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Usefulness of p16INK4a Immunocytochemical staining for the Differentiation between Atrophy and ASCUS in Diagnosis of Uterine Cervical Cancer
Biomed Sci Letters 2023;29:144-151
Published online September 30, 2023;  https://doi.org/10.15616/BSL.2023.29.3.144
© 2023 The Korean Society For Biomedical Laboratory Sciences.

Hye Ryoung Shin1,§,*, Taekil Eom2,§,** and Wan-Su Choi2,3,†,***

1Department of Smart Foods and Drugs Graduate School, Inje University, Gimhae 50834, Korea
2Department of Biomedical Laboratory Science, Inje University, Gimhae 50834, Korea
3Institute of Digital Anti-aging Healthcare, Inje University, Gimhae 50834, Korea
Correspondence to: Wan-Su Choi. Department of Biomedical Laboratory Science, Inje University, 197, Inje-ro, Gimhae-si, Gyeosangnam-do 50834, Korea.
Tel: +82-55-320-3481, Fax: +82-55-334-3426, e-mail: wansuchoi@inje.ac.kr
*Graduate student, **Researcher, ***Professor.
§Hye Ryoung Shin and Taekil Eom contributed equally.
Received September 6, 2023; Revised September 22, 2023; Accepted September 22, 2023.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 Abstract
A Pap smear is the most important screening test for the diagnosis of cervical cancer. However, subjective judgment by the operator cannot be excluded, and replicability may greatly be reduced if uncertain specimens are examined. Examiners often experience difficulties in differentiating atrophy with inflammatory changes and ASCUS when diagnosing squamous epithelial lesions from a pap smear. Reports often vary between cytologists and pathologists, and misdiagnosis may result in delayed follow-ups and advanced diseases. Hence, auxiliary examinations are necessary when confusing results between atrophy and ASCUS are obtained. The importance of p16INK4a activation due to HPV infection, which is an important factor in the outbreak of cervical cancer, has been highlighted. Recent studies have reported that p16INK4a immunocytochemical staining and HPV high-risk type tests using liquid-based cervical specimens are effective to detect the presence of lesions of grade HSIL or higher in patients with ASC-H. However, no research exists on the utility of HPV and p16INK4a tests on the differential diagnosis of atrophy and ASCUS. This study focused on whether p16INK4a immunocytochemical staining and HPV tests can help diagnose borderline lesions between atrophy and ASCUS. The results reported that p16INK4a activation can significantly (P<0.001) differentiate atrophy from ASCUS in atrophic lesions infected with High risk-HPV. Therefore, it may be concluded that p16INK4a immunocytochemical staining is an effective auxiliary test in lesions infected with HR-HPV when atrophic lesions are difficult to differentiate by morphology. Such results are expected to help decide on adequate follow-up and treatment.
Keywords : Bethesda system, Atrophy, ASCUS (atypical squamous cells of undermined significance), ASC-H (atypical squamous cells, excluded HSIL), HSIL (high grade squamous intraepithelial lesion)
꽌 濡

옄沅곴꼍遺븫 쟾 꽭怨꾩쟻쑝濡 몢 踰덉㎏濡 留롮씠 諛쒖깮븯硫 슦由щ굹씪 뿬꽦뿉꽌 쑀諛⑹븫, 媛묒긽꽑븫, 옣븫, 쐞븫, 룓븫, 媛꾩븫뿉 씠뼱 씪怨 踰덉㎏濡 諛쒖깮븯뒗 븫씠떎(Parkin et al., 1993). 씠윭븳 李⑥씠뒗 슦由щ굹씪쓽 寃쎌슦 援媛 븫 寃吏 議곌린 諛쒓껄 궗뾽 떆뻾쓣 넻빐 옄沅곸븫 諛쒖깮鍮덈룄瑜 以꾩씠뒗뜲 以묒텛쟻씤 뿭븷쓣 뻽湲 븣臾몄씠떎. 삉븳 꽭룷蹂묐━궗瑜 뼇꽦븯뿬 1李⑥쟻씤 꽑蹂꾧궗濡 옄沅곴꼍遺븫쓽 諛쒖깮瑜좉낵 洹몄뿉 뵲瑜 궗留앸쪧쓽 媛먯냼瑜 媛졇솕떎(Denny, 2005). 옄沅곴꼍遺븫 옄沅곴꼍遺 긽뵾궡 醫낆뼇(cervical intraepithelial neoplasia, CIN)씠 吏꾪뻾릺뼱 諛쒖깮릺湲 븣臾몄뿉 議곌린뿉 諛쒓껄븯뿬 移섎즺븯硫 븫쑝濡쒖쓽 吏꾪뻾쓣 삁諛⑺븷 닔 엳떎. 긽뵾궡 醫낆뼇 鍮꾩젙삎꽭룷媛 移⑤쾾븳 踰붿쐞뿉 뵲씪 3벑湲됱쑝濡 遺꾨쪟븳떎. 긽뵾궡 醫낆뼇 I (CIN 1) 씠븯뒗 씪諛섏쟻쑝濡 移섎즺 뾾씠 二쇨린쟻 寃吏꾩쓣 슂븯怨, 긽뵾궡 醫낆뼇 II (CIN 2), 긽뵾궡 醫낆뼇 III (CIN 3) 옄沅곴꼍遺 썝異 젅젣닠씠굹 깋룞 슂踰 벑쓣 넻빐 移섎즺븳떎(Rouzier, 2008). 洹몃윭誘濡 옄沅곴꼍遺 蹂묐뿉 븳 떊냽븯怨 젙솗븳 吏꾨떒 솚옄쓽 移섎즺 삁썑뿉 룄쓣 以 닔 엳떎.

