REVIEW ARTICLE ISSN: 2384-3799 Int J Thyroidol 2015 November 8(2): 153-160 http://dx.doi.org/10.11106/ijt.2015.8.2.153 세포학적으로진단이불확실한갑상선결절의분자진단 건국대학교의학전문대학원병리학교실 황태숙 Molecular Diagnosis for Cytologically Indeterminate Thyroid Nodules Tae Sook Hwang Department of Pathology, Konkuk University School of Medicine, Seoul, Korea An accurate diagnosis of cancer or benign disease is important for the effective clinical management of the patients. Thyroid fine needle aspiration cytology (FNAC) is a safe and cost effective technic for evaluating thyroid nodules. However, 20-30% of thyroid FNAC specimens are indeterminate and fall into one of the following categories; AUS/FLUS (atypical ceils of undetermined significance/follicular cells of undetermined significance), FN/SFN (follicular neoplasm/suspicious for follicular neoplasm), and SMC (suspicious for malignant cells). The AUS/FLUS, FN/SFN, and SMC diagnostic category is associated with a 5-15%, 15-30%, and 60-75% risk of malignancy, respectively. Of the indeterminate thyroid nodules that are surgically resected, 10-40% were confirmed to be malignant. A significant progress has been made in the development of molecular tests for cancer diagnosis in thyroid nodules. Most common molecular alteration in thyroid cancer is the activation of mitogen-activated protein kinase (MAPK) pathway. Activation of this pathway in thyroid cells results from point mutation of BRAF and RAS genes and rearrangement of RET/PTC and NTRK genes and these genetic alterations are mutually exclusive. Preoperative molecular diagnostic techniques could be applied in FNAC specimen when optimum dissection techniques are provided to collect sufficient numbers of target cells without contamination of blood cells, inflammatory cells including histiocytes, and stromal cells. The optimum number of cells for PCR is about 100 although as few 50 cells has been successful. To obtain a good DNA yield from a very limited number of target cells, avoid DNA loss as much as possible. Key Words: Molecular diagnosis, FNAC, Indeterminate nodule, Thyroid cancer 서론 갑상선암은모든암중약 1% 정도를차지하며우리나라의여성에서가장흔하게발생하는암이다. 대부분의갑상선암은소포세포에서기원하며일부는 C세포에서기원한다. 갑상선암은최근들어우리나라뿐만아니라서구에서도증가하고있으며 2011년보건복지부중앙암등록본부의통계에따르면우리나라갑상선 에서발생하는악성종양중가장흔한유형은유두암종 (papillary carcinoma) 으로갑상선암의약 95% 를차지한다. 