二代测序技术在儿童T淋巴母细胞淋巴瘤全外显子测序中的应用
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摘要
目的建立从石蜡包埋的儿童T淋巴母细胞淋巴瘤(T-LBL)病理组织中提取基因组DNA,采用二代测序技术行全外显子测序检测的方法并分析其可行性。方法从10例儿童T-LBL石蜡包埋组织中提取基因组DNA,PCR反应扩增后采用二代测序技术行全外显子测序检测并确定致病位点,所获得的致病突变采用Sanger测序法测序确认,比较两种方法的一致性。结果 10例石蜡包埋T-LBL组织标本中均成功提取到基因组DNA,采用二代测序技术开展全外显子测序测序均检测到致病突变;二代测序检测结果经Sanger测序法验证,证实致病突变确实存在,且与Sanger测序结果一致。结论基于二代测序技术的全外显子测序测序可用于检测石蜡包埋的儿童T-LBL病理标本的致病突变。
Objective To establish the methods of extract DNA and detect whole exome sequencing(WES) by using next generation sequencing(NGS) technique from paraffin embedded samples for children with T lymphoblastic lymphoma(TLBL) and to explore its feasibility. Methods Genomic DNA was extracted from 10 cases of paraffin embedded samples with pediatric T-LBL. Genomic DNA samples were amplified by polymerase chain reactions, NGS technique was used for WES detection and pathogenic mutations were founded. The pathogenic mutations were confirmed by Sanger sequencing,comparison of consistency between NGS technique and Sanger sequencing was evaluated. Results Genomic DNA samples were successfully extracted from the 10 T-LBL specimens of paraffin embedded tissues. The pathogenic mutations were detected by NGS technique and the result was consistent with the Sanger sequencing method. Conclusion NGS technology can be used for WES and detection of pathogenic mutation for paraffin embedded pediatric T-LBL specimens.
Objective To establish the methods of extract DNA and detect whole exome sequencing(WES) by using next generation sequencing(NGS) technique from paraffin embedded samples for children with T lymphoblastic lymphoma(TLBL) and to explore its feasibility. Methods Genomic DNA was extracted from 10 cases of paraffin embedded samples with pediatric T-LBL. Genomic DNA samples were amplified by polymerase chain reactions, NGS technique was used for WES detection and pathogenic mutations were founded. The pathogenic mutations were confirmed by Sanger sequencing,comparison of consistency between NGS technique and Sanger sequencing was evaluated. Results Genomic DNA samples were successfully extracted from the 10 T-LBL specimens of paraffin embedded tissues. The pathogenic mutations were detected by NGS technique and the result was consistent with the Sanger sequencing method. Conclusion NGS technology can be used for WES and detection of pathogenic mutation for paraffin embedded pediatric T-LBL specimens.
引文
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[3]Sanmann JN,Pickering DL,Stevens JM,et al.Microarray studies in pediatric T-Cell acute lymphoblastic leukemia/lymphoma:a report of four cases[J].Cancer Genet,2013,206(5):213-9.
[4]Mussolin L,Buldini B,Lovisa F,et al.Detection and role of minimal disseminated disease in children with lymphoblastic lymphoma:the AIEOP experience[J].Pediatr Blood Cancer,2015,62(11):1906-13.
[5]Zhang MY,Churpek JE,Keel SB,et al.Germline ETV6 mutations in familial thrombocytopenia and hematologic malignancy[J].Nat Genet,2015,47(2):180-5.
[6]Campo E,Swerdlow SH,Harris NL,et al.The 2008 WHOclassification of lymphoid neoplasms and beyond:evolving concepts and practical applications[J].Blood,2011,117(19):5019-32.
[7]Oyola SO,Otto TD,Gu Y,et al.Optimizing illumina nextgeneration sequencing library preparation for extremely AT-biased genomes[J].BMC Genomics,2012,13(1):1-8.
[8]Bodi K,Perera AG,Adams PS,et al.Comparison of commercially available target enrichment methods for next-generation sequencing[J].J Biomol Tech,2013,24(2):73-86.
