《(4.7)--Next-generationsequencinginthyro病理学生理学课件.pdf》由会员分享，可在线阅读，更多相关《(4.7)--Next-generationsequencinginthyro病理学生理学课件.pdf（10页珍藏版）》请在taowenge.com淘文阁网|工程机械CAD图纸|机械工程制图|CAD装配图下载|SolidWorks_CaTia_CAD_UG_PROE_设计图分享下载上搜索。
1、Cha and Koo J Transl Med (2016)14:322 DOI 10.1186/s12967-016-1074-7REVIEWNext-generation sequencing inthyroid cancerAbstract Next-generation sequencing(NGS)in thyroid cancer allows for simultaneous high-throughput sequencing analysis of variable genetic alterations and provides a comprehensive under
2、standing of tumor biology.In thyroid cancer,NGS offers diagnostic improvements for fine needle aspiration(FNA)cytology of thyroid with indeterminate features.It also contributes to patient management,providing risk stratification of patients based on the risk of malignancy.Furthermore,NGS has been a
3、dopted in cancer research.It is used in molecular tumor classification,and molecular prediction of recurrence and metastasis in papillary thyroid carcinoma.This review covers previous NGS analyses in variable types of thyroid cancer,where samples including FNA cytology,fresh frozen tissue,and formal
4、in-fixed,paraffin-embedded tissues were used.This review also focuses on the clinical and research implications of using NGS to study and treat thyroid cancer.Keywords:Next-generation sequencing,Cancer,Cytology,Thyroid The Author(s)2016.This article is distributed under the terms of the Creative Com
5、mons Attribution 4.0 International License(http:/creativecommons.org/licenses/by/4.0/),which permits unrestricted use,distribution,and reproduction in any medium,provided you give appropriate credit to the original author(s)and the source,provide a link to the Creative Commons license,and indicate i
6、f changes were made.The Creative Commons Public Domain Dedication waiver(http:/creativecommons.org/publicdomain/zero/1.0/)applies to the data made available in this article,unless otherwise stated.BackgroundAn understanding of the molecular mechanisms of tumor formation is mandatory for accurate dia
7、gnoses and per-sonalized treatments.Previously,single gene assays were commonly used for finding molecular alterations in tumors.Presently,NGS technology provides the simulta-neous analysis of hundreds of genes of interest,using tar-geted sequencing panels 1.Thus,NGS-based molecular tests for oncolo
8、gy research and clinical practice appear to be rapidly evolving.Thyroid cancer is the most common malignancy of the endocrine organs;its prevalence is increasing,more than tripling during the last three decades 2.Papillary thy-roid carcinoma(PTC)is the most common type of thy-roid cancer,followed by
9、 follicular carcinoma,medullary carcinoma,poorly differentiated carcinoma,and anaplas-tic carcinoma.NGS assays can allow improvements in diagnostic accuracy and precise personalized treatments.Thyroid cancers harbor characteristic genetic alterations,including point mutations for proto-oncogenes(BRA
10、F,NRAS,HRAS,KRAS)and chromosomal rearrangements(RET/PTC1,RET/PTC3,PAX8/PPARG),which vary with histologic subtype 3.This review outlines the results of NGS assays in thyroid cancer,and highlights their clinical implications.NGS application inthe diagnosis ofindeterminate cytologyA majority of previou
11、s studies using NGS in thyroid can-cer analyzed variable specimen sample types and histo-logic subtypes(Table1).In clinical practice,NGS assays have been used in the diagnosis of indeterminate cytol-ogy of thyroid nodules.FNA is an efficient method for evaluating thyroid nodules that has high sensit
12、ivity and specificity.However,FNA has some limitations,since 2030%of FNA samples fall into categories of indeter-minate cytology.These categories include atypia of unde-termined significance/follicular lesion of undetermined significance(AUS/FLUS,category III);follicular or onco-cytic(Hurthle cell)n
