003) was more significant than that of CH itself (p = 0.012) (Table 13 above). Moreover, unlike CH itself, the negative prognostic effect of CH-PD gradually increased with increasing mutation number, indicating that CH-PD is a more sensitive prognostic factor for OS than CH itself (Table 13 above). In particular, the proportion of CH-PD in this study (79.1%) was higher than that in other studies (52%-67%), probably because of the higher proportion of current/former smokers among lung cancer patients. will be. We believe this is the main reason why CH-PD as well as CH itself appeared as independent prognostic factors for OS in our study (Fig. 2c).

In view of the above, physicians should manage lung cancer patients with CH mutations for whom adjuvant therapy is prescribed as follows. First, it is necessary to distinguish between patients who are at high risk of adverse outcomes and those who are not. There is no clear definition of high-risk CH, but significant blood count abnormalities, single CH mutations at high VAF (> 10%), multiple CH mutations, variants of TP53 and/or PPM1D, DNMT3A variants, and hotspots of IDH1/2 ( hotspot mutations are considered to classify patients into a high-risk group. Second, the elaboration of the patient group for which adjuvant therapy has a favorable prognosis despite the risk of loss of survival due to CH should proceed. Considering this, the present inventors stratified patients who received adjuvant therapy according to the comprehensive stage and then performed subgroup analysis. Regardless of overall stage, patients with CH mutations had poorer survival outcomes compared to patients without CH ( FIGS. 6A to 6C ), but a particularly significant difference was observed in patients with stage IIB (5-year rate: 48.5 % vs. 73.8%, p < 0.001) (Fig. 4a), which was still significant after PSM (5-year ratio: 48.5% vs. 74.3%) (Fig. 4b). However, the presence of CH did not affect the significant difference in OS in stage IIB patients who did not receive adjuvant therapy after PSM (5-year rate: 41.6% vs. 53.7%) (FIG. 4D). These results indicate that preoperative CH mutations can amplify adverse outcomes associated with CH with adjuvant therapy, leading to poor survival outcomes. Therefore, considering that the survival benefit of adjuvant therapy ranges from 4% to 15%, we believe that adjuvant therapy should be more carefully determined with a multidisciplinary approach in stage II NSCLC patients. On the other hand, treatment that inhibits the progression of associated adverse outcomes may be a good supplemental therapy for managing lung cancer patients with CH mutations. For example, the anti-inflammatory drug canakinumab, a humanized monoclonal antibody to IL-1β, has been reported to reduce the incidence of cardiovascular events and lung cancer in high-risk patients with atherosclerotic disease. For patients with stage III disease who require systemic therapy for treatment, aggressive use of molecularly targeted interventions may be an alternative for patients with high-risk CH mutations. Finally, for patients with high-risk CH mutations, an individualized follow-up period and thorough monitoring for adverse outcomes of CH, such as cardiovascular disease or secondary malignancies, are required.

In conclusion, presurgery CH mutations have important clinical implications for NSCLC patients receiving adjuvant therapy following surgery, which reduces survival outcomes.

According to the present invention, when it is confirmed that there is a mutation in a gene related to clonal hematopoiesis (CH) of an individual, it is possible to predict the prognosis according to the treatment of lung cancer, particularly non-small cell lung cancer, of the individual. In addition, the present invention can provide useful information for determining the application of adjuvant therapy following surgical resection in relation to lung cancer treatment, information useful for determining whether to administer a therapeutic agent for lung cancer treatment, and the like, and furthermore, lung cancer It is expected to have great industrial value as it can provide useful information for evaluating the efficacy and safety of drug candidates for patients in clinical trials of drug candidates for treatment.

Claims (32)

1. Confirming whether clonal hematopoiesis exists in an individual through genetic analysis of a biological sample isolated from an individual being treated for lung cancer

   A method of providing information for predicting the prognosis of lung cancer treatment in a subject.
2. According to claim 1,

   The step of determining whether clonal hematopoiesis exists is DNMT3A, ASXL1, TET2, PPM1D, ATM, BCL11B, CARD11, CBL, CD79B, CHEK2, CUX1, ETV6, FOXP1, JAK2, KMT2D, MAP3K1, MPL, NF1, NOTCH1, NOTCH2, PHIP, PRPF40B, RAD21, SETD2, SF3B1, TET1, TNFAIP3, TP53, U2AF1 and ZRSR2 Including determining whether one or more mutations exist in one or more genes selected from the group consisting of

   How to Provide Information.
3. According to claim 2,

   The one or more genes include one or more selected from the group consisting of DNMT3A, ASXL1, TET2, PPM1D, SF3B1, ATM and TNFAIP3

   How to Provide Information.
4. According to claim 2,

   The one or more genes include DNMT3A

   How to Provide Information.
5. According to claim 2,

   Further comprising determining whether clonal hematopoiesis exists based on whether one or more mutations exist in the one or more genes and variant allele frequency (VAF)

   How to Provide Information.
6. According to claim 5,

   Further comprising determining that clonal hematopoiesis exists when one or more mutations are present in the one or more genes and the variant allele frequency (VAF) is about 1.8% or more

   How to Provide Information.
7. According to claim 1,

   When clonal hematopoiesis exists in the subject, compared to the case where clonal hematopoiesis does not exist, indicating that the prognosis of lung cancer treatment of the subject is not good

   How to Provide Information.
8. According to claim 1,

   The prognosis includes overall survival or recurrence-free survival after tumor removal surgery or after application of adjuvant therapy following tumor removal surgery.