1) 그외에빈도는낮으나소포암종 (follicular carcinoma), 수질암종 (medullary carcinoma), 분화나쁜암종 (poorly differentiated carcinoma), 역형성암종 (anaplastic carcinoma) 등의유형이있는데일부유형사이에는조직학적유사성이높아감별이어려운경우가있다. 특히유두암종은 10가지이상의아형이있는데이중대부분이평범한형 (conventional 혹은 classical) 이고소포 Received July 13, 2015 / Revised August 5, 2015 / Accepted August 6, 2015 Correspondence: Tae Sook Hwang, MD, PhD, Department of Pathology, Konkuk University School of Medicine, 120-1 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea Tel: 82-2-2030-5641, Fax: 82-2-2030-5629, E-mail: tshwang@kuh.ac.kr Copyright c 2015, the Korean Thyroid Association. All rights reserved. This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 153
Tae Sook Hwang 형 (follicular variant), 큰키세포형 (tall cell variant) 이그다음으로흔하다고알려져있다. 갑상선암종은조직학적유형뿐아니라각각의아형에따라재발, 전이혹은방사성요오드치료에대한반응등임상적양상에차이가있어정확한진단이요구된다. 갑상선암은대부분갑상선결절로나타나게되는데실제갑상선결절의일부만이수술후갑상선암으로판명된다. 갑상선결절의양상을평가하는데가장중요한수단인초음파유도하세침흡인세포검사 (ultrasonography guided fine needle aspiration cytology) 는임상소견이나영상소견에비하여상당히높은예측도 (predictive value) 를제공할수있는간편하고도안전한검사이다. 그러나그간세침흡인세포검사후세포판독결과를보고하는양식이다양하여병리의사와임상의사, 심지어는병리의사들사이에도의사소통이명확하지않다는문제점들이여러차례제기된바있었다. 이러한이유로 2009년갑상선세포검사보고를위한베데스타체계 (Bethesda system) 가발표되었고이후대부분의병원에서이에기반을두어판독을하고있다. 2) 갑상선결절의세침흡인세포검사는악성종양을진단하는데매우중요한정보를제공하나이검사의 25% 정도에서갑상선암과양성결절성증식을감별하지못하는 불확실한 (indeterminate) 소견을보여재검사를하거나진단을위한수술이불가피하게된다. 3,4) 베데스타체계에의하면양성도악성도아닌 불확실한 (indeterminate) 카테고리에는 AUS/FLUS (atypical cells of undetermined significance/follicular cells of undetermined significance), FN/SFN (follicular neoplasm/suspicious for follicular neoplasm) 과 SMC (suspicious for malignant cells) 가해당되나우리나라의경우 SMC는거의악성으로간주하는경향이있다. Cibas 등 5) 의보고에의하면 AUS/FLUS로진단된결절의약 5-15%, FN/SFN으로진단된결절의 15-30%, SMC로진단된결절의 60-75% 가수술후조직학적검사에서악성으로판명될가능성이있다고하였다. 실제 불확실한 세포소견을보였던결절을수술로절제한경우 10-40% 정도에서만악성으로확인되어, 6,7) 대부분환자가진단을위해불필요한수술을하게되며이로인해치료방침결정이지연되고, 환자의정신적불안및반복적인검사와불필요한수술등에의한의료비지출증가등이차적인문제들이수반되고있는실정이다. 반면에진단을목적으로엽절제술을받은환자의경우상당수에서남아있는갑상선조직을제거하기위한이차수술이불가피하다. 최근형태학적변화에의거한진단의한계점을보완하여갑상선암을진단하는데도움을주고예후에영향을미치는요인들을찾아내고자분자유전학적인연구가활발하게진행되어약 90% 정도의갑상선암에서발생과진행에관여하는분자학적변화가확인되었으며, 이러한분자유전학적지식을통해암의조기진단과표적치료제의개발및예방이가능하게되었다. 갑상선암의발암과정에관여하는분자학적변화 갑상선암의발생과관련된가장흔한기전은 mitogenactivated protein kinase (MAPK) 경로의활성화이며 BRAF와 RAS 유전자의점돌연변이및 RET/PTC 유전자재배열이대표적인변화이고이들은서로중복되어발생하지않는다. 8) 이중에서도 BRAF 유전자가가장널리연구되었으며또한가장흔한유전적변화이다. BRAF 점돌연변이 이중 BRAF 유전자돌연변이는유두암종에서가장흔히관찰되는유전적변화로대부분이 1799 뉴크레오티드부위에발생하여 thymine을 adenine으로치환시켜 valine을 glutamate로바꾸는 BRAF V600E 돌연변이이며갑상선암에서발생하는 BRAF 돌연변이의 98-99% 를차지한다. 