13、eoplasm/suspicious for a follicular or oncocytic(Hurthle cell)neoplasm(FN/SFN,category IV);and suspicious for malignant cells(SUSP)48.The average cancer risk for these categories is 15.9%in AUS/FLUS,26.1%in FN/SFN,and 75.2%in SUSP 9.NGS contributes to diagnostic decision making in patients with inde
14、terminate cytology.Findings from previous Open AccessJournal of Translational Medicine*Correspondence:kjs1976yuhs.ac Department of Pathology,Yonsei University College of Medicine,50-1 Yonsei-ro,Seodaemun-gu,Seoul 03722,South KoreaPage 2 of 10Cha and Koo J Transl Med (2016)14:322 Table 1 Studies ofth
15、yroid cancer using next-generation sequencing platformsAuthor(year)DiagnosisPatient numberSample typePlatformMutational panelAnalytic process toolImportant findingsNikiforova et al.10Classic PTC27Fresh frozen tissueFFPE tissueIon Torrent PGMThyroSeqTorrent Suit version 3.4.21.70%of classic PTCs harb
16、oring mutations:BRAF(59%)PIK3A (11%)TP53(7%)NRAS(4%)FVPTC302.83%of FVPTC harboring mutations:RAS(73%)BRAF(7%)TSHR(3%)Classic FC183.Mutations of FC in order of frequency Conventional type:NRAS TSHR KRAS Oncocytic type:TP53 HRAS KRAS PTENOncocytic FC184.30%of PDCs harboring mutations of NRAS,PIK3CA,GN
17、AS,BRAF and 74%of ACs harboring mutations of TP53,BRAF,RAS,PIK3CA,PTEN,CTNNB15.11 MCs(73%)harboring mutations:7 RET(47%),3 HRAS(20%),1 KRAS(7%)PDC10AC27MC15Smallridge et al.11PTC,BRAF-mutant12Fresh frozen tissueIllumina HiSeq 2000RNA-Seq(13085 genes)TopHat tool package1.51 genes related with immune
18、function pathway are downregulated in BRAF V600E-mutant PTCs2.HLAG,CXCL14,TIMP1,IL1RAP are overexpressed in BRAF V600E-mutant PTCs mutationPTC,BRAF-wild type8Leeman-Neil et al.12PTC,radiation-associated62Fresh frozen tissueIllumina HiSeq 2000RNA-SeqChimeraScan and defuse programETV6-NTRK3 rearrangem
19、ent are present in 14.5%of radiation-associated PTCs and 2%of sporadic PTCsPTC,sporadic151Simbolo et al.32MC20FFPE tissueIon Torrent PGMIon AmpliSeq Hot Spot Cancer Panel v2(50 genes)Torrent Suit version 3.61.85%of MCs have mutations:13 RET(60%),3 HRAS(15%),1 KRAS(5%),1 STK11(5%),and 3 undetected(15
20、%)2.Three RET mutations are found by NGS test,which are negative by Sanger sequencingNikiforov et al.34FN/SFN143(retrospective group,N=91;prospective group,N=52)FNAIon Torrent PGMThyroSeq v2N/APerformance of NGS test in cancer detection among nodules with FN/SFN cytology:sensitivity 90%,specificity
21、93%,PPV 83%,NPV 96%,accuracy 92%Cancer genome atlas research network 13PTC496Fresh frozen tissueIllumina HiSeq 2000Whole genome sequencingPicard pipeline1.Identification of potential new tumor-initiating mutation in PTC lacking known driver mutation(EIF1AX,PPMID,CHEK2)2.TERT promoter mutation(1%)ass
22、ociated with high risk of recurrence3.Categorization of PTC into BRAF-like and RAS-like types based on the multi-level molecular dataSykorova et al.27PDC3Fresh frozen tissueIllumina MiSeqThe TruSight Cancer Panel(94 genes)MiSeq Reporter v.2.41.All PDC and AC harbor more than one genetic alteration,a
23、nd TP53 mutation is commonly present except for 2 cases2.Altered genes in PDCs:CDH1,FANCD2,CHECK2,ADH1B,GPC3,TP53,PTEN3.Altered genes in ACs:ATM,HNF1A,MET,NF1,TP53,PTEN,MSH2,RB1,NBN,NF1,MUTYH,TSC2,HRAS,EGFRAC5Le Mercier et al.35Indeterminate cytology34Cell blocks(FFPE)and smearsIon torrent PGMAmpliS
24、eq Cancer hotspot panel version 2Torrent Suit version 3.6.2Performance of NGS test in cancer detection among nodules with indetermi-nate cytology:sensitivity 71%,specificity 89%,PPV 62%,NPV 92%,accuracy 85%Ion AmpliSeq HiFi Master MixVariant Caller plugin ver-sion 3.6Page 3 of 10Cha and Koo J Transl
25、 Med (2016)14:322 Table 1 continuedAuthor(year)DiagnosisPatient numberSample typePlatformMutational panelAnalytic process toolImportant findingsNikiforov et al.44AUS/FLUS465FNAIon torrent PGMIon protonThyroSeq v2.1N/APerformance of NGS test in cancer detection among nodules with AUS/FLUS cytology:se
