KR20200064891A - Method of providing the information for predicting of hematologic malignancy prognosis after peripheral blood stem cell transplantation - Google Patents
Method of providing the information for predicting of hematologic malignancy prognosis after peripheral blood stem cell transplantation Download PDFInfo
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Abstract
Description
본 발명은 조혈모세포 이식 후 혈액암 예후 예측을 위한 정보 제공 방법에 관한 것이다. The present invention relates to a method for providing information for predicting the prognosis of blood cancer after hematopoietic stem cell transplantation.
혈액 악성종양 또는 혈액암은 다양하지만, 골수 또는 림프 조직으로부터 기원하여 혈액 기능에 영향을 미치는 연관된 암이다. 매년, 백혈병, 호지킨 및 비-호지킨 림프종 및 골수종의 새로운 사례가, 미국에서 진단되는 모든 새로운 암 사례의 거의 10%를 차지한다. 항체 및 키나제 억제제(예: 이마티니브 - BCR-ABL 억제제)를 사용하는 표적 치료요법이 개발되었지만, 혈액암을 다루는데 있어서 여전히 화학 치료요법 및 방사선 치료요법에 많이 의존한다. 이들은 통상적으로 상당한 부작용을 나타내며, 낮은 효능을 생성한다. 골수형성이상 증후군(Myelodysplastic syndromes, MDS)은 클론성 줄기세포 장애에 의한 골수성 종양으로, 비효율적인 조혈 작용을 일으키고 급성 골수성 백혈병(Acute myeloid leukemia, AML)의 진행 위험성을 증가시킨다. 이 증후군은 무기력증부터 AML의 진행 또는 심각한 혈구 감소증과 같이 생명을 위협하는 다양한 임상적 증상을 나타낸다. 아자시티딘(Azacitidine) 또는 데시타빈(Decitabine)과 같은 저메틸화 억제제(Hypomethylating agents, HMA)를 이용한 치료는 고위험 MDS 환자 또는 혈구 감소증 증상을 가지는 저위험 MDS 환자에게 첫 번째 치료 방법이다. 그러나, 임상 시험자의 HMT에 따른 생존율 및 저메틸화 치료(Hypomethylating therapy, HMT)에 대한 응답을 예측하는 것은 여전히 해결해야 할 문제로 남아있다. Hematologic malignancies or hematologic cancers are various, but associated cancers originating from bone marrow or lymphoid tissues that affect blood function. Each year, new cases of leukemia, Hodgkin's and non-Hodgkin's lymphoma and myeloma account for almost 10% of all new cancer cases diagnosed in the United States. Targeted therapies using antibodies and kinase inhibitors (eg, Imatinib-BCR-ABL inhibitors) have been developed, but they still rely heavily on chemotherapy and radiation therapy to treat blood cancer. They usually exhibit significant side effects and produce low efficacy. Myelodysplastic syndromes (MDS) are myeloid tumors caused by clonal stem cell disorders, which cause inefficient hematopoietic action and increase the risk of progression of acute myeloid leukemia (AML). This syndrome exhibits a variety of life-threatening clinical symptoms, from lethargy to progression of AML or severe hemopenia. Treatment with hypomethylating agents (HMA), such as azacitidine or decitabine, is the first method of treatment for high-risk MDS patients or low-risk MDS patients with hemopenia symptoms. However, predicting the survival rate and response to hypomethylating therapy (HMT) according to HMT in clinical trials remains a problem to be solved.
치료법 선택을 돕기 위하여 현재까지 다양한 예후 점수 시스템이 개발되어 왔다. 그 중에서도, 가장 최근 시스템인 개정된 국제 예후 점수 시스템(Revised International Prognostic Scoring System, IPSS-R)은 환자의 생존율을 예측하는데 유용하나, HMT에 대한 치료 반응성을 예측하기에는 효과적이지 않았다. 따라서, 신뢰할 수 있는 분자유전학적 마커를 확인한다면, 이를 통해 MDS에 대한 추가적 예후 정보를 제공할 수 있을 것이다. 대상자가 골수에서 5% 미만의 모구를 보이고 분자적으로 검출 가능한 암을 나타내는 경우 대상자는 최소 잔류 질병(MRD: minimum residual disease)을 나타낸다고 일컬어진다. 어떤 MRD도 검출되지 않을 때 (<10-4, 즉 104 골수 세포 당 하나의 백혈병 세포 미만이 검출가능할 때) 완전한 분자적 완화가 달성된다. 지난 몇 년간 일련의 후향적 연구들은, 소아 백혈병에서 이미 밝혀진 바와 같이 성인 급성 림프구성 백혈병에서 MRD가 독립적인 예후인자임을 보였다 (Bruggemann et al. Blood 107 (2006), 1116-1123; Raff et al. Blood 109 (2007), 910-915).Various prognosis scoring systems have been developed to assist in the selection of treatment methods. Among them, the most recent system, the Revised International Prognostic Scoring System (IPSS-R), is useful for predicting patient survival, but was not effective in predicting treatment response to HMT. Therefore, if reliable molecular genetic markers are identified, this may provide additional prognostic information for MDS. A subject is said to exhibit a minimum residual disease (MRD) if the subject has less than 5% of the hair cells in the bone marrow and indicates molecularly detectable cancer. Complete molecular relaxation is achieved when no MRD is detected (<10 -4 , ie less than one leukemia cell per 10 4 bone marrow cells is detectable). In the past few years, a series of retrospective studies have shown that MRD is an independent prognostic factor in adult acute lymphocytic leukemia, as already revealed in pediatric leukemia (Bruggemann et al. Blood 107 (2006), 1116-1123; Raff et al. Blood 109 (2007), 910-915).
한편, 혈액암 환자에서 강력한 항암 화학 요법 단독 혹은 방사선 요법과 함께 암세포와 환자 자신의 조혈모세포를 제거한 다음 새로운 조혈모세포를 이식해 주는 치료법인 조혈모세포이식(hematopoietic stem cell transplantation, HSCT)은 과거 골수를 활용하던 골수이식(bone marrow transplantation, BMT)의 영역을 넘어서 현재는 말초혈액(peripheral blood, PB)과 제대혈(cord blood, CB) 내에 존재하는 모든 형태의 조혈모세포를 이식원으로 활용하여 이식하는 것을 말한다. 동종 조혈모세포 공여자(조혈모세포 기증자)는 환자와 성별, 혈액형이 달라도 상관 없으나, 인백혈구항원(HLA, human leukocyte antigen) 형(type)이 전부 또는 일부가 맞아야 하며, 이후 전처치 요법으로 인한 골수억제 기간을 지나 타인의 조혈모세포가 환자의 골수에 자리를 잡고 새로운 혈액세포를 만들어낼 때까지 (이를 생착이라 함) 감염 등의 부작용을 유의하며 보전적 치료를 유지한다. 조혈모세포 이식 후 추적 관찰을 위해 골수의 세포 형태 관찰, 세포유전검사, 분자유전검사와 적혈구 표현형 검사 등을 시행하고 있으나, 골수 검사나 적혈구 표현형 검사는 예민도가 낮고 조기에 임상 양상의 변화를 예측하기 어려운 단점이 있다. 세포유전검사는 진단 당시 특이한 염색체 이상이 없었던 정상 핵형인 경우 또는 같은 성별의 조혈모세포이식을 받은 경우에는 유용한 정보를 얻기 어렵다. 또한, 분자유전학검사도 예민도는 높지만 진단 당시 특이한 재배열이 없었던 경우 추적 관찰의 지표로 사용할 수 없는 문제점이 있어왔다. 조혈모세포 이식은 주로 혈연 관계에서 이루어지므로 대립인자의 양상이 유사한 경우가 많아 정보 제공에 유용한 표지자를 선택하기 위해서는 되도록 여러 표지자에 대한 검사를 동시에 시행할 필요가 있다. 공여자와 환자의 유전학적 다형성 부위의 변화 양상을 비교하는 키메리즘(chimerism) 검사는 현재 환자 조혈세포의 구성 상태를 파악할 수 있으므로 추적 관찰시 유용한 지표가 될 수 있다. 혼합 키메리즘(mixed chimerism)은 조혈모세포 이식후 환자의 골수세포가 공여자의 골수 세포로 완전히 치환되지 않고 남아 공여자와 환자의 세포가 함께 존재하는 상태를 의미한다. 완전 키메리즘(complete chimerism)은 환자의 골수가 완전히 공여자의 세포로만 구성된 상태를 의미하며, 성공적인 이식이란 완전 키메리즘 상태가 되는 것을 목표로 한다. On the other hand, hematopoietic stem cell transplantation (HSCT), a treatment that removes cancer cells and their own hematopoietic stem cells and then transplants new hematopoietic stem cells with powerful anti-cancer chemotherapy alone or radiation therapy in patients with hematologic cancer, has previously Transplanting by utilizing all types of hematopoietic stem cells present in peripheral blood (PB) and cord blood (CB) beyond the scope of bone marrow transplantation (BMT) Speak. Allogeneic hematopoietic stem cell donors (hematopoietic stem cell donors) may have different sex and blood types from patients, but all or part of the human leukocyte antigen (HLA) type must be correct, and bone marrow suppression due to pretreatment therapy After a period of time, until the hematopoietic stem cells of other people settle in the bone marrow of the patient and create new blood cells (called engraftment), keep in mind the side effects of infection and maintain conservative treatment. For follow-up after hematopoietic stem cell transplantation, bone marrow cell morphology, cytogenetic testing, molecular genetic testing, and erythrocyte phenotyping are performed. However, bone marrow or erythrocyte phenotyping tests have low sensitivity and predict changes in clinical features early. There are difficult disadvantages. Cytogenetic testing is difficult to obtain useful information in the case of a normal karyotype with no specific chromosomal abnormality at the time of diagnosis, or in the case of hematopoietic stem cell transplantation of the same sex. In addition, the molecular genetics test also has a high sensitivity, but there has been a problem that cannot be used as an indicator of follow-up when there is no specific rearrangement at the time of diagnosis. Since hematopoietic stem cell transplantation is mainly performed in a blood-related relationship, there are many cases in which alleles are similar, so it is necessary to simultaneously perform tests on multiple markers to select useful markers for information provision. The chimerism test, which compares donor-patient genetic polymorphism changes, can be a useful indicator for follow-up because it can determine the current state of hematopoietic cells. Mixed chimerism (mixed chimerism) refers to a state in which the bone marrow cells of a patient after hematopoietic stem cell transplantation are not completely replaced by the bone marrow cells of the donor, but the donor and the patient's cells are present together. Complete chimerism refers to a state in which the patient's bone marrow is composed entirely of donor cells, and a successful transplant aims to become a complete chimerism state.
본 발명에서는 조혈모세포 이식 후 혈액암 예후 예측을 위한 정보 제공 방법을 개발하는 것을 목적으로 한다.An object of the present invention is to develop a method for providing information for predicting the prognosis of blood cancer after hematopoietic stem cell transplantation.