9) 유두암종에서 BRAF K601E의빈도는 45% 정도로보고되어왔으나 10,11) 미국의경우최근그빈도가 75% 이상으로높아졌다는보고들이있으며, 12,13) 한국은그빈도가 70-90% 정도로서구에비해비교적높다. 14-18) 이돌연변이는유두암종에서주로관찰되나 8) 유두암종외에는유두암종에서발생한분화나쁜암종이나역형성암종에국한되어관찰된다. 10,11) 유두암종에서는평범한형이나큰키세포형에서주로관찰되고소포형에서는비교적드물게관찰되며다른유형의종양이나비종양성병변에서는관찰되지않아갑상선유두암종의진단적표지자로여겨진다. 10,19) 유두암종에서관찰되는다른 BRAF 변이는 BRAF K601E 점돌연변이, 코돈 600 근처의작은삽입 (in-frame insertion) 혹은결손 (deletion) 과방사능에노출되어생긴유두암종에서흔히관찰되는 AKAP9-BRAF 유전자재배열등이있다. 20,21) 이중 BRAF K601E 돌연변이는소포형유두암종에서만거의특이적으로관찰되는데이종양의약 9% 정도까지나타나그진단적가치는그리높지않다. 22,23) Vol. 8, No. 2, 2015 154
Molecular Diagnosis for Cytologically Indeterminate Thyroid Nodules RAS 점돌연변이조직학적으로소포형패턴을보이는갑상선종양에서흔히발견되는 RAS 유전자점돌연변이는갑상선암에서 BRAF 돌연변이다음으로흔히관찰되는유전적변화이다. RAS 돌연변이는평범한 (conventional) 소포암종의 40-50%, 평범한소포샘종의 20-40% 정도에서관찰되며 24,25) 소포샘종에서 RAS 유전자돌연변이가있는경우소포암종이나소포형유두암종으로전환될가능성이있다고한다. 26-28) 이돌연변이는유두암종에서도관찰되는데대부분이소포형 (follicular variant) 인경우에해당된다. 25,29-31) RAS 유전자돌연변이는소포세포에서기원한종양에서는 NRAS, HRAS 및 KRAS 유전자의 12, 13 및 61번코돈에서흔히일어나며 NRAS 61번코돈부위의변이가가장흔하고다음으로 HRAS 61번코돈변이이며다른부위의변이는드문것으로알려져있다. 32,33) 반면에수질암에서는 HRAS 유전자나 KRAS 유전자의돌연변이가흔하다고한다. 34) RET/PTC 재배열 RET/PTC 재배열은 RET 유전자의 receptor tyrosine kinase (RTK) 를코딩하는 3 - 말단부와 PTC 유전자의 5 -말단부가융합되어만들어지며 RET/PTC1 재배열이 60-70% 를차지하고 RET/PTC3 재배열이 20-30% 를차지한다. 35,36) RET/PTC 유전자는정상갑상선의소포세포에서는발현되지않으나재배열에의해발현되고활성화되어 MAPK 경로를활성화시키게된다. 이유전자변화의빈도는지리적요인이나검색방법에따라차이가있으며갑상선유두암종의약 10-20% 정도에서관찰된다고보고되었으나그빈도가점차감소하고있으며, 12,37,38) 어린아이에서발생하는유두암종이나방사능에노출된과거력이있는환자에서발생한유두암종의경우에높은빈도로보고되고있다. 39,40) 또한유두암종의한아형인미만성경화형 (diffuse sclerosing variant) 에서흔하며, 41) 분화나쁜암종이나역형성암종에서는아주드물게발견된다. 42) PAX8/PPARγ 재배열 PAX8/PPARγ 재배열은 t(2;3)(q13;p25) 전위에의해일어나며그결과로 PAX8 전사인자와스테로이드 / 갑상선핵수용체의일종인 PPARγ 유전자의접합 (fusion) 을일으킨다. 이유전자재배열은평범한소포암종의약 35% 에서관찰되며호산성형 (oncocytic variant) 에서는좀더낮은빈도로관찰된다. 43,44) 소포샘종의약 2-10% 정도에서관찰되긴하나주로소포암종에만국한되어관찰되므로악성종양을진단하는데활용할수있다. 43,44) 또한소포샘종에서 PAX8/PPARγ 재배열이관찰되는경우는침습전단계의상피내소포암종이거나침습형소포암종인데침습부위를확인하지못한경우라는의견이제시되고있다. 28) PAX8/PPARγ 재배열은전통적으로소포암종에서관찰되는것으로알려져왔으나최근연구결과에의하면이유전자재배열의대부분이소포형유두암종에서발견된다고한다. 45) 이런결과로볼때이유전자변화가확인되는결절은수술로제거하는것이좋을것으로생각한다. 분자진단검사의현재와미래 우리나라에서현재는연구목적으로만시행할수있는분자진단검사 1) mrna나 mirna마커발현 mrna 발현양상이나 mirna의정량적검사는미래에활용될전망이밝다. 대부분의중요한 mirna는 mrna에비하여변성이잘일어나지않기때문에진단적활용가치가더크다. 46) 최근연구결과에의하면유두암종이나소포암종의경우 mirna 발현양상이양성결절의발현양상과는차이가있다고하였으며, 47-49) Pallante 등 47) 은유두암종과양성결절의세침흡인검사검체에서 mir-221, -222와 -181b의발현양상이현저히다르다고하였다. 