26、nsitivity 90.9%,specificity 92.1%,PPV 76.9%,NPV 97.2%,accuracy 91.8%Picarsic et al.14PTC17(age 18 years)FNAFresh frozen tissueFFPE tissueIon Torrent PGMThyroSeq v2(14 gene and 42 types gene fusions)N/A1.Mutation detection rate is increased from 60%with 7-gene mutation panel to 80%with NGS2.Chromosom
27、al rearrangement is more common than point mutation in pediatric PTC(53 vs.33%)3.ETV6-NTRK3 fusions are present in 18%and associated with unfavorable histology such as solid,insular,or trabecular patternsBallester et al.15PTC25(age range,1019 years)FNAFFPE tissueIon torrent PGMAmpliSeq Cancer Hotspo
28、t Panel v2(50 genes)Torrent Suit version 4.2No additional mutation detected by NGS in cases lacking mutations in BRAF,RET/PTC,TERT promoter mutation at initial analysisLanda et al.28PDC34Fresh frozen tissue(N=37)FFPE tissue(N=80)N/AMSK-IMPACT cancer exome panel(341 genes)MSK-IMPACT pipeline1.Mutatio
29、n number is greater in AC(6 5)than PDC(2 3),and predomi-nantly affected genes are TP53,TERT promoter,PI3K/AKT/mTOR pathway effector,SWI/SNF subunit,and histone methyltransferase2.92%of RAS mutations are found in PDCs met Turin criteria;81%BRAF muta-tions are present in PDCs met MSKCC criteria3.BRAF-
30、mutant PDCs are smaller and frequently metastasize to lymph nodes;RAS-mutant PDCs are larger and have higher frequency of distant metastasis4.EIF1AX mutations are detected in 11%of PDCs and 9%of ACs,and 93%of which are associated with RAS mutations5.Chromosomal rearrangement is detected in 14%of PDC
31、s(RET/PTC,PAX8/PPARG,and ALK/EML4)and absent in ACAC33Swierniak et al.20FA26Fresh frozen tissueIllumina HiSeq 1500TruSeq panel(372 genes)Varscan2CODEX packageBreakDancerFACTERA1.Common somatic alterations:oncogenes(MDM2,FLI1),transcription factors and repressors(MITF,FLI1,ZNF331),epigenetic enzymes(
32、KMT2A,NSD1,NCOA1,NCOA2),and protein kinases(JAK3,CHEK2,ALK)2.Single nucleotide variant is the most common and large structural variants are the least3.Identification of novel translocation,DERL/COX6CFC20PDC2Paired normal thyroid tissue34AUS/FLUS atypia of undetermined significance/follicular lesion
33、of undetermined significance,FNA fine needle aspiration,N/A not applicable,PPV positive predictive value,NPV negative predictive value,FN/SFN follicular or oncocytic(Hurthle cell)neoplasm/suspicious for a follicular or oncocytic(Hurthle cell)neoplasm,FFPE formalin-fixed,paraffin-embedded,PTC papilla
34、ry thyroid carcinoma,FVPTC follicular variant papillary thyroid carcinoma,FC follicular carcinoma,PDC poorly differentiated carcinoma,AC anaplastic carcinoma,MSKCC Memorial Sloan-Kettering Cancer Center,MC medullary carcinomaPage 4 of 10Cha and Koo J Transl Med (2016)14:322 studies,which used NGS to
35、 analyze thyroid nodules with indeterminate cytology,are summarized in Table2.Genetic alteration ofthyroid cancer andNGSPapillary carcinomaSeveral studies have applied NGS to variable subgroups of PTCs.Nikiforova etal.analyzed FFPE or frozen tissue from 27 classic PTCs and 30 FVPTCs,using the ThyroS
36、eq NGS panel targeting 12 genes with 34 amplicons on the Ion Torrent PGM sequencer 10.The results showed that 70%of classic PTCs harbored mutated genes:BRAF(59%)was the most frequent,followed by PIK3A(11%),TP53(7%),and NRAS(4%).In contrast,83%of FVPTCs had mutated genes:RAS(73%)was the most frequent
37、,followed by BRAF(7%)and TSHR(3%)10.Smallridge etal.performed RNA sequencing(RNA-Seq)using the Illumina HiSeq 2000 platform on frozen tissue from 12 BRAF V600E-mutated PTCs and 8 BRAF-wild type PTCs 11.Among the 13,085 genes interrogated,560 were differentially expressed between BRAF V600E-mutated P