상기 목적의 달성을 위해, 본 발명은 조혈모세포 이식 후 혈액암 예후 진단용 NGS 패널을 제공한다.To achieve the above object, the present invention provides an NGS panel for prognostic diagnosis of hematologic cancer after hematopoietic stem cell transplantation.
또한, 본 발명은 조혈모세포 이식 후 혈액암 예후 진단용 조성물을 제공한다.In addition, the present invention provides a composition for diagnosing blood cancer prognosis after hematopoietic stem cell transplantation.
또한, 본 발명은 조혈모세포 이식 후 혈액암 예후 진단용 키트를 제공한다.In addition, the present invention provides a kit for diagnosing blood cancer prognosis after hematopoietic stem cell transplantation.
또한, 본 발명은 NGS 기반 조혈모세포 이식 후 미세잔존질환 및 키메리즘 분석방법을 제공한다.In addition, the present invention provides a method for analyzing microscopic residual disease and chimerism after transplantation of NGS-based hematopoietic stem cells.
아울러, 본 발명은 조혈모세포 이식 후 혈액암 예후 예측을 위한 정보 제공 방법을 제공한다.In addition, the present invention provides a method for providing information for predicting the prognosis of blood cancer after hematopoietic stem cell transplantation.
본 발명에 따르면, 본 발명의 NGS 패널은 조혈모세포 이식된 개체의 미세잔존질환 (Minimal Residual Disease; MRD) 및 조혈모세포 이식된 개체와 공여 개체의 키메리즘을 동시에 분석 가능하므로, 정확하고 빠르게 백혈병 및 림프종 등을 포함하는 혈액암의 예후 예측할 수 있어, 관련 산업에 유용하게 이용할 수 있다.According to the present invention, the NGS panel of the present invention can accurately and rapidly analyze the microscopic residual disease (MRD) of the hematopoietic stem cell transplanted individual and the chimerism of the hematopoietic stem cell transplanted and donor individual, so as to accurately and rapidly leukemia. And it can predict the prognosis of blood cancer, including lymphoma, etc., it can be usefully used in related industries.
도 1은 미세잔존질환 평가를 위한 질병 연관 유전자 돌연변이 분석 알고리즘을 모식화한 도이다.
도 2는 미세잔존질환 평가를 위하여, 혈액종양질환의 진단 및 예후와 연관된 유전자의 돌연변이를 선별하기 위한 차세대염기서열분석 패널을 나타낸 도이다.
도 3은 키메리즘 분석을 위한 NGS 분석용 SNP를 이용한 키메리즘 분석 알고리즘을 모식화한 도이다.
도 4는 Korean Genome Database (KRGDB)의 한국인 대립 유전자 빈도 자료(allele frequency data)를 나타낸 도이다.
도 5는 키메리즘 분석에 유용한 것으로 선정한 SNP의 크로모좀(chromosome, chr), coordinate, DB SNP ID, KRGDB 정보를 나타낸 도이다.
도 6은 본 발명의 개발된 알고리즘을 이용한 질병 연관 유전자 돌연변이 분석한 결과를 나타낸 도이다.
도 7은 조혈모세포 이식 후 질병 연관 돌연변이 정량 분석을 통한 미세잔존질환 평가한 결과를 나타낸 도이다.
도 8은 NGS 및 STR 분석 방법으로 키메리즘을 비교한 도이다:
A: NGS 및 STR 분석으로 얻은 공여자 키메리즘 값의 상관 관계;
B: 두 키메리즘 분석의 Blant-Altman 플롯;
C 내지 F: 환자 11(C), 12(D), 13(E) 및 14(F)의 조혈모세포 이식 전 후 NGS, STR 및 MAB(mutant allele burden)을 이용한 생착 모니터링; 및
G: 환자 1의 HSCT 전/후의 SETBP1 영역의 통합 지놈 뷰어.
도 9는 본 발명의 키메리즘 분석 알고리즘을 이용하여 분석한 결과를 나타낸 도이다.1 is a diagram schematically illustrating a disease-related gene mutation analysis algorithm for evaluating microscopic residual disease.
FIG. 2 is a diagram showing a next-generation base sequencing panel for screening mutations in genes associated with diagnosis and prognosis of blood tumor diseases for evaluating microscopic residual disease.
3 is a diagram schematically showing a chimeric analysis algorithm using SNP for NGS analysis for chimerism analysis.
4 is a diagram showing Korean allele frequency data of the Korean Genome Database (KRGDB).
5 is a diagram showing the chromosome (chromosome, chr), coordinate, DB SNP ID, KRGDB information of the SNP selected as useful for chimerism analysis.
6 is a diagram showing the results of analysis of disease-related gene mutations using the developed algorithm of the present invention.
7 is a view showing the results of evaluating microscopic residual disease through quantitative analysis of disease-related mutations after hematopoietic stem cell transplantation.
8 is a comparison of chimerism by NGS and STR analysis methods:
A: Correlation of donor chimerism values obtained by NGS and STR analysis;
B: Blant-Altman plot of two chimerism analysis;
C to F: Engraftment monitoring with NGS, STR and mutant allele burden (MAB) before and after hematopoietic stem cell transplantation of patients 11(C), 12(D), 13(E) and 14(F); And
G: Integrated genome viewer of SETBP1 region before/after HSCT in
9 is a diagram showing the results of analysis using the chimeric analysis algorithm of the present invention.
이하, 첨부된 도면을 참조하여 본 발명의 구현예로 본 발명을 상세히 설명하기로 한다. 다만, 하기 구현예는 본 발명에 대한 예시로 제시되는 것으로, 당업자에게 주지 저명한 기술 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략할 수 있고, 이에 의해 본 발명이 제한되지는 않는다. 본 발명은 후술하는 특허청구범위의 기재 및 그로부터 해석되는 균등 범주 내에서 다양한 변형 및 응용이 가능하다. Hereinafter, the present invention will be described in detail with reference to the accompanying drawings as an embodiment of the present invention. However, the following embodiments are presented as examples of the present invention, and when it is determined that a detailed description of a technique or configuration well known to those skilled in the art may unnecessarily obscure the subject matter of the present invention, the detailed description may be omitted. , Thereby, the present invention is not limited. The present invention is capable of various modifications and applications within the scope of the claims and the equivalents interpreted therefrom.
또한, 본 명세서에서 사용되는 용어(terminology)들은 본 발명의 바람직한 실시예를 적절히 표현하기 위해 사용된 용어들로서, 이는 사용자, 운용자의 의도 또는 본 발명이 속하는 분야의 관례 등에 따라 달라질 수 있다. 따라서, 본 용어들에 대한 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다. 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.In addition, terms used in the present specification (terminology) are terms used to properly represent a preferred embodiment of the present invention, which may vary according to a user, an operator's intention, or a custom in a field to which the present invention pertains. Therefore, definitions of these terms should be made based on the contents throughout the specification. Throughout the specification, when a part “includes” a certain component, it means that the component may further include other components, not to exclude other components, unless otherwise stated.
일 측면에서, 본 발명은 ABCA12, ABL1, ASXL1, ATM, ATRX, ATXN7L1, BCOR, BRAF, BRCC3, CALR, CBL, CBLB, CD101, CEBPA, CREBBP, CSF1R, CSF3R, CTCF, CUX1, DNMT1, DNMT3A, EGFR, EP300, ERG, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, HIPK2, IDH1, IDH2, INVS, IRF1, JAK2, KDM2B, KDM6A, KIT, KMT2A, KMT2D, KRAS, LAMB4, MECOM, MET, MLL3, MLL5, MN1, MPL, NCOR2, NF1, NLRP1, NOTCH1, NPM1, NRAS, NRD1, NUP98, OCA2, PDGFRA, PHF12, PHF6, PRPF40B, PRPF8, PTPN11, RAD21, RAD50, RINT1, ROBO1, ROBO2, RUNX1, RUNX1T1, SETBP1, SF3A1, SF3B1, SMC1A, SMC3, SRSF2, STAG2, TET1, TET2, TP53, TP53BP1, U2AF1, U2AF2, WT1 및 ZRSR2로 이루어지는 군으로부터 선택되는 어느 하나 이상의 유전자, 및 rs3736908, rs2304429, rs2289195, rs2276599, rs4685, rs16865262, rs788017, rs788018, rs788023, rs10498027, rs4673925, rs10498030, rs17501837, rs1523721, rs3821735, rs6795556, rs2271151, rs967454, rs2304503, rs11713094, rs2305037, rs2305036, rs2335052, rs12498609, rs2647243, rs2454206, rs10033601, rs2070731, rs2070730, rs9282762, rs9282761, rs216136, rs3829987, rs2228422, rs3830035, rs3830036, rs2072454, rs4947986, rs2227983, rs2227984, rs2293347, rs2240455, rs10953468, rs11976329, rs2074748, rs420753, rs2072407, rs10274535, rs2240819, rs74483926, rs6464211, rs10252263, rs2230722, rs2274649, rs34001282, rs35445683, rs1056171, rs2229971, rs10823229, rs12773594, rs12221107, rs11195199, rs1875, rs1799937, rs16754, rs664982, rs17210957, rs11168830, rs3741622, rs10849885, rs2293514, rs747443, rs4073630, rs17086226, rs2491223, rs2491227, rs2491231, rs1933437, rs1800414, rs1800404, rs550239, rs2602141, rs690367, rs689647, rs560191, rs2439831, rs60147683, rs11651270, rs884367, rs1642785, rs2952976, rs1801052, rs2905876, rs2285892, rs9894648, rs964288, rs663651, rs3744825, rs2290684, rs2114724, rs2228611, rs2241531, rs11672909, rs2228612, rs1049481, rs664684, rs2295454, rs2295765, rs4911231, rs7121, rs9608885, rs20552, rs2076577, rs2076578, rs5936062, rs6520618, rs2230018, rs20539, rs1264011, rs3088074 및 rs35268552로 이루어지는 군으로부터 선택되는 어느 하나 이상의 SNP를 특이적으로 검출하는 조혈모세포 이식 후 혈액암 예후 진단용 NGS(next generation sequencing) 패널에 관한 것이다.In one aspect, the present invention ABCA12, ABL1, ASXL1, ATM, ATRX, ATXN7L1, BCOR, BRAF, BRCC3, CALR, CBL, CBLB, CD101, CEBPA, CREBBP, CSF1R, CSF3R, CTCF, CUX1, DNMT1, DNMT3A, EGFR , EP300, ERG, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, HIPK2, IDH1, IDH2, INVS, IRF1, JAK2, KDM2B, KDM6A, KIT, KMT2A, KMT2D, KRAS, LAMB4, MECOM3, MELL, MET , MLL5, MN1, MPL, NCOR2, NF1, NLRP1, NOTCH1, NPM1, NRAS, NRD1, NUP98, OCA2, PDGFRA, PHF12, PHF6, PRPF40B, PRPF8, PTPN11, RAD21, RAD50, RINT1, ROBO1, ROBO2, RUNX1 , SETBP1, SF3A1, SF3B1, SMC1A, SMC3, SRSF2, STAG2, TET1, TET2, TP53, TP53BP1, U2AF1, U2AF2, WT1 and any one or more genes selected from the group consisting of ZRSR2, and rs3736908, rs2304429, rs22891, rs2304429, rs22891, rs4685, rs16865262, rs788017, rs788018, rs788023, rs10498027, rs4673925, rs10498030, rs17501837, rs1523721, rs3821735, rs6795556, rs2271151, rs967454, rs2304503, rs11713094, rs23050, rs23050 rs9282762, rs9282761, rs216136, rs3829987, rs2228422, r s3830035, rs3830036, rs2072454, rs4947986, rs2227983, rs2227984, rs2293347, rs2240455, rs10953468, rs11976329, rs2074748, rs420753, rs2072407, rs10274535, rs2240819, rs74483926, rs6464211, rs10252263, rs2230722, rs2274649, rs34001282, rs35445683, rs1056171, rs2229971, rs10823229, rs12773594, rs12221107, rs11195199, rs1875, rs1799937, rs16754, rs664982, rs17210957, rs11168830, rs3741622, rs10849885, rs2293514, rs747443, rs4073630, rs17086226, rs2491223, rs2427, 238 rs560191, rs2439831, rs60147683, rs11651270, rs884367, rs1642785, rs2952976, rs1801052, rs2905876, rs2285892, rs9894648, rs964288, rs663651, rs3744825, rs22906, rs2114724, 222 Hematopoiesis that specifically detects one or more SNPs selected from the group consisting of rs7121, rs9608885, rs20552, rs2076577, rs2076578, rs5936062, rs6520618, rs2230018, rs20539, rs1264011, rs3088074 and rs35268552 It relates to an NGS (next generation sequencing) panel for diagnosing blood cancer prognosis after cell transplantation.