2) 차세대염기서열분석 (targeted next generation sequencing) 최근에연구에의하면 BRAF, RAS 등을비롯한 13개유전자의점돌연변이와 PAX8/PPARγ나 RET/PTC를포함한 42개의접합유전자를차세대염기서열분석법으로확인한결과 145예의갑상선암중 68% 에서점돌연변이가확인되었고유전자접합까지포함할경우 80% 정도에서적어도하나의유전자변화가확인되었다고하여이검사법은향후세포학적으로진단이불확실한갑상선결절의분자진단법으로자리매김할가능성이높아보인다. 50) 이분석을위해서는 5-10 ng 정도의 DNA만필요하기때문에세침흡인검사검체로도분석이가능하다. 3) 유전자발현프로필 (1) Microarray/Immunohistochemistry CK-19, glectin-3, HBME-1, CD 56을비롯한다양한 155 Int J Thyroidol
Tae Sook Hwang 마커들이알려져있고이러한마커들을이용한면역조직화학기법이시행되고있다. 그러나현재로서는유두암종이나소포암종혹은양성결절에만특이적으로발현되는마커는관찰되지않아여러가지마커를조합해서진단에보조적으로활용하고있기는하나 불확실한 소견을보이는세침흡인검사검체에서수술전진단에적용하는데에는어려움이많다. (2) Gene Expression Classifier 상기한검사법들이세포학적으로양상이 불확실한 결절에서악성의가능성을제시할수있는검사라면 Gene Expression Classifier는양성결절을확인함으로써불필요한수술은줄이고자하는접근법이다. Alexander 등 51) 은 256예의 불확실한 세침흡인검사검체를대상으로유전자발현을확인한결과 AUS/FLUS; FN/SFN; SMC 카테고리에서음성예측도, 민감도, 특이도가각각 95%, 90%, 53%; 94%, 90%, 49%; 85%, 94%, 52% 로확인되었고 AUS/FLUS와 FN/SFN의경우음성예측도가 94% 이상으로높아갑상선결절이악성임을배제할수있다고하였다. 우리나라에서현재시행할수있는분자진단검사 가장흔하게사용되고임상적으로유용한검사는 BRAF 점돌연변이검사이며세침흡인세포검사검체와조직검체에시행할수있다. 다음으로 RAS 점돌연변이검사인데현재우리나라에선갑상선결절의진단목적으로는 NRAS 점돌연변이검사만가능하고 RAS 점돌연변이중두번째로흔한 HRAS 유전자검사는시행할수없는상태이다. 드물기는하나가족성갑상선수질암 (familial medullary thyroid carcinoma) 의 95% 이상에서관찰되는 RET 유전자돌연변이검사도시행할수있다. PAX8/PPARγ나 RET/PTC 재배열검사는현재로서는시행할수없다. 세침흡인세포검사검체를이용한분자진단검사에대한제언 앞에서언급한바와같이갑상선유두암종은전체갑상선암의 95% 를차지하며가장흔한아형인평범한형유두암종의 80-85% 정도와큰키세포형의대부분에서 BRAF 점돌연변이가발생한다. 52) 두번째로흔한소포형유두암종은전체유두암종의약 10-20% 로생각되며소포형의경우 60-70% 정도에서 BRAF 혹은 RAS 점돌연변이가확인되는데, 12,32,53) 각각의돌연변이가차지하는비율은보고자마다차이가있다. RET/PTC 재배 열은유두암의 10-20% 정도에서확인된다고보고된바있지만점차줄어드는추세이며지역에따라그빈도에차이를보이고과도한방사선에노출된병력이없는경우엔드물게나타나며우리나라에서발생하는암에선훨씬적게나타나는것으로추정된다. 저자의예비실험결과에의하면 BRAF와 RAS 돌연변이가음성인유두암종의약 1/3 정도에서 RET/PTC1 유전자재배열을확인할수있었다 (not published). 갑상선결절의양상을확인하기위해통상적으로시행하는세침흡인세포검사검체를이용한분자진단검사는세포학적으로 불확실한 소견을보이는갑상선결절의악성도를훨씬정확하게예측함으로써좀더정확한치료가이드라인을제공할수있다. Nikiforov 등 54) 은세침흡인세포검사결과만으로 AUS/FLUS군의 14%, FN/SFN군의 27%, SMC의 54% 에서암을예측할수있었으나 BRAF 돌연변이, RAS 돌연변이, RET/PTC 재배열및 PAX8/PPARγ 재배열을시행하였을때암예측도는 88%, 87%, 95% 로증가하였다고하였다. 저자의경험에의해서도비슷한결과를보여세침흡인세포검사검체를이용하여 BRAF와 RAS 유전자검사를보조적으로시행한경우 AUS/FLUS 환자의 84.1% 와 FN/ SFN 환자의 58.9% 에서수술후악성으로판명되었다 (not published). 우리나라의경우에는소포암종이 1.5% 정도로드물 고 1) 유두암에서 RET/PTC 재배열이일어나는빈도가 낮기때문에 BRAF와 RAS 점돌연변이확인만으로도대부분의갑상선암을진단할수있을것으로생각한다. BRAF 점돌연변이와 RAS 점돌연변이는상호배타적으로존재하고유두암종의대부분에서 BRAF 변이가관찰됨으로세포학적검사결과 불확실한 소견 ( 우리나라의경우는 AUS/FLUS와 FN/SFN만포함시키면될것임 ) 으로진단된환자에서 BRAF 점돌연변이검사를시행하고, 검사결과음성인환자를대상으로 RAS 유전자검사를시행한다면상당수의유두암종이나소포형종양을확인할수있으리라생각한다. 또한소포형유두암종에서확인된 RAS 유전자변이의 60% 이상이 NRAS 61번코돈에위치해있으므로 53,55) NRAS 61번코돈만을확인한다면경제적인부담도줄것으로생각한다. 수술전세침흡인세포검사검체를이용하여시행한유전자검사결과를보조적진단기법으로활용하기위해서는다음과같은사항을염두에두는것이좋으리라생각한다. Vol. 8, No. 2, 2015 156