38、TCs and BRAF-wild type PTCs 11.Among these 560 genes,67 were related to immune function pathways,51 were under-expressed in BRAF V600E-mutated PTCs,and HLAG,CXCL14,TIMP1,and IL1RAP were over-expressed.In BRAF-wild type PTCs,4 immune function genes(IL1B,CCL19,CCL21,and CXCR4)were most sig-nificantly
39、differentially expressed,and exhibited a high degree of correlation with lymphocytic infiltration 11.In a study by Leeman-Neill etal.fresh frozen tissue from 62 radiation-associated PTCs and 151 sporadic PTCs was analyzed using RNA-Seq on the Illumina HiSeq 2000 platform.This identified an ETV6-NTRK
40、3 rearrange-ment in 14.5%of radiation-associated PTCs and 2%of sporadic PTCs 12.The authors suggested that an ETV6-NTRK3 rearrangement may be a key mechanism of radi-ation-induced carcinogenesis 12.The Cancer Genome Atlas(TCGA)Network described the genomic characterization of 496 PTCs,and gener-ated
41、 data using whole genome sequencing.This was done on the NGS platform and by a multiplatform analysis of SNP arrays,DNA methylation,and reverse phase pro-tein arrays 13.In PTCs that lacked known driver muta-tions,alterations of EIF1AX,PPMID,and CHEK2 were discovered as potential new tumor-initiating
42、 mutations.The TCGA project identified the TERT promoter muta-tion,which accounts for approximately 1%of PTCs,but shows association with a high risk of recurrence.Based on the multi-level molecular data,PTCs were sepa-rated into two groups of distinct downstream signaling pathways:the BRAFV600E-like
43、 cohort and the RAS-like cohort.Genomic,epigenomic,and proteomic differences were revealed between these two groups,and most of the RAS-like PTCs were follicular variant PTCs(FVPTCs).Regarding pediatric thyroid carcinoma,Picarsic etal.analyzed 17 pediatric PTCs(age range 817 years,median 13 years)fr
44、om FNA,fresh frozen tissue,and FFPE samples.Mutation analysis with a 7-gene mutation panel using real-time PCR and ThyroSeq v2 on the Ion Torrent PGM sequencer showed that:(1)The detection rate of molecular alterations was increased by up to 87%by the ThyroSeq v2 NGS assay compared to an increase of
45、 60%by the 7-gene mutation panel.(2)In pediatric thyroid carcinoma,chromosomal rearrangement(53%)was more common than point mutation(33%).(3)ETV6-NTRK3 fusion was identified in 18%of samples,and was associated with aggressive histologic features such as non-encapsulation,solid/insular/trabecular pat
46、terns,extensive glandular involvement,and thick tumor fibro-sis 14.Ballester etal.analyzed FFPE and FNA samples from 25 pediatric PTCs(age range 1019years,median 14years)using the 50-gene Ion AmpliSeq Cancer Hot-spot Panel v2 on the Ion Torrent PGM sequencer 15.No additional mutations were detected
47、using the NGS assay on pediatric PTCs that initially were negative for BRAF V600E mutation,RET/PTC1/3 fusion,and TERT promoter mutation 15.Follicular carcinomaFollicular carcinoma(FC)is a well-differentiated thyroid carcinoma and the second-most common thyroid cancer after PTC.It accounts for 10%of
48、the total thyroid cancers 16.Although it is not firmly established,FC is classified into the minimally invasive type and the widely invasive type,according to the microscopic tumor extent 17.It can be classified into the conventional type and the onco-cytic type(Hurthle cell type)on the basis of cel
49、l type 18.A previous study retrospectively collected FFPE or fresh frozen tissue samples from 36 FCs to study 12 can-cer genes and 34 amplicons using the ThyroSeq panel on the Ion Torrent PGM sequencer.The analysis detected mutations of NRAS(N=9),KRAS(N=2),HRAS(N=1),TSHR(N=4),TP53(N=4),and PTEN(N=1)
50、10.Interestingly,conventional type(N=18)and oncocytic type(N=18)samples showed distinguished genetic alterations.In the conventional type FCs,NRAS was the most frequently affected gene,followed by TSHR and KRAS.TP53 was the most commonly mutated gene in the oncocytic type FCs,followed by HRAS,KRAS,a