옄沅곴꼍遺 긽뵾궡 醫낆뼇쓽 議곌린 諛쒓껄쓣 쐞빐 떎떆븯뒗 怨좎쟾쟻씤 옄沅곴꼍遺 꽭룷吏 寃궗諛⑸쾿씠 媛꾨떒븯怨 寃궗 鍮꾩슜룄 寃쎌젣쟻씤 씠쑀濡 옄沅곴꼍遺븫 꽑蹂꾧궗뿉 엳뼱 쑀슜븯寃 궗슜릺怨 엳쑝硫, 洹쇰옒뿉뒗 븸긽꽭룷寃궗踰(Liquid-based cytology)쓣 쟻슜븯뿬 옄沅곸꽭룷 룄留먮쾿쓽 쐞쓬꽦瑜좎쓣 以꾩씠뒗뜲 씪議고븯怨 엳떎. 洹몃윭굹 꽭룷븰쟻 寃궗뒗 二쇨쟻씤 꽦寃⑹쓣 셿쟾엳 諛곗젣븷 닔 뾾쑝硫 솗떎븳 媛먮퀎 諛 吏꾨떒씠 뼱젮슫 寃쎄퀎 쁺뿭쓽 利앸瑜 吏꾨떒븷 븣, 옱쁽꽦씠 겕寃 뼥뼱吏 닔 엳떎(Stoler and Schiffman, 2001; Lee et al., 2008). 씠윭븳 寃쎄퀎 쁺뿭쓣 Bethesada system뿉꽌뒗 鍮꾩젙삎 렪룊긽뵾(ASC-US) 怨좊벑湲됱쓣 諛곗젣븷 닔 뾾뒗 鍮꾩젙삎 긽뵾꽭룷(ASC-H)濡 遺꾨쪟븯떎. 씠 以 鍮꾩젙삎 렪룊긽뵾(ASC-US) 怨좊벑湲 렪룊 긽뵾궡 蹂묐(HSIL) 씠긽쑝濡 吏꾨떒릺뒗 鍮꾩쑉 11.6%씤뜲 諛섑빐, 怨좊벑湲됱쓣 諛곗젣븷 닔 뾾뒗 鍮꾩젙삎꽭룷(ASC-H)뿉꽌 吏꾨떒릺뒗 鍮꾩쑉 37.0~40.0%씠떎(Srodon et al., 2006; Sherman et al., 2001). 듅엳 쐞異뺤꽦(atrophic) 긽뵾꽭룷 솕깮긽뵾꽭룷뒗 怨좊벑湲 렪룊 긽뵾궡 蹂묐꽭룷 媛먮퀎吏꾨떒씠 뼱젮슱 닔 엳떎怨 蹂닿퀬릺뿀쑝硫(Nucci et al., 2003) 떎젣 엫긽뿉꽌 쐞異뺤꽦 蹂묐(atrophy)쓣 쁽誘멸꼍쑝濡 寃寃쏀븷 븣 꽭룷 援곗쭛쓽 以묒꺽 쁽긽, 꽭룷 룄留 怨쇱젙뿉꽌 빑쓽 쐞異뺤꽦 蹂솕, 닔蹂, 뿼利앹꽦 씠삎꽦, 誘몄꽦닕 솕깮 벑쑝濡 씤빐 鍮꾩젙삎 렪룊긽뵾(ASC-US) 媛먮퀎 吏꾨떒뿉 留롮 뼱젮쓣 寃り퀬 엳떎.

씤쑀몢醫낅컮씠윭뒪(human papilloma virus, HPV) 媛먯뿼 옄沅곴꼍遺븫쓣 씪쑝궎뒗 二쇱슂 썝씤쑝濡 븣젮졇 엳쑝硫, 以묓빀슚냼뿰뇙諛섏쓳(polymerase chain reaction, PCR)怨 媛숈 遺꾩옄 吏꾨떒踰뺤쓣 넻븳 HPV 寃異 뿬遺뒗 옄沅곴꼍遺븫 吏꾨떒 떆 蹂댁“쟻 吏몴濡 留롮씠 솢슜릺怨 엳떎(Lim et al., 2008). HPV뒗 쁽옱源뚯 100뿬 媛吏媛 꽆뒗 醫낅쪟媛 諛쒓껄릺뿀쑝硫 쐞뿕援(low-risk, LR)-HPV 怨좎쐞뿕援(high risk, HR)-HPV쑝濡 굹돇뼱 엳떎. 듅엳, 옄沅곴꼍遺븫뿉꽌 怨좎쐞뿕援 씤쑀몢醫낅컮씠윭뒪뒗 99% 씠긽 諛쒓껄릺怨 엳떎(Bibbo et al., 2002). 怨좎쐞뿕援 씤쑀몢醫낅컮씠윭뒪뒗 닕二쇱꽭룷쓽 쑀쟾옄뿉 궫엯릺뼱 E6, E7 諛쒖븫 떒諛깆쭏쓣 諛쒗쁽븯뒗뜲, E6뒗 p53 醫낆뼇뼲젣떒諛깆쭏쓽 湲곕뒫쓣 뼲젣븯怨, E7 retinoblastoma (Rb) 떒諛깆쭏쓣 鍮꾪솢꽦솕 떆耳, 꽭룷二쇨린瑜 G1 phase뿉꽌 S phase濡쒖쓽 씠뻾쓣 뼲젣븯吏 紐삵븯怨 꽭룷媛 臾댄븳 利앹떇븯寃 留뚮뱺떎(Mu챰oz et al., 2003: Keating et al., 2001).

p16INK4a 꽭룷二쇨린 議곗젅씤옄濡 G1 phase뿉꽌 S phase濡 씠룞쓣 議곗젅븳떎(Sano et al., 1998). p16INK4a Cyclin D Cyclin-dependent kinase (CDK)쓽 蹂듯빀泥 삎꽦쓣 諛⑺빐븯뿬 Rb 떒諛깆쭏쓽 씤궛솕瑜 뼲젣븯뒗 쑀쟾옄濡, 씤궛솕 릺吏 븡 Rb뒗 E2F씪뒗 쟾궗씤옄(transcription factor) 븞젙븳 蹂듯빀泥대 씠猷④퀬 엳떎. 씠윭븳 蹂듯빀泥대뒗 꽭룷遺꾩뿴怨 꽦옣뿉 븘슂븳 쑀쟾옄 쟾궗瑜 옞젙쟻쑝濡 뼲젣븯怨 꽭룷二쇨린瑜 뼲젣븳떎. 洹몃윭굹 Rb媛 씤궛솕 릺硫 쟾궗씤옄씤 E2F媛 솢꽦솕릺뼱 꽭룷利앹떇뿉 븘슂븳 떒諛깆쭏뱾쓽 빀꽦쓣 쑀룄븯怨, G1 phase뿉꽌 S phase濡쒖쓽 씠뻾쓣 議곗젅븳떎(Safwan-Zaiter and Wagner, 2022). HPV쓽 E7 諛쒖븫 떒諛깆쭏씠 Rb 寃고빀븯硫 E2F뒗 Rb濡쒕꽣 吏냽쟻쑝濡 쑀由щ릺뼱 G1 phase뿉꽌 S phase濡쒖쓽 씠뻾뿉 愿젴맂 쑀쟾옄 諛쒗쁽씠 珥됱쭊맂떎(Bringold and Serrano, 2000; Yoshida et al., 1995; Igaki et al., 1995).

