Metanephric adenoma: Molecular study and review of the literature

Onco a ge 387 www.onco a ge .com www.onco a ge .com Onco a ge , 2022, Vol. 13, pp: 387-392 Resea ch Pape Me aneph ic adenoma: molecula s udy and e iew o he li e a u e En ique Rod íguez-Za co1, Jesús Machuca-Aguado1, Lau a Macías-Ga cía2, Ana Vallejo-Bení ez3 and Juan José Ríos-Ma ín1 1Pa hology Depa men , Uni e si y Hospi al Vi gen Maca ena, Se ille, Spain 2School o Medicine, Uni e si y o Se ille, Se ille, Spain 3Pa hology Depa men , Regional Uni e si y Hospi al o Malaga, Malaga, Spain Co espondence o: En ique Rod íguez-Za co, email: [email p o ec ed] Keywo ds: me aneph ic adenoma; BRAF; enal neoplasms Recei ed: No embe 24, 2021 Accep ed: Janua y 17, 2022 Published: Feb ua y 17, 2022 Copy igh : © 2022 Rod íguez-Za co e al. This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC BY 3.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. ABSTRACT In oduc ion: Me aneph ic adenoma (MA) is an uncommon benign umo accoun ing o 0.2–0.7% o adul enal epi helial neoplasms. The clinical cou se is o en indolen , bu diagnosis should no be delayed since clinical symp oms (hema u ia, e e , palpable abdominal mass, and lank pain) may be non-speci ic and o e lap wi h hose o a malign enal neoplasm. We epo on 4 cases o AM, o which mo phological and mu a ional analysis we e pe o med. Ma e ial and Me hods: Immunohis ochemical s aining was pe o med on sec ions cu om pa a in blocks o assess exp ession o WT1, imen in, acemase, CK7, CD10 and RCC. Tes ing o he BRAF gene mu a ion V600 was ca ied ou using eal- ime PCR (Cobas® 4800). Resul s: In all ou cases, umo s we e isible as well-ci cumsc ibed, non- encapsula ed masses loca ed in he enal co ex and ex ending owa ds he medulla. A immunohis ochemical examina ion, umo cells s ained nega i e o CK7, CD10 and RCC and posi i e o bo h WT1 (nuclea , in ense) and imen in (cy oplasmic, in ense, and di use). Molecula analysis e ealed he BRAF gene mu a ion V600E in h ee cases and wild- ype BRAF in he ou h. Conclusions: BRAF molecula mu a ion analysis may aid diagnosis in cases wi h a ypical his ological ea u es, especially in small incisional biopsies when eassessmen o su gical ea men may be conside ed. INTRODUCTION Me aneph ic adenoma (MA) is a a e benign umo o he kidney, accoun ing o 0.2% o adul enal epi helial neoplasms [1]. The umo , which is composed o p imi i e me aneph ic cells [2], is o en asymp oma ic. Some au ho s ha e sugges ed ha MA may de i e om ma u ing neph oblas omas (Wilms umo ), since immunopheno ypic indings o e lap closely wi h hose o di e en ia ed neph oblas oma and neph ogenic es s [3]. Though i may also occu in child en, i is de ec ed mainly in adul s aged be ween 50 and 70 and is mo e common ( a io 2:1) in women [2, 4, 5]. Radical neph ec omy, c yoabla ion and adio equency ha e been used o ea his neoplasm [5]. While he clinical cou se is benign, his ological indings o en o e lap wi h hose o malignan umo s including Wilms umo and enal papilla y neoplasms, hus p omp ing he need o di e en ial diagnosis [6]. A be e unde s anding o his benign umo would undoub edly aid he de elopmen o less in asi e s a egies. Al hough mos au ho s ule ou he possibili y o MA becoming malignan , one case