일 구현예에서, 본 발명의 NGS 패널은 미세잔존질환(Minimal Residual Disease; MRD) 및 키메리즘(chimerism)을 동시에 분석할 수 있다.In one embodiment, the NGS panel of the present invention can simultaneously analyze Micro Residual Disease (MRD) and chimerism.
일 구현예에서, 키메리즘 분석에 가장 유용한 유전자 좌는 ABCA12 유전자 염색체 2번의 215928972 위치의 rs1523721일 수 있다.In one embodiment, the most useful locus for chimeric analysis may be rs1523721 at
일 구현예에서, 본 발명의 NGS 패널은 상기 유전자들을 특이적으로 검출하기 위한 표적 영역(target regions)에 대한 위치 정보를 포함할 수 있다.In one embodiment, the NGS panel of the present invention may include location information for target regions for specifically detecting the genes.
일 구현예에서, 상기 유전자들의 표 2에 표시된 영역에 특이적인 프로브들을 포함할 수 있다.In one embodiment, probes specific to the region indicated in Table 2 of the genes may be included.
일 구현예에서, 표적 캡쳐 방법을 이용한 NGS 분석 방법용 NGS 패널일 수 있다.In one embodiment, it may be a NGS panel for NGS analysis method using a target capture method.
일 구현예에서, 혈액암은 만성 골수단구성 백혈병(CMMoL), 발덴스트롬 마크로글로불린혈증, 호지킨 병(HD), 비-호지킨 림프종(NHL), 급성 림프성 백혈병(ALL), 급성 골수성 백혈병(AML), 급성 전골수성 백혈병(APL), 만성 림프성 백혈병(CLL), 만성 골수성 백혈병(CML), 만성 호중구성 백혈병(CNL), 급성 미분화 백혈병(AUL), 역성형 대세포림프종(ALCL), 전림프성 백혈병(PML), 청소년 골수단구성 백혈병(JMML), 성인 T-세포 ALL, 삼계열 골수 이형성증을 지니는 AML(AML/TMDS), 혼합된 계통백혈병(MLL), 골수이형성 증후군(MDS), 골수증식성 장애(MPD) 및 다발성 골수종(MM)으로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있으며, 급성 골수성 백혈병 및 골수이형성 증후군인 것이 더욱 바람직하다.In one embodiment, the blood cancer is chronic osteomyelocytic leukemia (CMMoL), Waldenstrom macroglobulinemia, Hodgkin's disease (HD), non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic neutrophil leukemia (CNL), acute undifferentiated leukemia (AUL), anaplastic large cell lymphoma (ALCL) , Prelymphocytic leukemia (PML), juvenile bone marrow constitutive leukemia (JMML), adult T-cell ALL, AML with trilineal bone marrow dysplasia (AML/TMDS), mixed lineage leukemia (MLL), myelodysplastic syndrome (MDS) ), myeloproliferative disorder (MPD) and multiple myeloma (MM) may be any one or more selected from the group consisting of acute myeloid leukemia and myelodysplastic syndrome is more preferred.
본 발명에서 용어, "NGS(Next-generation sequencing, 차세대 염기서열 시퀀싱)"은 Massive parallel sequencing, 대용량 염기서열 분석법, 또는 대규모 병렬형 염기서열 분석법이라고 한다. 하나의 유전체를 무수히 많은 조각으로 분해하여 각 조각을 동시에 읽어낸 뒤, 얻은 데이터를 생물 정보학적 기법을 이용하여 조합함으로써 방대한 유전체 정보를 빠르게 해독하는 분석법을 의미한다. 기존의 Sanger 방식과 달리 대량의 병렬 데이터 생산이 가능하고, DNA 서열에 대한 증폭을 하고 그 후 형광 표식 등을 카메라로 찍어 이미지 처리를 하는 과정을 거쳐 염기를 읽어내는 분석법이다. 상기 차세대 염기서열 시퀀싱의 분석 장비는 사용하는 화학 반응 및 염기서열 검출 원리 등 세부 기술에 따라 고유의 특성 및 장단점에 따라 다양한 플랫폼이 출시되었다.In the present invention, the term, "NGS (Next-generation sequencing, next-generation sequencing)" is referred to as Massive parallel sequencing, large-scale sequencing, or large-scale parallel sequencing. It refers to an analysis method that rapidly deciphers a large amount of genomic information by decomposing one genome into countless pieces, reading each piece simultaneously, and combining the obtained data using bioinformatics techniques. Unlike the conventional Sanger method, it is possible to produce a large amount of parallel data, and it is an analysis method that reads the base through a process of image processing by amplifying the DNA sequence and then photographing a fluorescent marker with a camera. As for the analysis equipment of the next-generation sequencing, various platforms have been released according to unique characteristics and advantages and disadvantages according to detailed technologies such as the chemical reaction and the sequence detection principle used.
본 발명에서 "진단"은 특정 질병 또는 질환에 대한 한 객체의 감수성(susceptibility)을 판정하는 것, 한 객체가 특정 질병 또는 질환을 현재 가지고 있는 지 여부를 판정하는 것, 특정 질병 또는 질환에 걸린 한 객체의 예후(prognosis)(예컨대, 전-전이성 또는 전이성 암 상태의 동정, 암의 단계 결정 또는 치료에 대한 암의 반응성 결정)를 판정하는 것, 또는 테라메트릭스(therametrics)(예컨대, 치료 효능에 대한 정보를 제공하기 위하여 객체의 상태를 모니터링 하는 것)을 포함한다."Diagnosis" in the present invention is to determine the susceptibility of an object to a specific disease or condition, to determine whether an object currently has a specific disease or condition, as long as the disease or condition Determining an object's prognosis (e.g., identification of a pre-metastatic or metastatic cancer state, determining the stage of the cancer or determining the responsiveness of the cancer to treatment), or terrametrics (e.g., for therapeutic efficacy) And monitoring the state of the object to provide information).
본 발명에서 "예후(prognosis)"는 질병을 진단하여 판단된 장래의 증세 또는 경과에 대한 전망을 말한다. 암 환자에 있어서 예후는 통상적으로 암 재발 또는 외과적 시술 후 일정기간 내의 전이 여부 또는 생존기간을 뜻한다. 예후의 예측(또는 예후의 진단)은 특히 초기 암 환자의 화학치료 여부를 비롯하여 향후 암 치료의 방향에 대한 단서를 제시하므로 매우 중요한 임상적 과제이다. 예후 예측은 질환 치료제에 대한 환자의 반응, 치료 경과에 대한 예측도 포함된다.In the present invention, "prognosis" refers to a prospect for future symptoms or progress determined by diagnosing a disease. In cancer patients, the prognosis usually refers to the recurrence of cancer or the metastasis or survival within a certain period after surgical procedure. Prognosis prediction (or diagnosis of prognosis) is a very important clinical task, as it provides clues on the direction of cancer treatment in the future, including chemotherapy in early cancer patients. Prognosis predictions also include patient response to disease treatments and predictions of treatment progress.
본 발명에서 용어, "프로브"란 DNA와 특이적 결합을 이룰 수 있는 짧게는 수 염기 내지 길게는 수백 염기에 해당하는 RNA 또는 DNA 등의 핵산 단편을 의미하며 라벨링 되어 있어서 특정 mRNA의 존재 유무를 확인할 수 있다. 프로브는 올리고뉴클레오타이드(oligonucleotide) 프로브, 단쇄 DNA(single stranded DNA) 프로브, 이중쇄 DNA(double stranded DNA) 프로브, RNA 프로브 등의 형태로 제작될 수 있다. 본 발명에서는 표 2의 유전자의 특정 영역에 상보적인 RNA 프로브를 이용하여 표적 캡쳐 방법으로 NGS를 수행할 수 있다. 적당한 프로브의 선택 및 혼성화 조건은 통상의 기술분야에 공지된 것을 기초로 변형할 수 있으므로 본 발명에서는 이에 대해 특별히 한정하지 않는다.In the present invention, the term "probe" means a nucleic acid fragment such as RNA or DNA corresponding to a few bases to a few hundred bases, which can achieve specific binding with DNA, and is labeled to confirm the presence or absence of a specific mRNA. Can be. The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe. In the present invention, NGS can be performed by a target capture method using an RNA probe complementary to a specific region of the gene of Table 2. Suitable probe selection and hybridization conditions can be modified based on those known in the art, and the present invention is not particularly limited.