p16INK4a HPV쓽 E7 諛쒖븫 떒諛깆쭏뿉 쓽빐 쓬꽦 릺癒뱀엫 湲곗쟾뿉 쓽빐 諛쒗쁽씠 利앷릺怨, 硫댁뿭꽭룷솕븰뿼깋 寃곌낵뿉꽌 諛쒗쁽씠 利앷맂 p16INK4a HPV 吏꾨떒뿉 룄쓣 以떎(Sano et al., 1998). 삉븳 꽭룷 궡 p16INK4a쓽 諛쒗쁽 뼇긽 HPV쓽 議댁옱 뿬遺瑜 꽆뼱꽌 HPV쓽 솢룞꽦쓣 룊媛븷 닔 엳뒗 吏몴濡 솢슜맂떎(Stanley, 2002). 理쒓렐 옄沅곴꼍遺븫 諛쒖깮 怨쇱젙뿉꽌 二쇱슂 썝씤씤 HPV 媛먯뿼뿉 뵲瑜 p16INK4a쓽 諛쒗쁽쓽 以묒슂꽦씠 遺媛곷릺硫댁꽌 옄沅곴꼍遺 븸긽꽭룷 寃泥대줈 怨좎쐞뿕援 HPV 媛먯뿼 뿬遺 寃궗 p16INK4a 硫댁뿭꽭룷솕븰뿼깋씠 ASC-H 씠긽쓽 蹂묒냼 쑀臾대 삁痢≫븯뒗뜲 룄씠 맂떎뒗 蹂닿퀬媛 엳怨, 씠 뿰援щ뱾 CIN2 씠긽쓽 利앸뿉꽌 p16INK4a 怨쇰컻쁽씠 吏꾨떒뿉 룄쓣 以 닔 엳떎怨 젣떆븳떎(Keating et al., 2001; Klaes et al., 2002; Kim et al., 2003). 洹몃윭굹 삎깭븰쟻 吏꾨떒씠 뼱젮슫 쐞異뺤꽦 蹂묐뿉꽌 ASC-US쓽 媛먮퀎뿉 븳 p16INK4a 硫댁뿭꽭룷솕븰뿼깋씠 엫긽뿉꽌 吏꾨떒뿉 룄쓣 以 닔 엳뒗吏뿉 븳 뿰援щ뒗 蹂닿퀬릺吏 븡怨 엳뒗 떎젙씠떎. 씠뿉 蹂 뿰援ъ뿉꽌뒗 옄沅곴꼍遺 븸긽꽭룷寃궗뿉꽌 꽭룷븰쟻쑝濡 媛먮퀎씠 뼱젮슫 쐞異뺤꽦 蹂묐 ASC-US쓽 寃쎄퀎 쁺뿭쓣 吏꾨떒븯뒗뜲 p16INK4a 硫댁뿭꽭룷솕븰뿼깋쓽 엫긽쟻 쑀슜꽦뿉 빐 븣븘蹂닿퀬, HPV 媛먯뿼쓽 뿰愿꽦뿉 빐 諛앺엳怨좎옄 븯떎.

옱猷 諛 諛⑸쾿

뿰援 긽

2019뀈 1썡遺꽣 2021뀈 12썡源뚯 李쎌썝븳留덉쓬蹂묒썝 궛遺씤怨쇱뿉꽌 옄沅곴꼍遺 븸긽꽭룷寃궗瑜 諛쏄퀬 atrophy 吏꾨떒쓣 諛쏆 100紐낆쓣 긽쑝濡 븯떎.

옄沅곴꼍遺 븸긽꽭룷寃궗

옄沅곴꼍遺쓽 븸긽꽭룷寃궗뒗 Surepath (Tripath imaging, USA) 궗슜븯뿬 Papanicolaou stain쓣 떎떆븯뿬 寃궗븯떎. 뿰援 긽 솚옄쓽 議곗쭅 깦뵆뿉꽌 옄沅곴꼍遺븫씠 옒 諛쒖깮븯뒗 遺쐞씤 transitional zone뿉꽌 brush瑜 씠슜븯뿬 씤쐞쟻쑝濡 깉씫맂 꽭룷 寃泥대 뼸 떎쓬, brush 遺遺꾩쓣 遺꾨━븯怨 꽭룷쓽 蹂꽦쓣 諛⑹븯湲 쐞빐 怨좎젙븸씠 泥④맂 SurePath 쟾슜 슜湲곗뿉 꽔뼱 븸긽룄留 꽭룷寃궗瑜 떆뻾븯떎.

寃泥댁쓽 洹좎쭏솕瑜 쐞븯뿬 Muti-Vial Vortexer瑜 씠슜븯뿬 3,000 rpm쑝濡 50珥덇컙 洹좎쭏솕 떆궓 떎쓬, PreMate Auto-mated accessory (TriPath Imaging, USA)瑜 씠슜븯뿬 Prestain density reagent媛 떞湲 썝떖 뒠釉뚮줈 삷寃⑥죱떎. 洹몃━怨 鍮꾩쨷븸쓣 꽔뼱 寃泥댁쓽 꽭룷 諛룄瑜 넂씠怨 썝떖遺꾨━瑜 2쉶 떆뻾븳 뮘 꽭룷쓽 移⑥궗痢듭쓣 뼸뿀떎. 씠긽쓽 쟾泥섎━ 怨쇱젙쓣 留덉튇 寃泥대뒗 PreStain chamber濡 삷寃⑥졇 肄뷀똿 뒳씪씠뱶濡 移⑥쟾떆궓 썑, 옄룞뿼깋옣鍮꾩씤 AutostainerXL (Leica, Germany)濡 papanicolaou 뿼깋쓣 떆뻾븯떎. 빑 뿼깋 Harris Hematoxylin쑝濡 3遺 뿼깋븳 썑, washing븳 떎쓬 0.02% HCl濡 빑쓣 깉깋븯怨, 꽭룷吏 뿼깋 OG6 EA50 슜븸뿉 媛 1遺꾧컙 떎떆븯떎. 洹몃━怨 뿼깋씠 셿猷뚮맂 뒳씪씠뱶뒗 옄룞 遊됱엯湲곗 遊됱엯젣(Synthetic Mountant, Shandon, USA)瑜 궗슜븯뿬 뒳씪씠뱶瑜 젣옉븳 썑 Aperio AT2 뒳씪씠뱶 뒪罹먮꼫(Leica, Germany)瑜 씠슜븯뿬 뒪罹뷀븯떎. 꽭룷寃궗쓽 吏꾨떒 꽭怨꾪몴以踰뺤씤 Bethesda system쓣 넻빐 寃곌낵瑜 蹂닿퀬븯怨 엳떎.