has been epo ed o a me aneph ic adenoma in associa ion wi h a high-g ade sa coma (me aneph ic adenosa coma) [5, 7]. Immunohis ochemical analysis is a use ul ool o di e en ial diagnosis. The li e a u e con ains ew epo s o in eg a ed diagnosis o MA using molecula echniques [8]. Onco a ge 388 www.onco a ge .com Mu a ion o he BRAF gene p omp s cons i u i e ac i a ion o he ERK-media ed signaling pa hway, a o ing cell p oli e a ion and di e en ia ion. Ac i a ion o RAF, in bo h i s homodime and he e odime o ms, igge s he phospho yla ion o MAPK kinase (MEK), which in u ns p omp s he phospho yla ion o ex acellula signal- egula ed kinase (ERK); ERK ac i a ion p omo es cell p oli e a ion and signal ans o ma ion h ough in e ac ion wi h se e al molecules c ucial o umo pa hogenesis [9, 10]. The speci ic BRAF gene mu a ion V600E has been epo ed in o e hal o all cu aneous melanomas and papilla y hy oid ca cinomas, as well as in a numbe o blood cance s including hai y cell leukemia; i is also p esen in indolen and benign umo s such as melanocy ic ne us [11]. This speci ic mu a ion has also been s udied in malignan neoplasms such as enal cell ca cinoma in esponse o a ge ed he apies [12]. In benign neoplasms like MA, se e al au ho s ha e no ed ha es ing o he BRAF V600E mu a ion may be a aluable ool o he diagnosis [1, 2, 11]. RESULTS G oss examina ion e ealed well-ci cumsc ibed, non-encapsula ed umo s measu ing be ween 1.5 and 6 cm (mean 3.7 cm), wi h ocal a eas o blood- con aining cys s and solid, in some cases p esen ing calci ica ions. His ologically, MAs we e composed o small, monomo phic epi helial cells displaying no signi ican a ypia o mi o ic igu es, showing papilla y o acina pa e ns, and edema ous o hyalinized s oma wi h calci ica ion (psammoma bodies) (Figu e 1A). Immunohis ochemical examina ion showed posi i e s aining o WT1 and imen in, and nega i e s aining o acemase, CK7, CD10, CD57 and RCC (Figu e 1B). Mu a ion analysis by eal- ime PCR e ealed he BRAF gene mu a ion V600E in h ee cases and wild- ype BRAF in he ou h. Based on mo phological ea u es and he indings o immunohis ochemical and molecula analysis, all ou cases we e diagnosed as MA. Th ee o he pa ien s a e ali e and well 12, 5 and 2 yea s a e su ge y, while he ou h died 13 yea s a e he p ocedu e due o o he causes. DISCUSSION MA is an uncommon, benign umo o he kidney composed o spindle cells associa ed wi h epi helial cells. In 1988, Mos o i e al. [13] desc ibed MA o he i s ime as a dis inc nosologic en i y among enal neoplasms, wi h ubula -like epi helial cells. This umo is cu en ly classi ied among he me aneph ic neoplasms, which also include me aneph ic adenoma and me aneph ic s omal umou [4]. MA, which accoun s o 0.2–0.7% o adul enal epi helial neoplasms, de i es om emnan s o emb yonic enal issue [1, 3, 4]. A g oss examina ion, MA appea s as a well- ci cumsc ibed neoplasm wi h a yellowish su ace, o en displaying e idence o seconda y changes including ocal nec osis, hemo hage and/o cys ic degene a ion. Coa se calci ica ion may also be p esen . The umo mass gene ally anges in size be ween 3 and 6 cm in diame e , al hough umo s o up o 15 cm ha e been epo ed [4]. His ologically, i comp ises an acina a angemen o small cells. Di e en ial