Molecular Diagnosis for Cytologically Indeterminate Thyroid Nodules 어떤분자세포진단을하는경우라도수준높은세포검사판독이근간이되어야한다. 대부분연구자들은갑상선결절의양상을확인하는데도움이되는분자진단을독립적으로사용하기보다는형태학적소견에근거한세포학적진단의보조적기법으로사용하는것을권장하고있어분자진단을시행하기전에세포병리의사가세침흡인세포검체를판독한후다음단계로분자검사를시행하는것이바람직하다. 점돌연변이나유전자재배열과같은분자학적검사는공기중에서말리거나 (air dried) 혹은알코올에고정된세침흡인도말을이용하여시행할수있다. 이경우분자검사를위하여따로검체를채취해야하는번거로움과비용을절약할수있으며분자검사의타겟이되는비정상세포를현미경하에서확인할수있다는것이큰장점이다. 목적에맞는검사방법의선택이중요하다. 갑상선암의분자유전학적변화를확인하는다양한검사법들이소개되고있으나임상에서환자의진료목적으로시행하는검사는정확하고재연성이높아야하며임상적으로도움이되는정보를줄수있어야한다. 일반적으로검사방법은검사결과를사용하기위한목적에따라선택되어야한다. 어떤검사결과로갑상선결절이악성일가능성을배제하고자한다면민감도와음성예측도가높은방법을선택하는것이바람직하나검사결과의양성여부가암의가능성을높이는경우에는특이도와양성예측도가높은방법을선택하는것이바람직하다. BRAF나 RAS 점돌연변이는비교적간단해서 conventional polymerase chain reaction (PCR) 을비롯하여 Sanger sequencing, pyrosequencing, real-time PCR amplification and post-pcr melting curve analysis, and allele-specific PCR 등다양한기법으로쉽게확인할수있다. 다만한가지주의할점은세포주를대상으로분석해보았을때최소한타겟세포가 1% 가넘는경우에만양성으로확인되는방법을선택하는것이바람직하다. 저자의경우는타겟세포가 10% 이상이되는경우를 cutoff value로사용하고있다. 타겟세포를선별적으로충분히확보하고 DNA 혹은 RNA 소실을최소화하는것이중요하다. 모든검사에있어서가장중요한과정은 DNA나 RNA 추출과정인데이중에서도유전자변화를알고자하는타겟세포를선별적으로충분히확보하는것이가장중요한단계이다. 갑상선세침흡인세포검사검체는대부분혈액세포, 조직구를비롯한염증세포및기질세포 (stromal cell) 들이섞여있으며세포검사에서 불확실한 소견을보이는경우에는타겟세포의비율이 10% 이하인경우가흔하다. 따라서검사의정확도를높이기위해서는분자학적변화를확인하고자하는타겟세포를다른세포가섞이지않게채집하는것이중요하다. 저자의실험실에서는세포병리의사가세포학적진단을한후에펜을이용하여타겟세포를표시하고다이아몬드펜으로슬라이드뒷면에표시한후슬라이드를 xylene 용액에담가두었다가커버슬라이드를벗긴후슬라이드가마르지않고세포가잘보이도록 DNA 혹은 RNA 추출용액을한두방울떨어뜨리고광학현미경하에서 26-gauge 바늘로세포를긁어내어 DNA 혹은 RNA를추출한다. 점돌연변이검사를시행하는데필요한 DNA를확보하기위해서는 100개정도의세포를채집하는것이바람직하나경험에따르면 50개정도의세포로도가능하다. 유전자재배열검사를시행하기위한 RNA를확보하기위해서는 100개정도의세포가필요하다. 세침흡인세포검사검체중에서도세포학적으로 불확실한소견 으로진단되었을경우는타겟세포의수가충분하지않은경우가대부분이므로 DNA 혹은 RNA 소실을최대한막을수있도록하는것이무엇보다도중요하다. 저자의실험실에서는 ammonium sulfate DNA extraction 방법을사용하고있는데소량의검체에서 DNA를추출하는데효율적이다. 56) 추출이나전처리과정에서튜브의벽에묻는양을줄이기위해최대한작은튜브를이용하고필터링이나튜브를옮기는과정을최소화하는것도도움이된다. 결론 갑상선암은대부분갑상선결절로나타나게되는데실제갑상선결절의일부만이수술후갑상선암으로판명된다. 갑상선결절의양상을평가하는데가장중요한수단인초음파유도하-세침흡인세포검사 (ultrasonography guided fine needle aspiration cytology) 는임상소견이나영상소견에비하여상당히높은예측도 (predictive value) 를제공할수있는간편하고도안전한검사이다. 그러나이검사의 25% 정도에서갑상선암과양성결절성증식을감별하지못하는 불확실한 (indeter- 157 Int J Thyroidol