Real-time Polymerase Chain reaction (RT-PCR)쓣 씠슜븳 HPV 媛먯뿼 뿬遺 寃궗

HPV 媛먯뿼 寃궗뒗 쇅옒 寃吏 怨쇱젙뿉꽌 transitional zone뿉꽌 梨꾩랬맂 꽭룷 寃泥대 씠슜븯뿬 real-time PCR쓣 떆뻾븯뿬 媛먯뿼 뿬遺瑜 痢≪젙븯쑝硫, 떎쓬怨 媛숈 怨쇱젙쓣 嫄곗퀜꽌 痢≪젙븯떎. 슦꽑 寃泥댁뿉꽌 NimBUS 쟾泥섎━ 옣鍮꾨줈 DNA瑜 異붿텧븯怨, Anyplex II HPV 28 Detection kit (Seegene, Korea)瑜 씠슜븯뿬 CFX96 Real-time thermocycler (Bio-Rad, USA)瑜 씠슜븯뿬 PCR쓣 吏꾪뻾븯떎. Anyplex II HPV 28 Detection kit뒗 몢 媛쒖쓽 primer set瑜 씠슜븯뿬 珥 28媛吏쓽 HPV gene type쓣 19媛吏쓽 HR-HPV type 9媛吏쓽 LR-HPV type쑝濡 젙꽦 諛 젙웾 遺꾩꽍븷 닔 엳떎.

옄沅곴꼍遺 븸긽꽭룷 p16INK4a Immunocytochemistry (IHC)

100紐낆쓽 atrophy 吏꾨떒씠 굹삩 솚옄쓽 꽭룷 뒳씪씠뱶瑜 0.05% HCl뿉 30遺 깉깋 썑, xylene-100%, alcohol-95% 諛 alcohol 怨쇱젙쓣 嫄곗퀜 닔꽭瑜 30遺 븳 썑 뒳씪씠뱶瑜 셿쟾엳 깉깋떆耳곕떎. IHC뿉 궗슜릺뼱吏 p16INK4a 빆泥대뒗 Roche Diagnostict (Rotkreuz, Switzerland)궗쓽 빆泥대 씠슜븯쑝硫, 硫댁뿭뿼깋寃궗옣鍮(Bond III, Germany)瑜 궗슜븯뿬 뿼깋쓣 떎떆븯떎. IHC쓽 怨쇱젙 빆썝蹂듦뎄, Primary antibody, Envision/HRP, Substrate-Chromogen (DAB) solution瑜 10遺꾧컙 쟻슜떆궓 썑, Mayer hematoxylin쑝濡 議곗뿼깋쓣 1遺꾧컙 븯쑝硫, p16INK4a 硫댁뿭뿼깋寃곌낵뒗 醫낆뼇꽭룷쓽 빑怨 꽭룷吏덉뿉 媛덉깋쑝濡 뿼깋릺뒗 긽뵾꽭룷瑜 꽭뼱 뙋룆븯떎.

옄沅곴꼍遺議곗쭅 p16INK4a Immunocytochemistry

옄沅곴꼍遺議곗쭅쓣 10% Neutral Buffered Formalin (NBF)쑝濡 怨좎젙븳 썑, 옄룞議곗쭅泥섎━湲(Sakura, Tissue Tec-5 VIP)瑜 씠슜븯뿬 깉닔, 닾紐, 移⑦닾 怨쇱젙쓣 嫄곗튇 썑, 뙆씪 釉붾줉쓣 留뚮뱾뿀떎. 씠썑, 4 關m 몢猿섎줈 諛뺤젅븳 떎쓬 옄룞硫댁뿭寃궗湲곌린(Bondmax, Germany)瑜 궗슜븯뿬 뿼깋쓣 떎떆븯떎. p16INK4a 빆泥(Roche, RTU)瑜 씠슜븯쑝硫, 硫댁뿭뿼깋寃궗옣鍮(Bond III, Germany)瑜 궗슜븯뿬 뿼깋쓣 떎떆븯떎. IHC쓽 怨쇱젙 빆썝蹂듦뎄, Primary antibody, Envision/HRP, Substrate-Chromogen (DAB) solution瑜 10遺꾧컙 쟻슜떆궓 썑, Mayer hematoxylin쑝濡 議곗뿼깋쓣 1遺꾧컙 븯쑝硫, p16INK4a 硫댁뿭뿼깋寃곌낵뒗 醫낆뼇꽭룷쓽 빑怨 꽭룷吏덉뿉 媛덉깋쑝濡 뿼깋릺뒗 긽뵾꽭룷瑜 꽭뼱 뙋룆븯떎.

옄沅곴꼍遺議곗쭅 H&E 뿼깋

옄沅곴꼍遺 punch 깮寃 議곗쭅쓣 10% NBF濡 怨좎젙븳 썑, 옄룞議곗쭅泥섎━湲(Sakura, Tissue Tec-5 VIP)瑜 씠슜븯뿬 깉닔, 닾紐, 移⑦닾 怨쇱젙쓣 嫄곗튇 떎쓬 뙆씪 釉붾줉쓣 留뚮뱾뼱 Histocore Multicut 諛뺤젅湲(Leica, Germany)濡 4 關m濡 諛뺤젅븯떎. 洹몃━怨 Tissue Tec Plasma plus 옄룞뿼깋湲(Sakura, Japan)瑜 씠슜븯뿬 Hematoxylin & Eosin (Leica, Germany)쑝濡 뿼깋븯怨, Synthetic Mountant (Leica, Germany)쑝濡 遊됱엯븯떎.