diagnosis o MA includes Wilms umo , neph ogenic es s and enal papilla y neoplasms [5]. MA ypically exp esses WT1 and CD57, bu s ains nega i e o CK7 and acemase. Posi i e in ense s aining o WT1 was eco ded in he ou cases epo ed he e, bu CD57 s aining was in mos o hem weak; so, BRAF mu a ion helped us o con i m he diagnosis o MA. Oncogenic BRAF no mally egula es cell di ision and di e en ia ion h ough he MAP kinase signaling pa hway. BRAF mu a ions, iden i ied in se e al solid umo s and blood cance s, p omp he cons i u i e ac i a ion o he pa hway, which has been widely documen ed in melanomas [9, 10, 14]. Mos BRAF mu a ions in ol e a hymine-adenine ans e sion, leading o he subs i u ion o aline by glu amic acid a codon V600 (V600E) [14]. Mos epo ed MAs display a no mal geno ype, lacking he simul aneous ch omosomes 7 and 17 gain and Y ch omosome loss cha ac e is ic o papilla y enal cell ca cinoma and common in neu oblas oma. The BRAF gene mu a ion V600E is epo ed in oughly 90% o me aneph ic adenomas [2, 4, 11, 15], wi h only 2 desc ibed cases o V600D mu a ion [2] and 1 o V600K [16]. Caliò [14] e al. iden i ied he V600E mu a ion in 41 ou o 48 MA pa ien s (85%) wi h a mean age o 54, while Chouei i e al. [11] epo ed i in 26/29 pa ien s (89%) also wi h a mean age o 54, and Ding e al. [17] desc ibed 27 MA pa ien s wi h mean age o 39 yea s and 22 (81%) wi h BRAF mu a ion. O he au ho s ha e epo ed he V600E mu a ion in smalle se ies [18–21]. In ou esea ch, as shown in Table 1, he mu a ion was iden i ied in h ee o he ou pa ien s s udied (75%), aged be ween 19 and 65 (mean 47.5). Se e al au ho s ha e d awn a en ion o he ela ionship be ween wild- ype BRAF and MA in younge adul s (i.e., unde 25). O he 29 cases epo ed by Choue i [11] e al., all h ee pa ien s ha bo ing wild- ype BRAF we e well below he mean age. By con as , in he ou pedia ic cases o MA desc ibed by Chami e al. [22], only one ha bo ed wild- ype BRAF. An epidemiological analysis was ca ied ou o he published cases o MA in which he BRAF mu a ion has been s udied. In Table 2 i can be obse ed ha , al hough he age ange o p esen a ion is simila in bo h g oups, he Onco a ge 389 www.onco a ge .com mean age is 19.6 yea s lowe in he wild- ype BRAF pa ien s (31.1 s. 50.7). The p esen s udy de ec ed he BRAF gene mu a ion V600E in pa ien s aged be ween 50 and 65, while he younges pa ien (aged 19) ha bo ed wild- ype BRAF. A ela ionship ha has no been p e iously highligh ed is he highe incidence o wild- ype BRAF in male pa ien s. As desc ibed abo e, me aneph ic adenoma is mo e common in emale pa ien s in a 2: 1 a io, jus like in mu a ed BRAF MA (Table 2). By con as , in wild- ype BRAF MA, he incidence in men is highe han women wi h a 1.45: 1 a io. A ema kable case is he se ies o 48 pa ien s by Calio e al. [14], whe e he F:M a io in mu a ed BRAF is 2.7: 1, while in wild- ype BRAF i is 1: 6. The e o e, mu a ed BRAF MA a e mo e equen in elde ly pa ien s and women, which is consis en wi h s udies in o he pa hologies. E en wi h hese esul s, i is necessa y o ca y ou s udies wi h a g ea e numbe o cases in o de o ensu e i . MATERIALS AND METHODS This pape epo s on MA in h ee men and one woman, aged be ween 19 and 65 (mean age 47.5); pa ien da a a e p o ided in