Tae Sook Hwang minate) 소견을보여재검사를하거나진단을위한수술이불가피하게된다. 최근형태학적변화에의거한진단의한계점을보완하여갑상선암을진단하는데도움을주고예후에영향을미치는요인들을찾아내고자분자유전학적인연구가활발하게진행되어약 90% 정도의갑상선암에서발생과진행에관여하는분자학적변화가확인되었으며, 이러한분자유전학적지식을통해암의조기진단과표적치료제의개발및예방이가능하게되었다. 이중에서도 BRAF와 RAS 유전자돌연변이가중요한역할을하며특히우리나라에서가장흔한유두암종과소포형종양에서상기유전자의돌연변이가흔히일어나는것이확인되어수술전세침흡인세포검사검체를대상으로한분자검사가널리이용되고있다. 갑상선결절에서세침흡인세포검사를시행하고그결과가 AUS/FLUS 혹은 FN/SFN으로보고된경우같은세침흡인검사검체를이용하여 BRAF V600E 돌연변이검사를시행하고돌연변이가확인되면수술로절제하고돌연변이가확인되지않는경우는 NRAS 61번코돈부위에대한돌연변이를확인하여치료방침을결정한다면갑상선암의발견과치료에도움이되리라생각한다. 중심단어 : 분자진단, 세침흡인세포검사, 불확실한결절, 갑상선암. References 1) http://www.cancer.go.kr [cited September 23, 2015] 2) Cibas ES, Ali SZ, Conference NCITFSotS. The Bethesda System for Reporting Thyroid Cytopathology. Am J Clin Pathol 2009;132(5):658-65. 3) Cooper DS, Doherty GM, Haugen BR, Kloos RT, Lee SL, Mandel SJ, et al. Management guidelines for patients with thyroid nodules and differentiated thyroid cancer. Thyroid 2006;16(2):109-42. 4) Greaves TS, Olvera M, Florentine BD, Raza AS, Cobb CJ, Tsao-Wei DD, et al. Follicular lesions of thyroid: a 5-year fine-needle aspiration experience. Cancer 2000;90(6):335-41. 5) Cibas ES, Ali SZ. The Bethesda System for Reporting Thyroid Cytopathology. Thyroid 2009;19(11):1159-65. 6) Baloch ZW, LiVolsi VA, Asa SL, Rosai J, Merino MJ, Randolph G, et al. Diagnostic terminology and morphologic criteria for cytologic diagnosis of thyroid lesions: a synopsis of the National Cancer Institute Thyroid Fine-Needle Aspiration State of the Science Conference. Diagn Cytopathol 2008;36(6):425-37. 7) Mazzaferri EL. Management of a solitary thyroid nodule. N Engl J Med 1993;328(8):553-9. 8) Bhaijee F, Nikiforov YE. Molecular analysis of thyroid tumors. Endocr Pathol 2011;22(3):126-33. 9) Kimura ET, Nikiforova MN, Zhu Z, Knauf JA, Nikiforov YE, Fagin JA. High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma. Cancer Res 2003;63(7):1454-7. 10) Xing M. BRAF mutation in thyroid cancer. Endocr Relat Cancer 2005;12(2):245-62. 11) Nikiforova MN, Kimura ET, Gandhi M, Biddinger PW, Knauf JA, Basolo F, et al. BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas. J Clin Endocrinol Metab 2003;88(11):5399-404. 