본 발명에서 “증폭”은 핵산 분자를 증폭하는 반응을 의미한다. 다양한 증폭 반응들이 당업계에 보고 되어 있으며, 이는 중합효소 연쇄반응(이하 PCR이라 한다)(미국 특허 제4,683,195, 4,683,202, 및 4,800,159호), 역전사-중합효소 연쇄반응(이하 RT-PCR로 표기한다)(Sambrook 등, Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press(2001)), Miller, H. I.(WO 89/06700) 및 Davey, C. 등 (EP 329,822)의 방법, 리가아제 연쇄 반응(ligase chain reaction; LCR)(17, 18), Gap-LCR(WO 90/01069), 복구 연쇄 반응(repair chain reaction; EP 439,182), 전사-중재 증폭(transcription-mediated amplification; TMA)(19) (WO 88/10315), 자가 유지 염기서열 복제(self sustained sequence replication)(20)(WO 90/06995), 타깃 폴리뉴클레오티드 염기서열의 선택적 증폭(selective amplification of target polynucleotide sequences)(미국 특허 제6,410,276호), 컨센서스 서열 프라이밍 중합효소 연쇄 반응(consensus sequence primed polymerase chain reaction; CP-PCR)(미국 특허 제4,437,975호), 임의적 프라이밍 중합효소 연쇄 반응 (arbitrarily primed polymerase chain reaction; AP-PCR)(미국 특허 제5,413,909호 및 제5,861,245호), 핵산 염기서열 기반 증폭(nucleic acid sequence based amplification; NASBA)(미국 특허 제5,130,238호, 제5,409,818호, 제5,554,517호, 및 제6,063,603호), 가닥 치환 증폭(strand displacement amplification) 및 고리-중재 항온성 증폭(loop-mediated isothermal amplification; LAMP)을 포함하나, 이에 한정되지는 않는다.In the present invention, "amplification" refers to a reaction that amplifies a nucleic acid molecule. Various amplification reactions have been reported in the art, which are polymerase chain reactions (hereinafter referred to as PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcriptase-polymerase chain reactions (hereinafter referred to as RT-PCR) (Sambrook et al., Molecular Cloning.A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al. (EP 329,822), ligase chain reaction ( ligase chain reaction (LCR) (17, 18), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA) (19) ( WO 88/10315), self sustained sequence replication (20) (WO 90/06995), selective amplification of target polynucleotide sequences (US Pat. No. 6,410,276) , Consensus sequence primed polymerase chain reaction (CP-PCR) (US Pat. No. 4,437,975), arbitrary priming polymerase chain reaction (AP-PCR) (US Pat. No. 5,413,909 And 5,861,245), nucleic acid sequence based amplification (NASBA) (US Pat. Nos. 5,130,238, 5,409,818, 5,554,517, and 6,063,603), strand disp amplification lacement amplification and loop-mediated isothermal amplification; LAMP).
일 측면에서, 본 발명은 본 발명의 NGS 패널을 포함하는 조혈모세포 이식 후 혈액암 예후 진단용 조성물 및 이를 포함하는 조혈모세포 이식 후 혈액암 예후 진단용 키트에 관한 것이다.In one aspect, the present invention relates to a composition for diagnosing blood cancer prognosis after hematopoietic stem cell transplantation comprising the NGS panel of the present invention and a kit for diagnosing blood cancer prognosis after transplantation of hematopoietic stem cells comprising the same.
일 측면에서, 본 발명은 (1) 대상으로부터 분리된 시료로부터 DNA를 분리하고; (2) 제 1항의 NGS 패널을 이용하여 ABCA12, ABL1, ASXL1, ATM, ATRX, ATXN7L1, BCOR, BRAF, BRCC3, CALR, CBL, CBLB, CD101, CEBPA, CREBBP, CSF1R, CSF3R, CTCF, CUX1, DNMT1, DNMT3A, EGFR, EP300, ERG, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, HIPK2, IDH1, IDH2, INVS, IRF1, JAK2, KDM2B, KDM6A, KIT, KMT2A, KMT2D, KRAS, LAMB4, MECOM, MET, MLL3, MLL5, MN1, MPL, NCOR2, NF1, NLRP1, NOTCH1, NPM1, NRAS, NRD1, NUP98, OCA2, PDGFRA, PHF12, PHF6, PRPF40B, PRPF8, PTPN11, RAD21, RAD50, RINT1, ROBO1, ROBO2, RUNX1, RUNX1T1, SETBP1, SF3A1, SF3B1, SMC1A, SMC3, SRSF2, STAG2, TET1, TET2, TP53, TP53BP1, U2AF1, U2AF2, WT1 및 ZRSR2로 이루어지는 군으로부터 선택되는 어느 하나 이상의 유전자의 돌연변이, 및 rs3736908, rs2304429, rs2289195, rs2276599, rs4685, rs16865262, rs788017, rs788018, rs788023, rs10498027, rs4673925, rs10498030, rs17501837, rs1523721, rs3821735, rs6795556, rs2271151, rs967454, rs2304503, rs11713094, rs2305037, rs2305036, rs2335052, rs12498609, rs2647243, rs2454206, rs10033601, rs2070731, rs2070730, rs9282762, rs9282761, rs216136, rs3829987, rs2228422, rs3830035, rs3830036, rs2072454, rs4947986, rs2227983, rs2227984, rs2293347, rs2240455, rs10953468, rs11976329, rs2074748, rs420753, rs2072407, rs10274535, rs2240819, rs74483926, rs6464211, rs10252263, rs2230722, rs2274649, rs34001282, rs35445683, rs1056171, rs2229971, rs10823229, rs12773594, rs12221107, rs11195199, rs1875, rs1799937, rs16754, rs664982, rs17210957, rs11168830, rs3741622, rs10849885, rs2293514, rs747443, rs4073630, rs17086226, rs2491223, rs2491227, rs2491231, rs1933437, rs1800414, rs1800404, rs550239, rs2602141, rs690367, rs689647, rs560191, rs2439831, rs60147683, rs11651270, rs884367, rs1642785, rs2952976, rs1801052, rs2905876, rs2285892, rs9894648, rs964288, rs663651, rs3744825, rs2290684, rs2114724, rs2228611, rs2241531, rs11672909, rs2228612, rs1049481, rs664684, rs2295454, rs2295765, rs4911231, rs7121, rs9608885, rs20552, rs2076577, rs2076578, rs5936062, rs6520618, rs2230018, rs20539, rs1264011, rs3088074 및 rs35268552로 이루어지는 군으로부터 선택되는 어느 하나 이상의 SNP를 NGS로 분석하며; (3) 대상의 조혈모세포 이식 후 미세잔존질환 및 키메리즘을 확인하는 것을 포함하는 NGS 기반 조혈모세포 이식 후 미세잔존질환 및 키메리즘 분석방법에 관한 것이다.In one aspect, the present invention provides (1) separating DNA from a sample isolated from a subject; (2) ABCA12, ABL1, ASXL1, ATM, ATRX, ATXN7L1, BCOR, BRAF, BRCC3, CALR, CBL, CBLB, CD101, CEBPA, CREBBP, CSF1R, CSF3R, CTCF, CUX1, DNMT1 , DNMT3A, EGFR, EP300, ERG, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, HIPK2, IDH1, IDH2, INVS, IRF1, JAK2, KDM2B, KDM6A, KIT, KMT2A, KMT2D, KRAS , MET, MLL3, MLL5, MN1, MPL, NCOR2, NF1, NLRP1, NOTCH1, NPM1, NRAS, NRD1, NUP98, OCA2, PDGFRA, PHF12, PHF6, PRPF40B, PRPF8, PTPN11, RAD21, RAD50, RINT1, ROBO1 , Mutation of any one or more genes selected from the group consisting of RUNX1, RUNX1T1, SETBP1, SF3A1, SF3B1, SMC1A, SMC3, SRSF2, STAG2, TET1, TET2, TP53, TP53BP1, U2AF1, U2AF2, WT1 and ZRSR2 , and rs37908 rs2304429, rs2289195, rs2276599, rs4685, rs16865262, rs788017, rs788018, rs788023, rs10498027, rs4673925, rs10498030, rs17501837, rs1523721, rs3821735, rs6795556, rs2271151, rs96450 rs10033601, rs2070731, rs2070730, rs9282762, rs9282761, rs216136, rs382998 7, rs2228422, rs3830035, rs3830036, rs2072454, rs4947986, rs2227983, rs2227984, rs2293347, rs2240455, rs10953468, rs11976329, rs2074748, rs420753, rs2072407, rs10274535, rs222 rs2229971, rs10823229, rs12773594, rs12221107, rs11195199, rs1875, rs1799937, rs16754, rs664982, rs17210957, rs11168830, rs3741622, rs10849885, rs2293514, rs747443, rs4073630, rs2424 rs690367, rs689647, rs560191, rs2439831, rs60147683, rs11651270, rs884367, rs1642785, rs2952976, rs1801052, rs2905876, rs2285892, rs9894648, rs964288, rs66365, rs96448 any one or more SNPs selected from the group consisting of rs2295765, rs4911231, rs7121, rs9608885, rs20552, rs2076577, rs2076578, rs5936062, rs6520618, rs2230018, rs20539, rs1264011, rs3088074 and rs35268552 Analyzed by NGS; (3) A method for analyzing microsurvival disease and chimerism after transplantation of an NGS-based hematopoietic stem cell, which includes identifying microsurvival disease and chimerism after hematopoietic stem cell transplantation.
일 구현예에서, 상기 시료는 조직, 세포, 전혈, 혈청, 혈장, 정액, 질 세포, 모발, 타액, 객담, 뇌척수액, 골수 및 뇨로부터 선택되는 어느 하나 이상일 수 있으며, 말초 혈액 또는 골수인 것이 더욱 바람직하다.In one embodiment, the sample may be any one or more selected from tissue, cells, whole blood, serum, plasma, semen, vaginal cells, hair, saliva, sputum, cerebrospinal fluid, bone marrow and urine, and is more preferably peripheral blood or bone marrow. desirable.