Table 3. The diagnosis Figu e 1: (A) Monomo phic epi helial p oli e a ion, displaying no signi ican a ypia o mi o ic igu es, wi hin an edema ous s oma con aining psammoma bodies (Pano amic iew. HE). (B) In ense posi i e nuclea s aining o WT1. Onco a ge 390 www.onco a ge .com was con i med and subsequen ly e iewed ollowing WHO- ecommended c i e ia [4]. In all cases, MA p esen ed as a single, asymp oma ic mass disco e ed inciden ally du ing imaging p ocedu es; CT scan con i med he p esence o a soli a y, space-occupying, solid enal umo . A neph ec omy was pe o med in all pa ien s. Immunohis ochemical s aining was pe o med on H&E- s ained sec ions cu om pa a in blocks o assess exp ession o WT1, imen in, acemase, CK7, CD10 and RCC. Tes ing o he BRAF gene mu a ion V600 was ca ied ou by eal- ime PCR (Cobas® 4800) using he DNA Sample P epa a ion Ki and he BRAF Mu a ion Tes (Roche), which de ec s he BRAF gene mu a ions V600E, Table 1: BRAF mu a ions in me aneph ic adenomas: li e a u e e iew Resea ch and yea Numbe o cases Mean age (yea , ange) Gende Tumo size (cm, ange) BRAF mu a ion Type o mu a ion P e ious epo s in Caliò e al., 2016 99 52 (5–84) 71F 28M 3.4 (1.1–8) 87 (88%) V600E (97) V600D (2) Ding e al., 2018 27 39 (12–80) 9F 18M 3.1 (2–7) 22 (81%) V600E Wobke e al., 2019 10 42 (10–62) 6F 4M 2.7 (1.3–3.5) 8 (80%) V600E Ca ic e al., 2020 28 52 (9–73) 17F 10M 3 (0.5–12) 15 (53%) V600E Chan e al., 2020 12 54 (38–76) 11F 1M 2.9 (1–6) 12 (100%) V600E Lenci e al., 2021 1 73 F 3.2 1 (100%) V600K Cu en s udy 4 54 (19–65) 1F 3M 3,7 (1.5–6) 3 (75%) V600E Table adap ed om Caliò e al. [14]. Table 3: Epidemiological da a and BRAF s a us Case Gende Age (yea s) BRAF s a us (V600E) 1 M 19 WT 2 F 50 Mu 3 M 56 Mu 4 M 65 Mu Abb e ia ions: M: male; F: emale; WT: wild ype; Mu : mu a ed. Table 2: Compa ison o age and gende incidence in mu a ed BRAF and wild ype BRAF me aneph ic adenomas: li e a u e e iew Resea ch and yea Numbe o MUTATED/WT cases Mean age MUTATED/WT (yea s, ange) Gende MUTATED BRAF WT BRAF Male Female Male Female Chouei i e al., 2012 26/3 54.7 (36−78)/32 (25−38) 3 (12%) 23 (88%) −3 (100%) Dadone e al., 2013 1/0 61/− − 1 (100%) − Pin o e al., 2015 6/0 52/− − 6 (100%) − Udage e al., 2015 10/1 51.2 (16−84)/32 4 (40%) 6 (60%) 1 (100%) − Chami e al., 2015 3/1 5.6 (4−9)/10 2 (67%) 1 (33%) 1 (100%) − Caliò A e al., 2016 41/7 57 (5−84)/33 (10−74) 11 (27%) 30 (73%) 6 (86%) 1 (14%) Ding e al., 2018 22/5 40 (25−73)/29 (12−47) 17 (77%) 5 (23%) 4 (80%) 1 (20%) Wobke e al., 2019 8/2 46 (19−62)/26.5 (10−43) 3 (37%) 5 (63%) 1 (50%) 1 (50%) Ca ic e al., 2020 15/9 47 (5−75)/36 (9−71) 7 (46%) 8 (54%) 3 (37%) 5 (63%) Chan e al., 2020 12/0 54 (38−76)/− 1 (8%) 11 (92%) − Lenci e al., 2021 1/0 73/− − 1 (100%) − Cu en s udy 3/1 57 (50−65)/19 2 (67%) 1 (33%) 1 (100%) − 148 (84%)/29 (16%) 50.7 (4−84)/31.1 (9−74) 50 (34%) 98 (66%) 17 (58%) 12 (42%) Onco a ge 391 www.onco a ge .com V600K and V600D in o malin- ixed, pa a in-embedded issue. CONCLUSIONS These esul s bea ou he indings o p e ious s udies o BRAF gene mu a ions in MA, showing ha molecula mu a ion analysis may aid diagnosis in cases wi h a ypical his ological ea u es, especially in small biopsies when non-su gical ea men is planned. A highly accu a e de ini i e diagnosis