   How to Provide Information.
9. According to claim 1,

   The subject is treated with tumor removal surgery, chemotherapy, chemoradiation, anti-cancer immunotherapy, or a combination thereof.

   How to Provide Information.
10. According to claim 1,

    The lung cancer is non-small cell lung cancer

    How to Provide Information.
11. According to claim 10,

    The non-small cell lung cancer is stage I, stage II or later stage

    How to Provide Information.
12. According to claim 1,

    The subject is a lung cancer patient who has undergone tumor removal surgery

    How to Provide Information.
13. According to claim 12,

    The step of determining whether clonal hematopoiesis exists is performed before receiving tumor removal surgery, after receiving tumor removal surgery, before applying adjuvant therapy after tumor removal surgery, or after applying adjuvant therapy following tumor removal surgery.

    How to Provide Information.
14. According to claim 1,

    The mutation is a missense mutation, a frameshift mutation, a nonsense mutation, a splice mutation, a nucleotide addition, deletion or substitution, or a combination thereof

    How to Provide Information.
15. According to claim 1,

    The genetic analysis includes Next Generation Sequencing (NGS)

    How to Provide Information.
16. Containing as an active ingredient an agent for confirming the presence of clonal hematopoiesis using a biological sample isolated from an individual being treated for lung cancer

    A composition for predicting the prognosis of lung cancer treatment in a subject.
17. According to claim 16,

    DNMT3A, ASXL1, TET2, PPM1D, ATM, BCL11B, CARD11, CBL, CD79B, CHEK2, CUX1, ETV6, FOXP1, JAK2, KMT2D, MAP3K1, MPL, NF1, NOTCH1, NOTCH2, PHIP, PRPF40B, RAD21, SETD2 , SF3B1, TET1, TNFAIP3, TP53, U2AF1 and ZRSR2 comprising an agent for detecting the presence of one or more mutations in one or more genes selected from the group consisting of

    composition.
18. According to claim 16,

    The agent comprises an agent for detecting whether one or more mutations exist in one or more genes selected from the group consisting of DNMT3A, ASXL1, TET2, PPM1D, SF3B1, ATM and TNFAIP3

    composition.
19. According to claim 16,

    The lung cancer is non-small cell lung cancer

    composition.
20. According to claim 19,

    The non-small cell lung cancer is stage I, stage II or later stage

    composition.
21. According to claim 16,

    The presence of clonal hematopoiesis indicates poor lung cancer treatment prognosis.

    composition.
22. According to claim 16,

    The mutation is a missense mutation, a frameshift mutation, a nonsense mutation or a splice mutation, a nucleotide addition, deletion or substitution, or a combination thereof

    composition.
23. According to claim 16,

    The formulation comprises primers, probes or antisense nucleic acids for detecting genetic mutations in clonal hematopoiesis.

    composition.
24. Comprising the composition according to any one of claims 16 to 23

    A kit for predicting the prognosis of lung cancer treatment in a subject.
25. Comprising the composition according to any one of claims 16 to 23

    A panel for genetic analysis to detect genetic mutations in clonal hematopoiesis.
26. Prior to administration of a therapeutic agent for lung cancer treatment, confirming whether clonal hematopoiesis exists in the subject through genetic analysis of a biological sample isolated from the subject

    Lung cancer treatment methods.
27. The method of claim 26,

    The step of determining whether clonal hematopoiesis exists is DNMT3A, ASXL1, TET2, PPM1D, ATM, BCL11B, CARD11, CBL, CD79B, CHEK2, CUX1, ETV6, FOXP1, JAK2, KMT2D, MAP3K1, MPL, NF1, NOTCH1, NOTCH2, PHIP, PRPF40B, RAD21, SETD2, SF3B1, TET1, TNFAIP3, TP53, U2AF1 and ZRSR2 Including determining whether one or more mutations exist in one or more genes selected from the group consisting of

    method.
28. The method of claim 27,

    Further comprising determining that clonal hematopoiesis exists when one or more mutations are present in the one or more genes and the variant allele frequency (VAF) is about 1.8% or more

    method.
29. The method of claim 26,

    Further comprising the step of administering a lung cancer treatment when it is determined that clonal hematopoiesis does not exist in the subject

    method.
30. The method of claim 26,

    The therapeutic agent for the treatment of lung cancer includes a drug candidate for clinical trials.

    method.
31. The method of claim 26,

    Further comprising the step of administering a lung cancer treatment when it is determined that clonal hematopoiesis exists in the subject

    method.
32. According to claim 31,

    The therapeutic agent for the treatment of lung cancer includes a drug candidate for clinical trials.

    method.

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