넻怨 遺꾩꽍

SPSSWIN 25.0 봽濡쒓렇옩쓣 궗슜븯뿬 援먯감遺꾩꽍(chi-square test) 2 (P)쓣 떎떆븯뿬 吏묐떒 媛꾩쓽 李⑥씠瑜 궡렣蹂댁븯怨 쑀쓽닔以 P뒗 0.05 誘몃쭔쑝濡 븯떎.

寃 怨

옄沅곴꼍遺 븸긽꽭룷寃궗 吏꾨떒

옄沅곴꼍遺 븸긽룄留 꽭룷寃궗뿉꽌 atrophy 諛 ASCUS쓽 媛먮퀎 吏꾨떒씠 쁽誘멸꼍쓣 씠슜븳 뙋떒뿉꽌 삎깭븰쟻쑝濡 atrophy 諛 ASCUS쓽 뙋떒씠 뼱젮슫 븸긽꽭룷 뒳씪씠뱶瑜 꽭룷蹂묐━궗 蹂묐━쓽궗쓽 援먯감 뙋룆븯뿬 理쒖쥌쟻쑝濡 atrophy濡 吏꾨떒맂 100紐낆쓽 솚옄쓽 寃泥대 떎뿕 긽쑝濡 븯떎. Fig. 1뿉꽌 굹궡뒗 諛붿 媛숈씠 쁽誘멸꼍긽쑝濡 hyperchromatic cluster쓽 빑 궡뿉 chromatin쓽 뼇긽怨 빑쓽 겕湲곗 삎깭媛 ASCUS 媛먮퀎븯湲곗뿉 뼱젮씠 엳뿀쑝굹 理쒖쥌쟻쑝濡 atrophy濡 뙋떒릺뿀떎.

Fig. 1. Representative images of papanicolaou staining in cervical liquid cytology diagnosed with atrophy.
(A) Atrophic cluster in which intranuclear chromatin is difficult to distinguish (B) Hyperstained atrophic cluster. (C) Irregularity of the marginal nucleus of the atrophic cluster. (D) Atrophic cluster with enlarged nucleus. (E) Overlapping nucleus atrophic cluster. (F) Atrophic cluster of irregular nuclear membrane. Original magnification: 횞200. scale bar = 100 關m.

Atrophy뿉꽌 p16INK4a쓽 諛쒗쁽

Atrophy濡 뙋떒맂 옄沅곴꼍遺 븸긽꽭룷 寃泥대 씠슜븯뿬 0.1% HCl濡 깉깋 怨쇱젙쓣 嫄곗튇 썑, p16INK4a 빆泥대 씠슜븯뿬 硫댁뿭꽭룷솕븰뿼깋쓣 떎떆븯떎(Fig. 2). p16INK4a쓽 諛쒗쁽 빑怨 꽭룷吏덉뿉꽌 媛덉깋쑝濡 뿼깋맂 긽뵾꽭룷쓽 닔瑜 쁽誘멸꼍(100횞)뿉꽌 꽭뼱 10媛 씠긽씪 븣 p16INK4a 怨쇰컻쁽쑝濡 媛꾩<븯뿬 뼇꽦쑝濡 뙋룆븯怨, 꽭룷媛 쟾 뿼깋릺吏 븡븯嫄곕굹, 뿼깋씠 릺뜑씪룄 뿼깋 媛뺣룄媛 빟븳 꽭룷留 愿李곕릺嫄곕굹 10媛 誘몃쭔씪 븣 쓬꽦쑝濡 뙋룆븯쑝硫 Table 1뿉 굹궡뿀떎. 洹 寃곌낵, atrophy瑜 굹궡뒗 100媛쒖쓽 寃泥댁뿉꽌 p16INK4 諛쒗쁽씠 愿痢〓릺뒗 뼇꽦쓽 寃泥대뒗 35媛쒕줈 굹궗쑝硫, 굹癒몄 65媛쒖쓽 寃泥댁뿉꽌뒗 p16INK4 諛쒗쁽씠 愿痢〓릺吏 븡쓬쓣 굹궡뿀떎.

Fig. 2. Representative images of p16INK4a immunostaining in cervical liquid cytology.
(A)~(D) Atrophic cluster p16INK4a expression infected with high-risk HPV. (E, F) Atrophic cluster p16INK4a expression infected with low-risk HPV. (G, H) Atrophic cluster p16INK4a expression without HPV infection. Original magnification: 횞200. scale bar = 100 關m.

Proportion positive specimens for p16INK4a expression in liquid-based cytology samples of cervical tissue using immunohistochemical staining methods

Cytology diagnosis No. of cases Cytology p16INK4a
Negative (%) Positive (%)
Atrophy 100 65 (65%) 35 (35%)


Atrophy 寃泥댁뿉꽌 HPV 媛먯뿼怨 p16INK4a 諛쒗쁽怨쇱쓽 긽愿 愿怨

Atrophy濡 吏꾨떒맂 100媛쒖쓽 寃泥댁뿉꽌 transitional zone뿉꽌 DNA瑜 梨꾩랬븯뿬 HPV 媛먯뿼긽깭瑜 솗씤븯湲 쐞븯뿬 HPV뿉 꽑깮쟻쑝濡 利앺룺씠 릺뒗 primer瑜 궗슜븯뿬 Real-Time PCR 湲곌린瑜 씠슜븯뿬 PCR 諛섏쓳쓣 吏꾪뻾븯뿬 HPV 媛먯뿼 뿬遺瑜 뙋떒븯떎(Table 2). HPV 媛먯뿼쓣 솗씤븳 寃곌낵 p16INK4a씠 諛쒗쁽씠 愿痢〓릺뒗 35媛쒖쓽 寃泥 以묒뿉꽌 以 HR(high risk, 怨좎쐞뿕援) HPV 媛먯뿼뿉 븳 뼇꽦쓣 굹궡뒗 寃泥대뒗 32媛쒖쓽 寃泥(84.3%)濡 굹궗쑝硫, 3媛쒖쓽 寃泥(15%)뿉꽌뒗 LR(low risk, 쐞뿕援) HPV 媛먯뿼뿉 븳 뼇꽦쓣 굹궡뿀떎. p16INK4a씠 諛쒗쁽씠 愿痢〓릺뒗 寃泥댁뿉꽌 HPV뿉 빐꽌 媛먯뿼씠 굹굹吏 븡뒗 寃泥대뒗 議댁옱븯吏 븡쓬쓣 솗씤븯떎.