일 측면에서, 본 발명은 (1) 조혈모세포 이식된 대상으로부터 분리한 시료로부터 DNA를 분리하고; (2) ABCA12, ABL1, ASXL1, ATM, ATRX, ATXN7L1, BCOR, BRAF, BRCC3, CALR, CBL, CBLB, CD101, CEBPA, CREBBP, CSF1R, CSF3R, CTCF, CUX1, DNMT1, DNMT3A, EGFR, EP300, ERG, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, HIPK2, IDH1, IDH2, INVS, IRF1, JAK2, KDM2B, KDM6A, KIT, KMT2A, KMT2D, KRAS, LAMB4, MECOM, MET, MLL3, MLL5, MN1, MPL, NCOR2, NF1, NLRP1, NOTCH1, NPM1, NRAS, NRD1, NUP98, OCA2, PDGFRA, PHF12, PHF6, PRPF40B, PRPF8, PTPN11, RAD21, RAD50, RINT1, ROBO1, ROBO2, RUNX1, RUNX1T1, SETBP1, SF3A1, SF3B1, SMC1A, SMC3, SRSF2, STAG2, TET1, TET2, TP53, TP53BP1, U2AF1, U2AF2, WT1 및 ZRSR2로 이루어지는 군으로부터 선택되는 어느 하나 이상의 유전자의 돌연변이, 및 rs3736908, rs2304429, rs2289195, rs2276599, rs4685, rs16865262, rs788017, rs788018, rs788023, rs10498027, rs4673925, rs10498030, rs17501837, rs1523721, rs3821735, rs6795556, rs2271151, rs967454, rs2304503, rs11713094, rs2305037, rs2305036, rs2335052, rs12498609, rs2647243, rs2454206, rs10033601, rs2070731, rs2070730, rs9282762, rs9282761, rs216136, rs3829987, rs2228422, rs3830035, rs3830036, rs2072454, rs4947986, rs2227983, rs2227984, rs2293347, rs2240455, rs10953468, rs11976329, rs2074748, rs420753, rs2072407, rs10274535, rs2240819, rs74483926, rs6464211, rs10252263, rs2230722, rs2274649, rs34001282, rs35445683, rs1056171, rs2229971, rs10823229, rs12773594, rs12221107, rs11195199, rs1875, rs1799937, rs16754, rs664982, rs17210957, rs11168830, rs3741622, rs10849885, rs2293514, rs747443, rs4073630, rs17086226, rs2491223, rs2491227, rs2491231, rs1933437, rs1800414, rs1800404, rs550239, rs2602141, rs690367, rs689647, rs560191, rs2439831, rs60147683, rs11651270, rs884367, rs1642785, rs2952976, rs1801052, rs2905876, rs2285892, rs9894648, rs964288, rs663651, rs3744825, rs2290684, rs2114724, rs2228611, rs2241531, rs11672909, rs2228612, rs1049481, rs664684, rs2295454, rs2295765, rs4911231, rs7121, rs9608885, rs20552, rs2076577, rs2076578, rs5936062, rs6520618, rs2230018, rs20539, rs1264011, rs3088074 및 rs35268552로 이루어지는 군으로부터 선택되는 어느 하나 이상의 SNP를 분석하며; (3) 대상의 조혈모세포 이식 후 미세잔존질환 및 키메리즘을 확인하는 것을 포함하는 조혈모세포 이식 후 혈액암 예후 예측을 위한 정보 제공 방법에 관한 것이다.In one aspect, the present invention provides (1) DNA separation from a sample isolated from a hematopoietic stem cell transplanted subject; (2) ABCA12, ABL1, ASXL1, ATM, ATRX, ATXN7L1, BCOR, BRAF, BRCC3, CALR, CBL, CBLB, CD101, CEBPA, CREBBP, CSF1R, CSF3R, CTCF, CUX1, DNMT1, DNMT3A, EGFR, EP300, ERG , ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, HIPK2, IDH1, IDH2, INVS, IRF1, JAK2, KDM2B, KDM6A, KIT, KMT2A, KMT2D, KRAS, LAMB4, MECOM, MET, MLLN, MLLN, , MPL, NCOR2, NF1, NLRP1, NOTCH1, NPM1, NRAS, NRD1, NUP98, OCA2, PDGFRA, PHF12, PHF6, PRPF40B, PRPF8, PTPN11, RAD21, RAD50, RINT1, ROBO1, ROBO2, RUNX1, RUNX1T1, SF1 , SF3B1, SMC1A, SMC3, SRSF2, STAG2, TET1, TET2, TP53, TP53BP1, U2AF1, U2AF2, WT1 , and mutation of any one or more genes selected from the group consisting of ZRSR2, and rs3736908, rs2304429, rs2289195, rs2276599, rs2289195, rs2276599, rs16865262, rs788017, rs788018, rs788023, rs10498027, rs4673925, rs10498030, rs17501837, rs1523721, rs3821735, rs6795556, rs2271151, rs967454, rs2304503, rs11713094, rs2305037, rs23050, 260 rs9282761, rs216136, rs3829987, rs2228422, rs383 0035, rs3830036, rs2072454, rs4947986, rs2227983, rs2227984, rs2293347, rs2240455, rs10953468, rs11976329, rs2074748, rs420753, rs2072407, rs10274535, rs22408, rs74483926, rs rs12773594, rs12221107, rs11195199, rs1875, rs1799937, rs16754, rs664982, rs17210957, rs11168830, rs3741622, rs10849885, rs2293514, rs747443, rs4073630, rs17086226, rs2491223, rs2427, 238 rs560191, rs2439831, rs60147683, rs11651270, rs884367, rs1642785, rs2952976, rs1801052, rs2905876, rs2285892, rs9894648, rs964288, rs663651, rs3744825, rs22906, rs2114724, 222 analyze any one or more SNPs selected from the group consisting of rs7121, rs9608885, rs20552, rs2076577, rs2076578, rs5936062, rs6520618, rs2230018, rs20539, rs1264011, rs3088074 and rs35268552; (3) A method for providing information for predicting the prognosis of hematologic cancer after hematopoietic stem cell transplantation, including identifying microscopic residual disease and chimerism after transplantation of hematopoietic stem cells.
일 구현예에서, 상기 (2) 단계에서, 유전자 및 SNP 분석은 차세대 염기서열 시퀀싱(Next-generation sequencing, NGS), 생어 시퀀싱(Sanger sequencing), 단일-분자 실시간 분석법(Single-molecule real-time sequencing), 이온 반도체 분석(Ion semiconductor), 파이로 시퀀싱(Pyrosequencing), SBS(Sequencing by synthesis), SBL(Sequencing by ligation) 및 연쇄 정지반응(Chain termination)으로 이루어진 군에서 선택된 1종 이상의 방법을 이용할 수 있으며, 본 발명의 NGS 패널을 이용하여 차세대 염기서열 시퀀싱(Next-generation sequencing) 방법으로 분석하는 것이 더욱 바람직하다.In one embodiment, in the step (2), the gene and SNP analysis are next-generation sequencing (NGS), Sanger sequencing, and single-molecule real-time sequencing. ), Ion semiconductor analysis, Pyrosequencing, SBS (Sequencing by synthesis), SBL (Sequencing by ligation) and one or more methods selected from the group consisting of chain termination can be used. It is more preferable to analyze by the next-generation sequencing method using the NGS panel of the present invention.
일 실시예에서, 키메리즘 분석은 이식 전 SNP가 동형접합(homozygous; aa)이고, 이식 후 이형접합(heterozygous; Aa)으로 변화했을 때, wild type 염기는 공여자 세포에서 기원한 염기를 의미하는 것으로 확인하였다. 반면, 이식 전 SNP가 이형접합(heterozygous; Aa)이고, 이식 후 동형접합(homozygous; aa)으로 변화했을 때, 치환된 염기는 공여자 또는 환자의 세포에서 기원하였을 것이고, 검출되는(%가 낮더라도) wild type 염기는 환자의 남아있는 세포에서 기원한 염기를 의미하는 것으로 확인하였다.In one embodiment, chimeric analysis shows that when the SNP before transplantation is homozygous (aa) and after transplantation to heterozygous (Aa), the wild type base refers to the base originating from the donor cell. Was confirmed. On the other hand, when the SNP before transplantation was heterozygous (Aa) and changed to homozygous (aa) after transplantation, the substituted base would have originated in the donor or patient's cells and was detected (even if the percentage was low). ) The wild type base was confirmed to mean the base originating from the patient's remaining cells.
본 발명에서 용어, "SNP (single nucleotide polymorphism)"는 단일 뉴클레오티드에서의 다형성 (polymorphism)이다. 즉, 어느 집단에 있어 전체 게놈 (genomome)에 있는 하나의 염기가 염색체 마다 다른 경우가 존재하는데, 통상적으로 SNP는 300 내지 1000개의 염기에 하나 정도 존재하기 때문에 인간 게놈 DNA 전 체에는 적어도 300만개의 SNP가 존재하게 된다. The term "single nucleotide polymorphism (SNP)" in the present invention is polymorphism in a single nucleotide. That is, there is a case where one base in the entire genome of a certain genome is different for each chromosome in a certain group. Typically, at least 3 million are present in the entire human genome DNA because SNPs are present in about 300 to 1000 bases. SNP is present.
하기의 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 그러나 하기 실시예는 본 발명의 내용을 구체화하기 위한 것일 뿐 이에 의해 본 발명이 한정되는 것은 아니다. The present invention will be described in more detail through the following examples. However, the following examples are only intended to materialize the contents of the present invention, and the present invention is not limited thereto.
실시예 1. 차세대염기서열분석 패널 구성Example 1. Construction of next-generation base sequencing panel
1-1. 혈액종양질환 질병 연관 유전자 선발 및 NGS 패널 구성1-1. Blood tumor disease disease related gene selection and NGS panel composition
미세잔존질환 평가를 위해, MDS 및 골수 증식성 종양 형성(myeloproliferative neoplasia)을 가진 환자들에서 자주 돌연변이되는 유전자를 함유하는 차세대염기서열분석 패널을 구성하였다. 구체적으로, 도 1의 알고리즘에 따라 MDS 및 골수 증식성 종양 형성과 연관된 87개의 ABCA12, ABL1, ASXL1, ATM, ATRX, ATXN7L1, BCOR, BRAF, BRCC3, CALR, CBL, CBLB, CD101, CEBPA, CREBBP, CSF1R, CSF3R, CTCF, CUX1, DNMT1, DNMT3A, EGFR, EP300, ERG, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, HIPK2, IDH1, IDH2, INVS, IRF1, JAK2, KDM2B, KDM6A, KIT, KMT2A, KMT2D, KRAS, LAMB4, MECOM, MET, MLL3, MLL5, MN1, MPL, NCOR2, NF1, NLRP1, NOTCH1, NPM1, NRAS, NRD1, NUP98, OCA2, PDGFRA, PHF12, PHF6, PRPF40B, PRPF8, PTPN11, RAD21, RAD50, RINT1, ROBO1, ROBO2, RUNX1, RUNX1T1, SETBP1, SF3A1, SF3B1, SMC1A, SMC3, SRSF2, STAG2, TET1, TET2, TP53, TP53BP1, U2AF1, U2AF2, WT1 및 ZRSR2 유전자 (도 2, 표 1 및 2)를 선별하여 차세대염기서열분석(next-generation sequencing, NGS) 패널을 구성하였다.For the evaluation of micro-residual disease, a next-generation sequencing panel was constructed containing genes that are frequently mutated in patients with MDS and myeloproliferative neoplasia. Specifically, 87 ABCA12, ABL1, ASXL1, ATM, ATRX, ATXN7L1, BCOR, BRAF, BRCC3, CALR, CBL, CBLB, CD101, CEBPA, CREBBP, associated with MDS and myeloproliferative tumor formation according to the algorithm of FIG. CSF1R, CSF3R, CTCF, CUX1, DNMT1, DNMT3A, EGFR, EP300, ERG, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, HIPK2, IDH1, IDH2, INVS, IRF1, JAK2, KDM2, KDMA, KDM2, KDMA, KDM2, KMT2A, KMT2D, KRAS, LAMB4, MECOM, MET, MLL3, MLL5, MN1, MPL, NCOR2, NF1, NLRP1, NOTCH1, NPM1, NRAS, NRD1, NUP98, OCA2, PDGFRA, PHF12, PHF6, PRPF40B, PRPF8, PTP11 RAD21, RAD50, RINT1, ROBO1, ROBO2, RUNX1, RUNX1T1, SETBP1, SF3A1, SF3B1, SMC1A, SMC3, SRSF2, STAG2, TET1, TET2, TP53, TP53BP1, U2AF1, U2AF2, WT1 and ZR2 , Tables 1, 2 And 2) were selected to construct a next-generation sequencing (NGS) panel.