12) Jung CK, Little MP, Lubin JH, Brenner AV, Wells SA Jr, Sigurdson AJ, et al. The increase in thyroid cancer incidence during the last four decades is accompanied by a high frequency of BRAF mutations and a sharp increase in RAS mutations. J Clin Endocrinol Metab 2014;99(2):E276-85. 13) Mathur A, Moses W, Rahbari R, Khanafshar E, Duh QY, Clark O, et al. Higher rate of BRAF mutation in papillary thyroid cancer over time: a single-institution study. Cancer 2011;117(19):4390-5. 14) Kim TY, Kim WB, Rhee YS, Song JY, Kim JM, Gong G, et al. The BRAF mutation is useful for prediction of clinical recurrence in low-risk patients with conventional papillary thyroid carcinoma. Clin Endocrinol (Oxf) 2006;65(3):364-8. 15) Chung KW, Yang SK, Lee GK, Kim EY, Kwon S, Lee SH, et al. Detection of BRAFV600E mutation on fine needle aspiration specimens of thyroid nodule refines cyto-pathology diagnosis, especially in BRAF600E mutation-prevalent area. Clin Endocrinol (Oxf) 2006;65(5):660-6. 16) Kim SK, Hwang TS, Yoo YB, Han HS, Kim DL, Song KH, et al. Surgical results of thyroid nodules according to a management guideline based on the BRAF(V600E) mutation status. J Clin Endocrinol Metab 2011;96(3):658-64. 17) Hong AR, Lim JA, Kim TH, Choi HS, Yoo WS, Min HS, et al. The frequency and clinical implications of the BRAF(V600E) mutation in papillary thyroid cancer patients in Korea over the past two decades. Endocrinol Metab (Seoul) 2014;29(4):505-13. 18) Ahn D, Park JS, Sohn JH, Kim JH, Park SK, Seo AN, et al. BRAFV600E mutation does not serve as a prognostic factor in Korean patients with papillary thyroid carcinoma. Auris Nasus Larynx 2012;39(2):198-203. 19) Nikiforov YE, Ohori NP. Papillary carcinoma. In: Nikiforov YE, Biddinger PW, Thompson LDR, editors. Diagnostic pathology and molecular genetics of the thyroid. 2nd ed. Philadelphia: Lippincott Williams and Wilkins; 2012. p.183-246. 20) Cho U, Oh WJ, Bae JS, Lee S, Lee YS, Park GS, et al. Clinicopathological features of rare BRAF mutations in Korean thyroid cancer patients. J Korean Med Sci 2014;29(8):1054-60. 21) Ciampi R, Nikiforov YE. Alterations of the BRAF gene in thyroid tumors. Endocr Pathol 2005;16(3):163-72. 22) Trovisco V, Vieira de Castro I, Soares P, Maximo V, Silva P, Magalhaes J, et al. BRAF mutations are associated with some histological types of papillary thyroid carcinoma. J Pathol 2004; Vol. 8, No. 2, 2015 158
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