Comparative analysis of HPV infection and p16INK4a expression in atrophic lesions from liquid-based cytology specimens of cervical tissue

p16INK4a expression Total
Positive (%) Negative (%)
Atrophy HR-HPV 32 (84.2%) 6 (15.8%) 38
LR-HPV 3 (15%) 17 (85%) 20
No-HPV 0 42 (100%) 42

HR, High risk; LR, Low risk



p16INK4a씠 諛쒗쁽릺吏 븡 65媛쒖쓽 寃泥댁뿉꽌 HR-HPV 뼇꽦쓣 굹궡뒗 寃泥대뒗 3媛쒖쓽 寃泥(15.8%)뿉꽌 굹궡뿀쑝硫, LR-HPV 뼇꽦 17媛쒖쓽 寃泥(85%)뿉꽌 굹궡뿀떎. p16INK4a쓽 諛쒗쁽씠 愿痢〓릺뒗 寃泥댁뒗 떎瑜닿쾶 p16INK4a쓽 諛쒗쁽씠 굹굹吏 븡뒗 寃泥댁뿉꽌뒗 42媛쒖쓽 寃泥댁뿉꽌 HPV뿉 媛먯뿼씠 릺吏 븡쓬쓣 굹궡뿀떎. 씠 媛숈씠 HR-HPV뿉 媛먯뿼쓣 굹궡뒗 寃泥댁뿉꽌뒗 p16INK4a쓽 諛쒗쁽 鍮꾩쑉씠 넂寃 굹궗쑝硫, HPV뿉 媛먯뿼릺吏 븡 寃泥대뒗 p16INK4a쓽 諛쒗쁽씠 愿痢〓릺吏 븡쓬쓣 굹궡뿀떎. 뵲씪꽌 HR-HPV 媛먯뿼씠 솗씤맂 atrophy뿉꽌 p16INK4a 諛쒗쁽 꽌濡 諛젒븳 긽愿愿怨(P<0.001)瑜 굹궗쓬쓣 븣 닔 엳떎.

HR-HPV 媛먯뿼맂 옄沅곴꼍遺議곗쭅쓽 p16INK4a 諛쒗쁽

HR-HPV뿉 媛먯뿼릺뼱 엳쑝硫댁꽌 atrophy瑜 媛吏뒗 솚옄쓽 옄沅곴꼍遺뿉꽌 뼸뼱吏 15媛쒖쓽 寃泥대 씠슜븯뿬 옄沅곴꼍遺議곗쭅뿉꽌 p16INK4a 諛쒗쁽쓣 硫댁뿭議곗쭅솕븰뿼깋瑜 넻븯뿬 遺꾩꽍븯떎(Fig. 3). 議곗쭅寃궗 吏꾨떒 寃곌낵 Flat condyloma뒗 13媛쒖쓽 寃泥(86.7%)뿉꽌 굹궗쑝硫, 留뚯꽦 寃쎈뿼(chronic cervicitis) 2媛 寃泥(13.3%)뿉꽌 諛쒓껄릺뿀떎. 씠 以묒뿉꽌, 13媛쒖쓽 Flat condyloma瑜 굹궡뒗 寃泥댁뿉꽌 紐⑤몢 p16INK4a 諛쒗쁽씠 굹궓쓣 솗씤븯쑝硫, 2媛쒖쓽 留뚯꽦 寃쎈뿼쑝濡 吏꾨떒맂 寃泥댁뿉꽌뒗 p16INK4a 諛쒗쁽씠 굹굹吏 븡븯떎.

Fig. 3. Representative images of hematoxylin/eosin and immunostaining of p16INK4a expression in atrophic cervical tissue.
A, D, G: Hematoxylin & Eosin staining in atrophic cervical tissue infected in high-risk groups. B, C, E, F, H, I: p16INK4a expression in high-risk infected atrophic cervical tissue. Original magnification: 횞100. scale bar = 200 關m.
怨 李