[9]Roncero AM,Lopez-Nieva P,Cobos-Fernandez M,et al.Contribution of JAK2 mutations to T-cell lymphoblastic lymphoma development[J].Leukemia,2016,30(1):94-103.
[10]Ittel A,Jeandidier E,Helias C,et al.First description of the t(10;11)(q22;q23)/MLL-TET1 translocation in a T-cell lymphoblastic lymphoma,with subsequent lineage Switch to acute myelomonocytic myeloid leukemia[J].Haematologica,2013,98(12):E166-8.
[11]Bandapalli OR,Schuessele S,Kunz JB,et al.The activating STAT5B N642H mutation is a common abnormality in pediatric T-cell acute lymphoblastic leukemia and confers a higher risk of relapse[J].Haematologica,2014,99(10):E188-92.
[12]Trinquand A,Tanguy-Schmidt A,Ben Abdelali RA,et al.Toward a NOTCH1/FBXW7/RAS/PTEN-Based oncogenetic risk classification of adult T-Cell acute lymphoblastic leukemia:a group for research in adult acute lymphoblastic leukemia study[J].J Clin Oncol,2013,31(34):4333-42.
[13]Tomczak K,Czerwińska P,Wiznerowicz M.The cancer genome Atlas(TCGA):an immeasurable source of knowledge[J].Contemp Oncol,2015,19(1A):A68-77.
[14]Krasnov GS,Dmitriev AA,Melnikova NV,et al.CrossHub:a tool for multi-way analysis of The Cancer Genome Atlas(TCGA)in the context of gene expression regulation mechanisms[J].Nucleic Acids Res,2016,44(7):e62-8.
[15]Dellinger TH,Smith DD,Ouyang C,et al.L1CAM is an Independent predictor of poor survival in endometrial cancer-An analysis of The Cancer Genome Atlas(TCGA)[J].Gynecol Oncol,2016,141(2):336-40.
[16]Huang R,He N,Li Z.Recent progresses in DNA nanostructurebased biosensors for detection of tumor markers[J].Biosens Bioelectron,2018,109(1):27-34.
[17]Rosolen LA.Minimal disseminated disease in pediatric NonHodgkin lymphoma[J].J Carcinog Mutagen,2014,05(1):12-9.
[18]Mussolin L,Pillon M,Basso GC.Could(disseminated and residual)minimal disease be a useful prognostic marker in nonHodgkin paediatric lymphomas[J].Clin Managem Issues,2014,8(2):37-42.
[19]Hou S,Shen J,Tan J.Case report:multiple systemic disseminated tuberculosis mimicking lymphoma on 18F-FDG PET/CT[J].Medicine,2017,96(29):e7248-55.
[20]赵倩雯,司徒博,郑磊.循环肿瘤细胞检测与临床应用进展[J].南方医科大学学报,2017,37(10):1423-6.
[21]许洁,张科平,葛岩,等.低温冻存对非霍奇金淋巴瘤石蜡组织DNA质量的影响[J].诊断病理学杂志,2016,23(3):234-6.
[22]Chen Y,Lu J,Chen G.Genetic heterogeneity in B‐cell lymphoma detected by next‐Generation sequencing(NGS)technology in a cohort of 150 Chinese patients[J].Hematol Oncol,2017,35(S2):280-1.
[23]Lohr JG,Stojanov P,Lawrence MS,et al.Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma(DLBCL)by whole-exome sequencing[J].Proc Natl Acad Sci USA,2012,109(10):3879-84.
[24]Palmieri G,Cossu A,Caraco C,et al.Evaluation of genetic heterogeneity in paired lymph node metastases from melanoma patients using next-generation sequencing(NGS)approaches[J].PLoS One,2015,33(15,S):e19534-9.
[25]Tong AK,Neo SH,Kok TY.Disseminated lymphoma evolving into neurolymphomatosis during mid-cycle of chemotherapy detected by(18)F-FDG PET/CT[J].Ann Acad Med Singapore,2015,44(11):545-7.