o MA was achie ed by combining immunohis ochemical and molecula analysis; mu a ed BRAF MA a e mo e equen in elde ly pa ien s and women. Accu a e ea ly diagnosis may help o a oid unnecessa y agg essi e ea men s such as adical neph ec omy. Au ho con ibu ions ERZ: concep ualiza ion, me hodology and w i ing-o iginal d a ; JMA: w i ing-o iginal d a and in es iga ion; LMG: concep ualiza ion and in es iga ion; AVB: me hodology and w i ing- e iew & edi ing; JJRM: w i ing- e iew & edi ing and supe ision. CONFLICTS OF INTEREST Au ho s ha e no con lic s o in e es o decla e. FUNDING This esea ch did no ecei e any speci ic g an om any unding agency in he public, comme cial, o no - o -p o i sec o . REFERENCES 1. Sun Z, Kan S, Zhang L, Zhang Y, Jing H, Huang G, Yu Q, Wu J. Immunohis ochemical pheno ype and molecula pa hological cha ac e is ics o me aneph ic adenoma. In J Clin Exp Pa hol. 2015; 8:6031–36. [PubMed] 2. Udage AM, Pan J, Mage s MJ, Palapa u GS, Mo gan TM, Mon gome y JS, Weize AZ, Ha ez KS, Mille DC, Wol JS J , McHugh JB, Chinnaiyan AM, Dhanaseka an SM, Meh a R. Molecula and immunohis ochemical cha ac e iza ion e eals no el BRAF mu a ions in me aneph ic adenoma. Am J Su g Pa hol. 2015; 39:549–57. h ps://doi. o g/10.1097/PAS.0000000000000377. [PubMed] 3. Mui TE, Che ille JC, Lage DJ. Me aneph ic adenoma, neph ogenic es s, and Wilms’ umo : a his ologic and immunopheno ypic compa ison. Am J Su g Pa hol. 2001; 25:1290–96. h ps://doi.o g/10.1097/00000478-200110000- 00010. [PubMed] 4. Moch H, Humph ey PA, Ulb igh TM, Reu e VE. WHO Classi ica ion o Tumou s o he U ina y Sys em and Male Geni al O gans. Lyon, F ance: In e na ional Agency o Resea ch on Cance ; 2016. 5. Rod íguez-Za co E, Vallejo-Bení ez A, Fa án-López FJ, Vilches-A enas Á, Pe ei a-Galla do S, Vázquez- Ramí ez FJ. [Adenoma me aneph ic: Repo o 2 cases and bibliog aphic e iew]. A ch Esp U ol. 2017; 70:792–96. [PubMed] 6. A oyo MR, G een DM, Pe lman EJ, Beckwi h JB, A gani P. The spec um o me aneph ic adeno ib oma and ela ed lesions: clinicopa hologic s udy o 25 cases om he Na ional Wilms Tumo S udy G oup Pa hology Cen e . Am J Su g Pa hol. 2001; 25:433–44. h ps://doi. o g/10.1097/00000478-200104000-00002. [PubMed] 7. Picken MM, Cu y JL, Lindg en V, Cla k JI, Eble JN. Me aneph ic adenosa coma in a young adul : mo phologic, immunopheno ypic, ul as uc u al, and luo escence in si u hyb idiza ion analyses: a case epo and e iew o he li e a u e. Am J Su g Pa hol. 2001; 25:1451–57. h ps://doi. o g/10.1097/00000478-200111000-00016. [PubMed] 8. Kinney SN, Eble JN, Hes O, Williamson SR, G ignon DJ, Wang M, Zhang S, Bald ige LA, Ma ignoni G, B unelli M, Wang L, Compe a E, Fan R, e al. Me aneph ic adenoma: he u ili y o immunohis ochemical and cy ogene ic analyses in di e en ial diagnosis, including solid a ian papilla y enal cell ca cinoma and epi helial-p edominan neph oblas oma. Mod Pa hol. 2015; 28:1236–48. h ps:// doi.o g/10.1038/modpa hol.2015.81. [PubMed] 9. Bee am M, Pa naik A, Rowinsky EK. Ra : a s a egic a ge o he apeu ic de elopmen agains cance . J Clin Oncol. 