1-2. 조혈모세포 이식 후 키메리즘 분석을 위한 SNP 선발 및 NGS 패널 구성1-2. Selection of SNP and NGS panel for chimerism analysis after hematopoietic stem cell transplantation
상기 실시예 1-1의 질병연관 돌연변이 유전자와 연관이 없는 SNP(single nucleotide polymorphism)의 접합성(heterozygosity) 정보를 이용하여 조혈모세포 이식 후 키메리즘을 분석하기 위하여, 키메리즘 분석에 유용한 SNP 마커를 도 3의 알고리즘으로 선정하였다. 이를 위해, SNP은 인종별 차이를 보이므로 한국인에 적합한 SNP 조합을 구성하고, 해당 유전자가 질병과 연관된 후천적 돌연변이 (예, LOH)를 보일 수 있으므로, 다양한 유전자를 포함한 키메리즘 분석 SNP 조합을 구성하였다. 구체적으로, 밝혀진 모든 SNP들을 일반 한국인의 이형접합체 빈도 (the Korean reference genome database investigated 1722 Korean individuals)로 검토하였다 (도 4). 동형 접합체와 이형 접합체 대립 유전자는 각각 90~100 % 및 45 ~ 60 %의 염기수 빈도에 의해 결정되었고, 한국인 데이터베이스에서 0.2 내지 0.8의 이형접합 빈도를 가지는 153개의 SNP (도 5)를 조사하고 NGS 키메리즘 분석에 최적인 SNP들을 하기 기준에 따라 선택하였다: 1) > 500 mean read depth; 2) ≤ 0.2% background error (BE); 및 3) < 10% 이형 접합 대립유전자의 measurement error (ME, 참조 대립형질 및 대체 대립형질 사이의 판독수의 차). In order to analyze chimerism after hematopoietic stem cell transplantation using heterozygosity information of SNP (single nucleotide polymorphism) that is not associated with the disease-associated mutant gene of Example 1-1, a useful SNP marker for chimerism analysis Was selected as the algorithm of FIG. 3. To this end, since SNPs show differences by race, SNP combinations suitable for Koreans are constructed, and since the corresponding gene may exhibit acquired mutations (eg, LOH) associated with disease, chimeric analysis SNP combinations including various genes are constructed. Did. Specifically, all revealed SNPs were reviewed by the frequency of heterozygotes in the general Korean population (the Korean reference genome database investigated 1722 Korean individuals) (FIG. 4). Homozygous and heterozygous alleles were determined by base frequency of 90-100% and 45-60%, respectively, and 153 SNPs with heterozygous frequencies of 0.2 to 0.8 in the Korean database were examined and NGS keys SNPs that were optimal for the analysis of the merism were selected according to the following criteria: 1)> 500 mean read depth; 2) ≤ 0.2% background error (BE); And 3) <10% measurement error of heterozygous allele (ME, difference in readings between reference and alternative alleles).
그 결과, 121개의 SNP들이 NGS 키메리즘 분석용으로 선택되었으며, 선택된 SNP들의 판독 깊이, %BE 및 %ME는 각각 1398.3 ± 538.9, 0.082% ± 0.035% 및 3.31% ± 2.27%로 나타났다 (표 3). As a result, 121 SNPs were selected for NGS chimerism analysis, and the reading depths, %BE and %ME of the selected SNPs were 1398.3 ± 538.9, 0.082% ± 0.035% and 3.31% ± 2.27%, respectively (Table 3). ).
실시예 2. 미세잔존질환 및 키메리즘 분석Example 2. Analysis of micro residual disease and chimerism
2-1. 환자 선발 및 시료 준비2-1. Patient selection and sample preparation
골수이형성증후군(Myelodysplastic syndrome, MDS)로 진단받고 서울 성모 병원에서 allo-HSCT를 받은 14명의 환자들을 본 연구에 등록하였으며, 본 연구는 헬싱키 선언에 따라 수행되고 서울 성모 병원의 기관 검토위원회로부터 승인을 받았다 (KC16SISI0395). 환자들로부터 말초 혈액 또는 골수(bone marrow, BM) 흡인(aspirates)을 수집하고 (N = 53) 이로부터 DNA를 추출하였다. Fourteen patients who were diagnosed with myelodysplastic syndrome (MDS) and allo-HSCT at the St. Mary's Hospital in Seoul were enrolled in this study, which was conducted in accordance with the Helsinki Declaration and approved by the Institutional Review Board of the St. Mary's Hospital in Seoul. Received (KC16SISI0395). Peripheral blood or bone marrow (BM) aspirates were collected from patients (N = 53) and DNA was extracted therefrom.
2-2. STR 분석2-2. STR analysis
STR 분석은 AmpFlSTR Identifier PCR Amplification (Applied Biosystems, Warrington, UK)를 이용하여 종래의 16개의 마커 (염색체 8의 D8S1179, 21q11.2-q21의 D21S11, 7q11.2-22의 D7S820 , 5q33.3-34의 CSF1PO, 3p의 D3S1358, 11p15.5의 TH01, 13q22-31의 D13S317, 16q24-qter의 D16S539, 2q35-37.1의 D2S1338, 19q12-13.1의 D19S433, 12p12-pter의 vWA, 2p23-2per의 TPOX, 18q21.3의 D18S51, 5q21-31의 D5S818, 4q28의 FGA, 및 X (p22.1-22.3) 및 Y (p11.2) 염색체의 아멜로제닌(amelogenin) 유전자좌)로 수행되었다.STR analysis was performed using AmpFlSTR Identifier PCR Amplification (Applied Biosystems, Warrington, UK) using 16 conventional markers (D8S1179 of chromosome 8, D21S11 of 21q11.2-q21, D7S820 of 7q11.2-22, D7S820, 5q33.3-34 CSF1PO, 3p D3S1358, 11p15.5 TH01, 13q22-31 D13S317, 16q24-qter D16S539, 2q35-37.1 D2S1338, 19q12-13.1 D19S433, 12p12-pter vWA, 2p23-2per TPOX, 18q21 .3 D18S51, 5q21-31 D5S818, 4q28 FGA, and X (p22.1-22.3) and Y (p11.2) chromosome amelogenin loci).
2-3. NGS 수행을 통한 미세잔존질환 및 키메리즘 평가2-3. Micro residual disease and chimerism evaluation through NGS
2-3-1. NGS를 이용한 MDS 관련 유전자 변이 확인2-3-1. Confirmation of genetic mutation related to MDS using NGS
NGS 분석은 상기 실시예에서 도 1의 알고리즘에 따라 선별한 MDS 및 골수 증식성 종양 형성(myeloproliferative neoplasia)을 가진 환자들에서 자주 돌연변이되는 87개의 유전자 (도 2 및 표 1)를 함유하는 차세대염기서열분석 패널을 이용하여 수행되었다. 표적 캡쳐 시퀀싱(Target capture sequencing)은 맞춤형 표적 키트 (3039061, Agilent Technologies, Santa Clara, CA, USA)를 이용하여 수행되었다 DNA 라이브러리는 SureSelct Target Enrichment System Kit (G7530-90000) (Agilent, CA)를 이용하여 제작되었다. Covaris S2 (Covaris, MA) 장비를 이용해 각 샘플 당 500ng genomic DNA를 250bp 정도의 조각으로 shearing하고 target-specific Capture Library (Bait ID : 3039061)에 혼성결합시킨다. 준비된 라이브러리의 양은 KAPA Library Quantification Kit (KK4824, Kapa Biosystems)을 이용해 계량하였고, 아답터가 결합된 라이브러리의 품질과 사이즈 분포는 electrophoresis on Agilent Bioanalyzer High Sensitivity DNA microfluidic chips (Agilent, CA)을 이용해 측정하였다. Illumina HiSeq2500로 라이브러리의 서열판독을 진행하였는데, 장비 내에서 클러스터 생성이 진행되었으며, 이미지 분석은 HiSeq control Software version 1.8.4.을 이용해 수행되었다. 또한, 아답터 서열과 저품질 서열 판독을 제거하기 위해 cutadapt (https://doi.org/10.14806/ej.17.1.200) 및 sickle (https://github.com/najoshi/sickle)를 이용하였다. 서열 fead를 인간 참조 지놈 (hg19)에 얼라인하기 위해 Burrows-Wheeler aligner (https://doi.org/10.1093/bioinformatics/btp324)를 이용했다. local realignment, 점수 재조정 및 서열 데이터의 필터링을 위해 Genome Analysis ToolKit (GATK) (http://genome.cshlp.org/content/20/9/1297)를 이용하였다. 또한, Picard (https://github.com/broadinstitute/picard) 및 Samtools (https://doi.org/10.1093/bioinformatics/btp352)를 기본 프로세스, 서열 데이터의 관리 및 mpileup 생성을 위해 사용하였다. VarScan v.2.3.9 (http://varscan.sourceforge.net/)를 변이를 확인하는데 사용하였다. NGS analysis is a next-generation base sequence containing 87 genes (FIG. 2 and Table 1) that are frequently mutated in patients with MDS and myeloproliferative neoplasia selected according to the algorithm of FIG. 1 in the above example. This was done using an analytical panel. Target capture sequencing was performed using a custom target kit (3039061, Agilent Technologies, Santa Clara, CA, USA). DNA library was performed using the SureSelct Target Enrichment System Kit (G7530-90000) (Agilent, CA). Was produced. Using a Covaris S2 (Covaris, MA) instrument, 500 ng genomic DNA per sample is sheared into pieces of about 250 bp and hybridized to a target-specific Capture Library (Bait ID: 3039061). The amount of the prepared library was quantified using KAPA Library Quantification Kit (KK4824, Kapa Biosystems), and the quality and size distribution of the adapter-coupled library were measured using electrophoresis on Agilent Bioanalyzer High Sensitivity DNA microfluidic chips (Agilent, CA). The library was sequenced with Illumina HiSeq2500, cluster generation was performed in the instrument, and image analysis was performed using HiSeq control Software version 1.8.4. In addition, cutadapt (https://doi.org/10.14806/ej.17.1.200) and sickle ( https://github.com/najoshi/sickle ) were used to remove the adapter sequence and low quality sequence reading. The Burrows-Wheeler aligner ( https://doi.org/10.1093/bioinformatics/btp324 ) was used to align the sequence fead to the human reference genome (hg19). Genome Analysis ToolKit (GATK) ( http://genome.cshlp.org/content/20/9/1297 ) was used for local realignment, score readjustment and filtering of sequence data. In addition, Picard ( https://github.com/broadinstitute/picard ) and Samtools ( https://doi.org/10.1093/bioinformatics/btp352 ) were used for basic processes, management of sequence data and generation of mpileup. VarScan v.2.3.9 ( http://varscan.sourceforge.net/ ) was used to identify mutations.