蹂 뿰援ъ뿉꽌뒗 옄沅곴꼍遺븫 寃궗뿉꽌 룓寃 씠썑 뿬꽦쓽 atrophy ASCUS瑜 삎깭븰쟻 꽭룷쓽 듅吏뺣쭔쑝濡 援щ퀎븯뿬 媛먮퀎 吏꾨떒븯湲 뼱젮슫 寃쎌슦 p16INK4a 硫댁뿭솕븰꽭룷뿼깋寃궗瑜 떆뻾븯뿬 꽭룷寃궗쓽 吏꾨떒쓣 蹂댁셿븯湲 쐞븳 寃궗踰뺤쑝濡 쑀슜꽦쓣 솗씤븯떎. Atrophy뿉꽌 怨쇱뿼깋 릺뼱吏 꽭룷 援곗쭛뱾 빑쓽 chromatin 愿李곗씠 뼱졄怨, 븸긽룄留 寃궗踰뺤쓽 怨쇱젙뿉꽌 빑쓽 紐⑥뼇씠 異뺤냼릺뼱 옄沅곴꼍遺쓽 씠긽냼寃ъ쓣 諛쒓껄븯吏 紐삵븯뒗 寃쎌슦媛 諛쒖깮맂떎. 뵲씪꽌 솚옄媛 異붿쟻 寃궗쓽 떆湲곕 넃爾 옄沅곴꼍遺븫쓽 以묒쬆룄媛 吏꾪뻾맂 썑 諛쒓껄맆 닔룄 엳湲 븣臾몄뿉 atrophy怨 ASCUS쓽 媛먮퀎씠 紐⑦샇븳 寃쎌슦 吏꾨떒뿉 蹂댁“쟻씤 寃궗쓽 븘슂꽦쓣 뒓겮怨 엳뒗 떎젙씠떎. 옄沅곴꼍遺븫쓽 젙솗븳 吏꾨떒쓣 쐞빐 꽭룷寃궗 蹂묓뻾븯뿬 寃궗 媛뒫븳 諛붿씠삤 留덉빱뱾뿉 뿰援щ뱾씠 吏꾪뻾릺怨 엳쑝硫 떎뼇븳 諛붿씠삤 留덉빱뱾씠 젣떆릺怨 엳떎. 쐞異뺤꽦 긽뵾꽭룷 誘몄꽦닕 솕깮긽뵾꽭룷뒗 怨좊벑湲 렪룊긽뵾궡 蹂묐꽭룷 꽭룷븰쟻쑝濡 媛먮퀎씠 뼱젮슫 寃쎌슦 꽭룷二쇨린뿉꽌 諛쒗쁽릺뒗 꽭룷쓽 利앹떇뒫쓣 諛섏쁺븯뒗 꽭룷利앹떇쓽 몴吏吏닔 Ki-67 硫댁뿭議곗쭅솕븰뿼깋踰뺤씠 젙솗븳 吏꾨떒뿉 룄씠 맂떎뒗 蹂닿퀬媛 엳떎(Keating et al., 2001). 삉븳 떎뼇븳 젙룄쓽 옄沅곴꼍遺 긽뵾궡 醫낆뼇 솚옄뱾쓣 긽쑝濡 꽭룷二쇨린瑜 Ki-67, p53 諛 p63 諛쒗쁽쓣 硫댁뿭뿼깋쓣 넻븯뿬 鍮꾧탳 遺꾩꽍븳 寃곌낵 CIN II 諛 CIN I뿉 鍮꾪빐 CIN III 솚옄쓽 씠뱾 諛붿씠삤 留덉빱 諛쒗쁽씠 쑀쓽븯寃 利앷븯뒗 寃껋쑝濡 諛앺議뚮떎. 씠 쇅뿉룄 CIN I뿉꽌뒗 p53怨 p63쓽 諛쒗쁽씠 議곌뎔뿉 鍮꾪빐 썾뵮 뜑 紐낇솗엳 利앷븯뿬, CIN I쓽 諛섏쓳꽦 蹂솕瑜 援щ퀎븷 닔 엳떎怨 꽕紐낇븯떎(Mitildzans et al., 2016). 삉븳 Ki-67 쟾븫꽦 蹂묐 諛 옄沅곴꼍遺븫뿉꽌 Ki-67, p16INK4a 諛 p53쓽 硫댁뿭議곗쭅솕븰쟻 諛쒗쁽 룊媛뿉 뿰援ъ뿉꽌뒗 젙긽쟻씤 옄沅곴꼍遺 긽뵾뿉꽌뒗 理쒖냼 p16INK4a 諛 Ki-67쓽 諛쒗쁽씠 굹굹吏 븡嫄곕굹 븘二 쟻 諛쒗쁽쓣 蹂댁뿬二쇰굹, 蹂묐씠 떖븳 議곗쭅뿉꽌뒗 븘二 넂 諛쒗쁽쓣 굹궡怨 엳떎怨 蹂닿퀬븯떎(Silva et al., 2017). 씠긽뿉꽌 媛숈씠 꽭룷二쇨린瑜 議곗젅븯뒗 떎뼇븳 떒諛깆쭏뱾쓣 씠슜븯뿬 옄沅곴꼍遺븫 吏꾨떒뿉 蹂댁“쟻쑝濡 솢슜 媛뒫븿씠 留롮 뿰援 寃곌낵뱾濡 젣떆릺뼱吏怨 엳떎.

理쒓렐 뱾뼱 옄沅곴꼍遺븫 寃궗뿉꽌 p16INK4a 諛쒗쁽뿉 븳 꽑뻾뿰援ъ뿉꽌, 議곗쭅븰쟻 吏꾨떒쓽 grade媛 넂븘吏덉닔濡 諛쒗쁽쑉씠 利앷븯뿬 옄沅곴꼍遺 궡쓽 蹂묐쓣 媛먮퀎븯뒗 寃궗諛⑸쾿쑝濡 쑀슜븯떎怨 蹂닿퀬븯떎(Byun et al., 2013; Dona et al., 2012; Schmidt et al., 2011). 삉븳, HPV뿉 媛먯뿼맂 옄沅곴꼍遺 蹂묐뿉꽌 p16INK4씠 諛쒗쁽씠 利앷맂떎怨 뿰援щ릺뼱졇 엳쑝硫(Klaes et al., 2001; Keating et al., 2001), p16INK4쓽 怨쇰컻쁽 HPV쓽 媛먯뿼怨 룞떆뿉 솢룞쟻씤 긽깭뿉 議댁옱븿쓣 쓽誘명븳떎怨 븣젮졇 엳떎(Stanley, 2002).

씠뿉 蹂 뿰援ъ뿉꽌뒗 옄沅곴꼍遺븫 寃궗 솚옄瑜 긽쑝濡 븸긽꽭룷寃궗瑜 떎떆븯뿬 뒳씪씠뱶몴蹂몄쓣 젣옉븯怨 꽭룷븰쟻 吏꾨떒씠 뼱젮슫 atrophy뿉꽌 ASCUS瑜 媛먮퀎븯湲 쐞븳 蹂댁“寃궗濡 p16INK4a 硫댁뿭꽭룷솕븰寃궗媛 엫긽쟻쑝濡 쑀슜븳吏 븣븘蹂닿퀬, HPV뿉 쓽빐 옄沅곴꼍遺븫쓽 븫솕 怨쇱젙뿉꽌 HPV E6 諛 E7 떒諛깆쭏씠 p16INK4a쓽 怨쇰컻쁽怨쇱쓽 뿰愿꽦뿉 빐 뿰援щ 븯떎. Atrophy濡 吏꾨떒씠 굹媛 100紐낆쓣 긽쑝濡 p16INK4a 硫댁뿭꽭룷솕븰뿼깋쓣 떎떆븳 寃곌낵 p16INK4a 諛쒗쁽쓽 뼇꽦씠 23嫄(23%), 쓬꽦씠 65嫄(65%)濡 議곗궗릺뿀떎(Fig. 2, Table 1). 洹몃━怨 atrophy쑝濡 吏꾨떒맂 100紐낆쓽 紐⑤몢 HPV 媛먯뿼뿉 븳 Real time PCR쓣 떎떆븳 寃곌낵 p16INK4a 諛쒗쁽 뼇꽦뿉꽌 HR-HPV 뼇꽦씠 38嫄(84.2%) 굹솕怨, LR-HPV 뼇꽦씠 3嫄(15%), No HPV infection 0嫄(0%)濡 굹궗떎(Table 2). p16INK4a 諛쒗쁽 쓬꽦뿉꽌뒗 HR-HPV 뼇꽦씠 6嫄(15.8%), LR-HPV 뼇꽦씠 17嫄(85%), No HPV infection 42嫄(100%)濡 굹궗떎. 씠뒗 HPV 媛먯뿼 떆 HR-HPV 뼇꽦씪 寃쎌슦 p16INK4a 諛쒗쁽씠 利앷븯쑝硫 씠뒗 씠쟾 뿰援щ뱾怨 룞씪븳 寃곌낵瑜 蹂댁씤떎(Klaes et al., 2001; Sano et al., 2002; Simonescu et al., 2010).