2005; 23:6771–90. h ps://doi.o g/10.1200/ JCO.2005.08.036. [PubMed] 10. Ga ne MJ, Rana S, Pa e son H, Ba o d D, Ma ais R. Wild- ype and mu an B-RAF ac i a e C-RAF h ough dis inc mechanisms in ol ing he e odime iza ion. Mol Cell. 2005; 20:963–69. h ps://doi.o g/10.1016/j.molcel.2005.10.022. [PubMed] 11. Chouei i TK, Che ille J, Palescandolo E, Fay AP, Kan o PW, A kins MB, McKenney JK, B own V, Lamp on ME, Zhou M, Hi sch MS, Signo e i S. BRAF mu a ions in me aneph ic adenoma o he kidney. Eu U ol. 2012; 62:917–22. h ps://doi.o g/10.1016/j.eu u o.2012.05.051. [PubMed] 12. Bane jee N, Sachde E, Figlin RA. A Ra e Finding o a BRAF Mu a ion in Renal Cell Ca cinoma wi h Response o BRAF-Di ec ed Ta ge ed The apy. Cu eus. 2016; 8:e449. h ps://doi.o g/10.7759/cu eus.449. [PubMed] 13. Mos o i FK, Ses e henn IA, Da is CJ J . Benign umo s o he kidney. P og Clin Biol Res. 1988; 269:329–46. [PubMed] 14. Caliò A, Eble JN, Hes O, Ma ignoni G, Ha a i SE, Williamson SR, B unelli M, Osunkoya AO, Wang L, Compe a E, Lopez-Bel an A, Wang M, Zhang S, e al. Dis inc clinicopa hological ea u es in me aneph ic adenoma ha bo ing BRAF mu a ion. Onco a ge . 2016; 8:54096–105. h ps://doi.o g/10.18632/onco a ge .11117. [PubMed] Onco a ge 392 www.onco a ge .com 15. Pin o A, Signo e i S, Hi sch MS, Ba le a JA. Immunohis ochemical s aining o BRAF V600E suppo s he diagnosis o me aneph ic adenoma. His opa hology. 2015; 66:901–04. h ps://doi.o g/10.1111/his.12509. [PubMed] 16. Lenci N, F ancesco P, Sca ciglia E, Fio en ino V, Schino M, Pale mo G, Racioppi M, Bassi P, Ma ini M. Me aneph ic adenoma wi h BRAF V600K mu a ion and a doub ul adiological imaging: pi alls in he diagnos ic p ocess. Med Mol Mo phol. 2021; 54:187–91. h ps://doi.o g/10.1007/ s00795-020-00269-z. [PubMed] 17. Ding Y, Wang C, Li X, Jiang Y, Mei P, Huang W, Song G, Wang J, Ping G, Hu R, Miao C, He X, Chen G, e al. No el clinicopa hological and molecula cha ac e iza ion o me aneph ic adenoma: a s udy o 28 cases. Diagn Pa hol. 2018; 13:54. h ps://doi.o g/10.1186/s13000-018-0732-x. [PubMed] 18. Ca ic A, Ku o ic-Koza ic A, Sophian A, Mazu L, Skende i F, Hes O, Rohan S, Rakheja D, Kogan J, Pins MR. KANK1-NTRK3 usions de ine a subse o BRAF mu a ion nega i e enal me aneph ic adenomas. BMC Med Gene . 2020; 21:202. h ps://doi.o g/10.1186/s12881-020-01143-6. [PubMed] 19. Dadone B, Amb ose i D, Ca pen ie X, Du an on-Tanneu V, Bu el-Vandenbos F, Amiel J, Pedeu ou F. A enal me aneph ic adenoma showing bo h a 2p16e24 dele ion and BRAF V600E mu a ion: a syne gis ic ole o a umo supp esso gene on ch omosome 2p and BRAF ac i a ion? Cance Gene . 2013; 206:347–52. h ps://doi.o g/10.1016/j. cance gen.2013.09.002. [PubMed] 20. Wobke SE, Ma oso A, P a ilas CA, Mang ay S, Zheng G, Lin MT, Debeljak M, Eps ein JI, A gani P. Me aneph ic Adenoma-Epi helial Wilms Tumo O e lap Lesions: An Analysis o BRAF S a us. Am J Su g Pa hol. 2019; 43:1157–69. h ps://doi.o g/10.1097/ PAS.0000000000001240. [PubMed] 21. Chan E, S oh BA, C oom NA, Cho SJ, Ga g K, T oxell ML, Higgins JP, Bean GR. Molecula cha ac e isa ion o me aneph ic adenomas beyond BRAF: gene ic e idence o po en ial malignan e olu ion. His opa hology. 2020; 76:1084–90. h ps://doi.o g/10.1111/his.14094. [PubMed] 22. Chami R, Yin M, Ma ano P, Tee apakpinyo C, Shuangsho i S, Tho ne PS. BRAF mu a ions in pedia ic me aneph ic umo s. Hum Pa hol. 2015; 46:1153–61. h ps://doi. o g/10.1016/j.humpa h.2015.03.019. [PubMed]