그 결과, 환자에 동종이계의 조혈모세포 이식(allogeneic hematopoietic stem cell transplantation, allo-HSCT)을 수행하기 전(pre-allo-HSCT) 및 수행 후(post-allo-HSCT)의 ASXL1 및 EZH2 돌연변이에 대한 대립유전자 깊이(allele depth)를 분석함으로써 조혈모세포 이식 후 질병 연관 돌연변이 정량 분석을 통한 미세잔존질환을 평가하였다(도 6). 그 결과, 조혈모세포 이식 전의 환자에서는 ASXL1 및 EZH2 돌연변이가 존재하나, 조혈모세포 이식 후의 환자에서는 ASXL1 및 EZH2 돌연변이가 낮음을 확인하였다 (도 7).As a result, the pre-allo-HSCT and post-allo-HSCT mutations for ASXL1 and EZH2 mutations were performed in patients with allogeneic hematopoietic stem cell transplantation (allo-HSCT). By analyzing allele depth, microscopic residual disease was evaluated through quantitative analysis of disease-related mutations after hematopoietic stem cell transplantation (FIG. 6 ). As a result, it was confirmed that ASXL1 and EZH2 mutations exist in patients before hematopoietic stem cell transplantation, but that ASXL1 and EZH2 mutations are low in patients after hematopoietic stem cell transplantation (FIG. 7 ).
2-3-2. NGS를 이용한 조혈모세포 이식 후 키메리즘 확인2-3-2. Confirmation of chimerism after hematopoietic stem cell transplantation using NGS
121개의 SNP를 포함하는 NGS 패널을 이용하여 상기 실시예에서와 같이 NGS로 분석하고, 각 SNP의 공여자 allele burden은 하기 수학식 1 내지 4로 계산하였다.Using NGS panels containing 121 SNPs, NGS analysis was performed as in the above example, and the donor allele burden of each SNP was calculated by the following equations 1-4.
*공여자 키메리즘은 평균 공여자 allele burden으로 정의되었다.* Donor chimerism was defined as the average donor allele burden.
그 결과, 유용한 SNP들의 수는 가변적이었으며 (중앙값 25.5, 범위 8-41) STR (중앙값 10.5, 범위 5-14)보다 더 높게 나타났다. NGS 분석에서 공여자 키메리즘은 STR 분석 결과와 매우 높은 상관관계를 나타냈으며 (r 2 = 0.988 (P < 0.0001)) (도 8A), 모든 데이터 포인트는 Blant-Altman plot에서 평균 % 차이의 3 표준 편차(SD) 이내였다 (도 8B). MRD 역치는 표 3의 각 유전자좌에서 평균 %BE + 3SDs로 결정되었다 (표 4). 9명의 환자 (1-9)는 0.03 ± 0.05% 돌연변이 allele burden을 가지는 완전히 공여자 키메리즘 (STR 99.29 ± 0.76, NGS 99.60 ± 0.58)을 나타냈다 (표 5). 환자 10은 정상적인 혈액 소견과 혼합된 키메리즘을 나타냈다. 14 환자 중, 세 명의 환자 (11-13)가 allo-HSCT 후에 재발하였다. 재발에서 돌연변이 allele burden의 감소 및 공여자 키메리즘 감소를 검출하였다 (환자 11, 도 8C). 환자 12는 추적 관찰 첫 1년에 혼합된 키메리즘을 보였고 SF3B1 돌연변이가 낮은 수준 (0.44 ± 0.16)으로 지속되었다 (도 8D). 환자 13은 allo-HSCT 후 7개월까지 혼합된 키메리즘을 나타냈고, NRAS 돌연변이가 진단시 관찰되었으나 allo-HSCT 후 사라졌다. 상기 환자는 공여자 키메리즘이 감소된 allo-HSCT 후 9개월차에 AML이 재발하였다. 특히, 새로운 세포유전적 이상이 NRAS 돌연변이 재발생 없이 얻어졌다 (도 8E). 이를 통해 개체별 질병 진행이 동시 분석을 통해 효과적으로 입증될 수 있음을 나타냈다. 또한, 환자 14는 분석 알고리즘의 효과를 확인했다. 상기 AML 환자는 allo-HSCT를 11년 전에 받았다. 정기적인 추적 관찰 동안 범혈구감소증(pancytopenia)이 발생했으며, 혈청학적 측정 및 키메리즘 분석을 위한 BM 검사를 수행하여, 3-계통 이형성증 및 증가된 blast (5%)를 나타냈다. 특히, 완전한 공여자 키메리즘 (99.8%)과 PHF6 돌연변이가 NGS로 발혀졌으며, 이는 돌연변이가 공여자 세포로부터 유래된 것임을 나타냈다. 이는 1q 획득 및 7q 상실을 동반한 공여자-세포 유래된 MDS를 입증하는 세포유전학적 분석 //46,XY,+1,der(1;7)(q10;q10)[5]/46,XY[5]으로 뒷받침되었다.As a result, the number of useful SNPs was variable (median 25.5, range 8-41) and higher than STR (median 10.5, range 5-14). In the NGS analysis, the donor chimerism showed a very high correlation with the results of the STR analysis ( r 2 = 0.988 ( P <0.0001)) (FIG. 8A ), all data points were 3 standard of the mean% difference in the Blant-Altman plot. Within the deviation (SD) (Fig. 8B). The MRD threshold was determined as the average %BE + 3SDs at each locus in Table 3 (Table 4). Nine patients (1-9) showed fully donor chimerism (STR 99.29 ± 0.76, NGS 99.60 ± 0.58) with a 0.03 ± 0.05% mutant allele burden (Table 5).
즉, 상기 실시예에서 선발된 SNP 중 조혈모세포를 이식받은 공여자와 환자 개체별로 실제 차이를 나타내는 유용한 수여자 대립유전자 (Informative recipient alleles, IRA)는 하기의 기준으로 선정되었다:That is, among the SNPs selected in the above example, useful recipient alleles (IRAs) representing actual differences between donor and patient individuals transplanted with hematopoietic stem cells were selected based on the following criteria:
1) 환자 (수여자) (Recipient, Rw, Rv)의 SNP이 이형접합인 경우, 공여자(Donor, Dw)의 SNP는 동형접합이어야 한다; 및 1) If the SNP of the patient (recipient) (Recipient, Rw, Rv) is heterozygous, the SNP of the donor (Dnor) must be homozygous; And
2) 환자와 공여자의 SNP이 동형접합인 경우, 환자와 공여자는 서로 다른 염기의 동형접합이어야 한다.2) If the SNP of the patient and the donor is homozygous, the patient and the donor must be homozygous for different bases.
아울러, 키메리즘 계산을 위하여, (1) 환자(Recipient, Rw, Rv)의 SNP이 이형접합인 경우, 공여자(Donor, Dw)의 SNP는 동형접합이어야 하며, 이때 환자 키메리즘(Recipient Chimerism)(%)은 하기의 수학식 5로 계산된다.In addition, in order to calculate chimerism, (1) If the SNP of the patient (Recipient, Rw, Rv) is heterozygous, the SNP of the donor (Dnor, Dw) must be homozygous, and at this time, the patient chimerism (Recipient Chimerism) ) (%) is calculated by
또한, (2) 환자(Recipient, Rw)와 공여자(Donor, Dv)의 SNP가 서로 다른 염기의 동형접합인 경우 하기의 수학식 6으로 계산된다.In addition, (2) when the SNP of the patient (Recipient, Rw) and the donor (Donor, Dv) is homozygous for different bases, it is calculated by
이를 이용하여 환자(Recipient, Rw, Rv)에 조혈모세포 이식(PBSCT)을 수행하기 전(pre-PBSCT) 및 수행 후(post-PBSCT)와 공여자(Donor, Dv)의 키메리즘을 분석한 결과, 도 9에 나타낸 바와 같이, 환자의 키메리즘(Recipient Chimerism)은 7.21%인 것으로 확인되었다.Using this, the results of analyzing the chimerism of the pre-PBSCT and post-PBSCT and donor (Dv) of the hematopoietic stem cell transplantation (PBSCT) to the patient (Recipient, Rw, Rv) , As shown in Figure 9, the patient's chimerism (Recipient Chimerism) was found to be 7.21%.
상기 결과를 통해, 조혈모세포 이식 전 환자의 검체에서 시행한 SNP의 접합성(heterozygosity)가 이식 후 검체에서 변화했을 때 이를 이용하여 공여자 세포에서 기원한 염기인지 또는 남아있는 또는 재발한 세포에서 기원한 염기임을 예상할 수 있음을 확인하였다. 이는, 이식 전 SNP가 동형접합(homozygous; aa)이고, 이식 후 이형접합(heterozygous; Aa)으로 변화했을 때, wild type 염기는 공여자 세포에서 기원한 염기를 의미하는 것이며, 반면, 이식 전 SNP가 이형접합(heterozygous; Aa)이고, 이식 후 동형접합(homozygous; aa)으로 변화했을 때, 치환된 염기는 공여자 또는 환자의 세포에서 기원하였을 것이고, 검출되는(%가 낮더라도) wild type 염기는 환자의 남아있는 세포에서 기원한 염기를 의미하는 것임을 확인하였다. 이를 통해, 대립유전자 빈도 데이터(Allele frequency data)를 이식 전과 이식 후에 비교함으로써 키메리즘 분석에 이용할 수 있음을 확인하였다. Through the above results, when the heterozygosity of SNPs performed on a patient's sample before hematopoietic stem cell transplantation was changed in a sample after transplantation, it was used to determine whether it is a base originating from a donor cell or a base originating from a remaining or recurrent cell. It was confirmed that can be expected. This means that when the SNP before transplantation is homozygous (aa) and after conversion to heterozygous (Aa), the wild type base refers to a base originating from a donor cell, whereas SNP before transplantation When it is heterozygous (Aa) and changed to homozygous (aa) after transplantation, the substituted base would have originated in the donor or patient's cells, and the detected wild type base (even if the percentage is low) is the patient It was confirmed that it means the base originated from the remaining cells of. Through this, it was confirmed that allele frequency data can be used for chimeric analysis by comparing before and after transplantation.