洹몃━怨 atrophy뿉꽌 HR-HPV 媛먯뿼맂 솚옄쓽 옄沅곴꼍遺 議곗쭅寃궗瑜 떎떆븳 寃곌낵 p16INK4a 諛쒗쁽 13紐낆쑝濡 86.7%쓽 뼇꽦쓣 蹂댁떎. 洹몃윭굹 LR-HPV뿉 媛먯뿼맂 솚옄뱾 옄沅곴꼍遺 議곗쭅寃궗瑜 떎떆븯吏 븡븘 p16INK4a 諛쒗쁽쓣 솗씤븷 닔 뾾뿀떎. 移댁씠뒪섏뼱 寃利앹뿉 쓽븳 쑀쓽誘몄꽦쓣 궡렣蹂대㈃ Fisher쓽 젙솗븳 寃젙쓣 떎떆븳 寃곌낵 HR-HPV 媛먯뿼씠 맂 atrophy뿉꽌 p16INK4a쓽 諛쒗쁽 쑀쓽닔以 P<0.001 닔以뿉꽌 쑀쓽븯寃 굹궗쓬쓣 븣 닔 엳떎. 씠뒗 p16INK4a쓽 怨쇰컻쁽씠 怨좎쐞뿕援 HPV 媛먯뿼쓣 떆궗븯뒗 몴吏옄濡쒖꽌 궗슜맆 닔 엳뒗 媛뒫꽦쓣 떎떆 븳踰 솗씤븷 닔 엳뿀떎. 뵲씪꽌 HR-HPV뿉 媛먯뿼씠 릺뿀쓣 븣 atrophy뿉꽌 p16INK4a 諛쒗쁽 뼇꽦쓣 蹂댁씠硫 씠뒗 ASCUS쓽 媛먮퀎 吏꾨떒뿉 룄씠 맂떎뒗 젏쓣 븣젮二쇨퀬 엳떎. 31紐낆쓽 옄沅곴꼍遺븫 솚옄뿉꽌 뼸뼱吏 깮泥 寃泥대 씠슜븳 CIN grade p16INK4 諛 ki-67 諛쒗쁽怨쇱쓽 긽愿꽦쓣 뿰援ы븳 寃곌낵뿉꽌룄 怨좎쐞뿕援 HPV 媛먯뿼 떆, Ki-67怨쇱쓽 겙 뿰愿꽦 蹂닿린 뼱졄吏留, p16INK4 떒諛깆쭏쓽 諛쒗쁽 利앷븳떎뒗 寃곌낵瑜 蹂닿퀬븯쑝硫, 뵲씪꽌 CIN怨 怨좎쐞뿕援 HPV 媛먯뿼쓽 吏꾨떒뿉꽌 p16INK4a씠 슚쑉쟻씤 몴吏옄濡 궗슜맆 닔 엳떎怨 븯떎(Nam et al., 2008). 洹몃━怨 옄沅곴꼍遺 븸긽꽭룷뿉꽌 ASC-H 솚옄뿉寃 p16INK4a 뿼깋쓣 떆뻾븯뿬 HSIL 씠긽쓽 蹂묒냼뿉 넂 誘쇨컧룄 80.0% 듅씠룄 77.8%瑜 蹂댁씠硫, HPV 寃궗 媛숈씠 떎떆븳 寃쎌슦 HSIL 씠긽쓽 蹂묒냼뿉 誘쇨컧룄 71.2%, 듅씠룄 86.4%濡 HSIL 씠긽쓽 蹂묒냼 쑀臾대 삁痢≫븯뒗뜲 쑀슜븯떎뒗 뿰援ш 蹂닿퀬릺怨 엳떎(Yim et al., 2011). 삉븳, p16INK4a쓽 諛쒗쁽 利앷 湲곗쟾 HPV E7 떒諛깆쭏씠 pRb쓽 鍮꾪솢꽦쓣 珥덈옒븯뿬 긽쟻쑝濡 p16INK4a 諛쒗쁽씠 利앷븳떎怨 諛앺졇꽌 븣젮吏怨 엳떎(Sakaguchi et al., 1996; Hofmann et al., 1996; Hateboer et al., 1996). 삉븳 옄沅곴꼍遺븫쓽 二쇱슂 썝씤씤 HPV 媛먯뿼怨 愿젴씠 엳뒗 寃궗踰뺤쑝濡 p16INK4a Ki-67 硫댁뿭뿼깋쓣 蹂묓뻾븯뿬 떎떆븷 寃쎌슦 뼇꽦쑉씠 넂븘, 옄沅곴꼍遺븫 議곌린 吏꾨떒쓽 젙솗꽦, 誘쇨컧룄쓽 利앷 떆궗 닔 엳뒗 엳떎뒗 뿰援ш 吏꾪뻾릺怨 엳떎(Keating et al., 2001).

씠윭븳 寃곌낵瑜 넗濡 옄沅곴꼍遺븫 寃궗 솚옄瑜 긽쑝濡 븸긽꽭룷寃궗瑜 씠슜븳 寃궗뿉꽌 p16INK4a 硫댁뿭꽭룷솕븰寃궗瑜 蹂댁“쟻쑝濡 떎떆븳떎硫 꽭룷븰쟻 吏꾨떒씠 뼱젮슫 atrophy뿉꽌 ASCUS瑜 媛먮퀎븯湲 쐞븳 蹂댁“寃궗濡 p16INK4a 硫댁뿭꽭룷솕븰寃궗媛 엫긽쟻쑝濡 ASCUS 媛먮퀎뿉 쑀슜븳 蹂댁“ 닔떒쑝濡 솢슜씠 媛뒫븿쓣 젣떆븷 닔 엳떎.

ACKNOWLEDGEMENT

This work was supported by the National Research Foundation of Korea (NRF) funded by the Korea government (MSIT) (NRF-2020R1C1C1003718, RS-2023-00264369 and RS-2023-00211284).

AUTHOR CONTRIBUTIONS

H.R.S. and T.E designed, performed, and analyzed the experiments. H.R.S, T.E and W.-S.C, wrote the manuscript. W.-S.C. conceived, planned, and oversaw the study.

CONFLICT OF INTEREST

Authors declare no competing interests.

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