Claims (8)
(2) 제 1항의 NGS 패널을 이용하여 ABCA12, ABL1, ASXL1, ATM, ATRX, ATXN7L1, BCOR, BRAF, BRCC3, CALR, CBL, CBLB, CD101, CEBPA, CREBBP, CSF1R, CSF3R, CTCF, CUX1, DNMT1, DNMT3A, EGFR, EP300, ERG, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, HIPK2, IDH1, IDH2, INVS, IRF1, JAK2, KDM2B, KDM6A, KIT, KMT2A, KMT2D, KRAS, LAMB4, MECOM, MET, MLL3, MLL5, MN1, MPL, NCOR2, NF1, NLRP1, NOTCH1, NPM1, NRAS, NRD1, NUP98, OCA2, PDGFRA, PHF12, PHF6, PRPF40B, PRPF8, PTPN11, RAD21, RAD50, RINT1, ROBO1, ROBO2, RUNX1, RUNX1T1, SETBP1, SF3A1, SF3B1, SMC1A, SMC3, SRSF2, STAG2, TET1, TET2, TP53, TP53BP1, U2AF1, U2AF2, WT1 및 ZRSR2로 이루어지는 군으로부터 선택되는 어느 하나 이상의 유전자의 돌연변이, 및 rs3736908, rs2304429, rs2289195, rs2276599, rs4685, rs16865262, rs788017, rs788018, rs788023, rs10498027, rs4673925, rs10498030, rs17501837, rs1523721, rs3821735, rs6795556, rs2271151, rs967454, rs2304503, rs11713094, rs2305037, rs2305036, rs2335052, rs12498609, rs2647243, rs2454206, rs10033601, rs2070731, rs2070730, rs9282762, rs9282761, rs216136, rs3829987, rs2228422, rs3830035, rs3830036, rs2072454, rs4947986, rs2227983, rs2227984, rs2293347, rs2240455, rs10953468, rs11976329, rs2074748, rs420753, rs2072407, rs10274535, rs2240819, rs74483926, rs6464211, rs10252263, rs2230722, rs2274649, rs34001282, rs35445683, rs1056171, rs2229971, rs10823229, rs12773594, rs12221107, rs11195199, rs1875, rs1799937, rs16754, rs664982, rs17210957, rs11168830, rs3741622, rs10849885, rs2293514, rs747443, rs4073630, rs17086226, rs2491223, rs2491227, rs2491231, rs1933437, rs1800414, rs1800404, rs550239, rs2602141, rs690367, rs689647, rs560191, rs2439831, rs60147683, rs11651270, rs884367, rs1642785, rs2952976, rs1801052, rs2905876, rs2285892, rs9894648, rs964288, rs663651, rs3744825, rs2290684, rs2114724, rs2228611, rs2241531, rs11672909, rs2228612, rs1049481, rs664684, rs2295454, rs2295765, rs4911231, rs7121, rs9608885, rs20552, rs2076577, rs2076578, rs5936062, rs6520618, rs2230018, rs20539, rs1264011, rs3088074 및 rs35268552로 이루어지는 군으로부터 선택되는 어느 하나 이상의 SNP를 분석하며;
(3) 대상의 조혈모세포 이식 후 미세잔존질환 및 키메리즘을 확인하는 것을 포함하는 NGS 기반 조혈모세포 이식 후 미세잔존질환 및 키메리즘 분석방법.(1) DNA is separated from a sample separated from the subject;
(2) ABCA12, ABL1, ASXL1, ATM, ATRX, ATXN7L1, BCOR, BRAF, BRCC3, CALR, CBL, CBLB, CD101, CEBPA, CREBBP, CSF1R, CSF3R, CTCF, CUX1, DNMT1 , DNMT3A, EGFR, EP300, ERG, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, HIPK2, IDH1, IDH2, INVS, IRF1, JAK2, KDM2B, KDM6A, KIT, KMT2A, KMT2D, KRAS , MET, MLL3, MLL5, MN1, MPL, NCOR2, NF1, NLRP1, NOTCH1, NPM1, NRAS, NRD1, NUP98, OCA2, PDGFRA, PHF12, PHF6, PRPF40B, PRPF8, PTPN11, RAD21, RAD50, RINT1, ROBO1 , Mutation of any one or more genes selected from the group consisting of RUNX1, RUNX1T1, SETBP1, SF3A1, SF3B1, SMC1A, SMC3, SRSF2, STAG2, TET1, TET2, TP53, TP53BP1, U2AF1, U2AF2, WT1 and ZRSR2 , and rs37908 rs2304429, rs2289195, rs2276599, rs4685, rs16865262, rs788017, rs788018, rs788023, rs10498027, rs4673925, rs10498030, rs17501837, rs1523721, rs3821735, rs6795556, rs2271151, rs96450 rs10033601, rs2070731, rs2070730, rs9282762, rs9282761, rs216136, rs382998 7, rs2228422, rs3830035, rs3830036, rs2072454, rs4947986, rs2227983, rs2227984, rs2293347, rs2240455, rs10953468, rs11976329, rs2074748, rs420753, rs2072407, rs10274535, rs222 rs2229971, rs10823229, rs12773594, rs12221107, rs11195199, rs1875, rs1799937, rs16754, rs664982, rs17210957, rs11168830, rs3741622, rs10849885, rs2293514, rs747443, rs4073630, rs2424 rs690367, rs689647, rs560191, rs2439831, rs60147683, rs11651270, rs884367, rs1642785, rs2952976, rs1801052, rs2905876, rs2285892, rs9894648, rs964288, rs66365, rs96448 any one or more SNPs selected from the group consisting of rs2295765, rs4911231, rs7121, rs9608885, rs20552, rs2076577, rs2076578, rs5936062, rs6520618, rs2230018, rs20539, rs1264011, rs3088074 and rs35268552 Analyze;
(3) NGS-based method for analyzing micro-survival disease and chimerism after transplantation of a target hematopoietic stem cell, comprising confirming micro-survival disease and chimerism after transplantation of hematopoietic stem cells.
(2) ABCA12, ABL1, ASXL1, ATM, ATRX, ATXN7L1, BCOR, BRAF, BRCC3, CALR, CBL, CBLB, CD101, CEBPA, CREBBP, CSF1R, CSF3R, CTCF, CUX1, DNMT1, DNMT3A, EGFR, EP300, ERG, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, HIPK2, IDH1, IDH2, INVS, IRF1, JAK2, KDM2B, KDM6A, KIT, KMT2A, KMT2D, KRAS, LAMB4, MECOM, MET, MLL3, MLL5, MN1, MPL, NCOR2, NF1, NLRP1, NOTCH1, NPM1, NRAS, NRD1, NUP98, OCA2, PDGFRA, PHF12, PHF6, PRPF40B, PRPF8, PTPN11, RAD21, RAD50, RINT1, ROBO1, ROBO2, RUNX1, RUNX1T1, SETBP1, SF3A1, SF3B1, SMC1A, SMC3, SRSF2, STAG2, TET1, TET2, TP53, TP53BP1, U2AF1, U2AF2, WT1 및 ZRSR2로 이루어지는 군으로부터 선택되는 어느 하나 이상의 유전자의 돌연변이, 및 rs3736908, rs2304429, rs2289195, rs2276599, rs4685, rs16865262, rs788017, rs788018, rs788023, rs10498027, rs4673925, rs10498030, rs17501837, rs1523721, rs3821735, rs6795556, rs2271151, rs967454, rs2304503, rs11713094, rs2305037, rs2305036, rs2335052, rs12498609, rs2647243, rs2454206, rs10033601, rs2070731, rs2070730, rs9282762, rs9282761, rs216136, rs3829987, rs2228422, rs3830035, rs3830036, rs2072454, rs4947986, rs2227983, rs2227984, rs2293347, rs2240455, rs10953468, rs11976329, rs2074748, rs420753, rs2072407, rs10274535, rs2240819, rs74483926, rs6464211, rs10252263, rs2230722, rs2274649, rs34001282, rs35445683, rs1056171, rs2229971, rs10823229, rs12773594, rs12221107, rs11195199, rs1875, rs1799937, rs16754, rs664982, rs17210957, rs11168830, rs3741622, rs10849885, rs2293514, rs747443, rs4073630, rs17086226, rs2491223, rs2491227, rs2491231, rs1933437, rs1800414, rs1800404, rs550239, rs2602141, rs690367, rs689647, rs560191, rs2439831, rs60147683, rs11651270, rs884367, rs1642785, rs2952976, rs1801052, rs2905876, rs2285892, rs9894648, rs964288, rs663651, rs3744825, rs2290684, rs2114724, rs2228611, rs2241531, rs11672909, rs2228612, rs1049481, rs664684, rs2295454, rs2295765, rs4911231, rs7121, rs9608885, rs20552, rs2076577, rs2076578, rs5936062, rs6520618, rs2230018, rs20539, rs1264011, rs3088074 및 rs35268552로 이루어지는 군으로부터 선택되는 어느 하나 이상의 SNP를 분석하며;
(3) 대상의 조혈모세포 이식 후 미세잔존질환 및 키메리즘을 확인하는 것을 포함하는 조혈모세포 이식 후 혈액암 예후 예측을 위한 정보 제공 방법.
(1) DNA was isolated from a sample isolated from a hematopoietic stem cell transplanted subject;
(2) ABCA12, ABL1, ASXL1, ATM, ATRX, ATXN7L1, BCOR, BRAF, BRCC3, CALR, CBL, CBLB, CD101, CEBPA, CREBBP, CSF1R, CSF3R, CTCF, CUX1, DNMT1, DNMT3A, EGFR, EP300, ERG , ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, HIPK2, IDH1, IDH2, INVS, IRF1, JAK2, KDM2B, KDM6A, KIT, KMT2A, KMT2D, KRAS, LAMB4, MECOM, MET, MLLN, MLLN, , MPL, NCOR2, NF1, NLRP1, NOTCH1, NPM1, NRAS, NRD1, NUP98, OCA2, PDGFRA, PHF12, PHF6, PRPF40B, PRPF8, PTPN11, RAD21, RAD50, RINT1, ROBO1, ROBO2, RUNX1, RUNX1T1, SF1 , SF3B1, SMC1A, SMC3, SRSF2, STAG2, TET1, TET2, TP53, TP53BP1, U2AF1, U2AF2, WT1 , and mutation of any one or more genes selected from the group consisting of ZRSR2, and rs3736908, rs2304429, rs2289195, rs2276599, rs2289195, rs2276599, rs16865262, rs788017, rs788018, rs788023, rs10498027, rs4673925, rs10498030, rs17501837, rs1523721, rs3821735, rs6795556, rs2271151, rs967454, rs2304503, rs11713094, rs2305037, rs23050, 260 rs9282761, rs216136, rs3829987, rs2228422, rs383 0035, rs3830036, rs2072454, rs4947986, rs2227983, rs2227984, rs2293347, rs2240455, rs10953468, rs11976329, rs2074748, rs420753, rs2072407, rs10274535, rs22408, rs74483926, rs rs12773594, rs12221107, rs11195199, rs1875, rs1799937, rs16754, rs664982, rs17210957, rs11168830, rs3741622, rs10849885, rs2293514, rs747443, rs4073630, rs17086226, rs2491223, rs2427, 238 rs560191, rs2439831, rs60147683, rs11651270, rs884367, rs1642785, rs2952976, rs1801052, rs2905876, rs2285892, rs9894648, rs964288, rs663651, rs3744825, rs22906, rs2114724, 222 analyze any one or more SNPs selected from the group consisting of rs7121, rs9608885, rs20552, rs2076577, rs2076578, rs5936062, rs6520618, rs2230018, rs20539, rs1264011, rs3088074 and rs35268552;
(3) A method of providing information for predicting the prognosis of hematologic cancer after hematopoietic stem cell transplantation, including identifying microscopic residual disease and chimerism after transplantation of hematopoietic stem cells.
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