Germline RET 634 Mutation Positive MEN 2A-related C-Cell Hyperplasias Have Genetic Features Consistent with Intraepithelial Neoplasia

Ge mline RET 634 Mu a ion Posi i e MEN 2A- ela ed C-Cell Hype plasias Ha e Gene ic Fea u es Consis en wi h In aepi helial Neoplasia SALVADOR J. DIAZ-CANO, MANUEL DE MIGUEL, ALFREDO BLANES, ROBERT TASHJIAN, AND HUBERT J. WOLFE Depa men s o Pa hology, Tu s Uni e si y–New England Medical Cen e (S.J.D.-C., R.T., H.J.W.), Bos on, Massachuse s 02111; Ba s and The London Queen Ma y’s School o Medicine and Den is y (S.J.D.-C.), E1 1BB London, Uni ed Kingdom; Uni e si y Hospi al o Se ille (M.d.M.), 41009-Se ille, Spain; and Uni e si y Hospi al o Malaga (A.B.), 29010-Malaga, Spain C-cell hype plasias a e no mally mul i ocal in mul iple endo- c ine neoplasia ype 2A. We compa ed clonali y, mic osa el- li e pa e n o umo supp esso genes, and cellula kine ics o C-cell hype plasia oci in each hy oid lobe. We selec ed 11 emales om mul iple endoc ine neoplasia ype 2A kind ed ea ed wi h hy oidec omy due o hype cal- ci oninemia. C-cell hype plasia oci we e mic odissec ed o DNA ex ac ion o analyze he me hyla ion pa e n o and o- gen ecep o alleles and mic osa elli e egions (TP53,RB1, WT1, and NF1). Consecu i e sec ions we e selec ed o MIB-1, pRB1, p53, Mdm-2, and p21 WAF1 immunos aining, DNA con en analysis, and in si u end labeling. App op ia e issue con ols we e un. Only wo pa ien s had medulla y hy oid ca cinoma oci. Nine in o ma i e C-cell hype plasia pa ien s showed ge m- line poin mu a ion in RET, eigh o hem wi h he same an- d ogen ecep o allele p e e en ially me hyla ed in bo h lobes. C-cell hype plasia oci showed he e ogeneous DNA de- le ions e ealed by loss o he e ozygosi y o TP53 (12 o 20), RB1 (6 o 14), and WT1 (4 o 20) and hypodiploid G 0 /G 1 cells (14 o 20), low cellula u no e (MIB-1 index 4.5%, in si u end labeling index 0.03%), and signi ican ly high nuclea a ea o DNA index a io. MEN 2A (ge mline poin mu a ion in RET codon 634) C-cell hype plasias a e monoclonal and gene ically he e ogeneous and show down- egula ed apop osis, indings consis en wi h an in aepi helial neoplasia. Conco dan X-ch omosome in- ac i a ion and in e s i ial gene dele ions sugges clone ex- pansions o p ecu so s occu ing a a poin in emb yonic de- elopmen be o e di e gence o each hy oid lobe and may ep esen a pa adigm o o he ge mline mu a ions. (J Clin Endoc inol Me ab 86: 3948–3957, 2001) THE IDENTIFICATION OF RET ge mline mu a ions in mul iple endoc ine neoplasia ype 2 (MEN 2) has e- sul ed in ca ie de ec ion and ea ly diagnosis o C-cell hy- pe plasias (CCHs) and medulla y hy oid ca cinomas (MTCs) (1–3). RET mu a ion seems o be necessa y, bu o he al e a ions should be equi ed o explain he MEN 2 phe- no ypic he e ogenei y and he di e ences be ween CCH and ad enal medulla y hype plasias. Clonali y is s ill he hallma k o neoplasia and s ongly sugges s acqui ed soma ic mu a ions, p o iding p oli e a- i e ad an age o a gi en cell popula ion (4, 5). Clonali y has been s udied by X-ch omosome inac i a ion (XCI) assays, which a e based on he DNA me hyla ion o many genes ha ende ma e nal and pa e nal ch omosome unc ionally non- equi alen (6–8). The same poin mu a ion o single nucle- o ide polymo phism (SNP) wi hin all cells also implies a common p ogeni o , (4), and i has been ound associa ed wi h loss o he e ozygosi y (LOH) o ce ain loci (9). LOH should be linked o inac i a ion o umo supp esso genes (TSG) by DNA dele ions, con ibu ing o umo cell selec ion (4). These non-X-linked ma ke s es clonal expansions as- socia ed o selec i e g ow h ad an ages (high p oli e a ion and/o abno mally low apop osis) (4, 10–13), bu hey ail o iden i y clones occu ing p io o he p esence o he gene ic lesion (9, 10). This s udy in es iga es he clonal pa e ns o CCH asso- cia ed wi h MEN 2A (RET poin mu a ion in codon 634) in bo h hy oid lobes, based on he analysis o bo h he me h- yla ion pa e n o and ogen ecep o alleles and TSG mic- osa elli e pa e ns using mic odissec ed issue samples. The kine ic ea u es o such lesions a e also analyzed. Ma e ials and Me hods Case selec ion MEN 2A kind ed wi h a Cys634 o Ty subs i u ion o RET p o o- oncogene (177 membe s, i e gene a ions) we e sc eened o CCH/MTC by basal and pen agas in-induced calci onin le els (14–16). All pa ien s ul illed he clinical and molecula c i e ia p oposed by he In e na ional RET Mu a ion Conso ium (17). Pa ien s wi h pen agas in-induced hy- pe calci oninemia and/o RET poin mu a ions unde wen o al hy- oidec omy, he specimens being se ially sec ioned and comple ely em- bedded o his opa hologic diagnosis. App op ia e a chi al ma e ial om bo h lobes was a ailable om 11 emales. We ini ially selec ed cases showing 50 o mo e C cells in a leas one ⫻100 mic oscope ield pe lobe (18, 19). C cells we e la ge, mildly o mode a ely a ypical, and con ined wi hin he ollicula basemen memb ane, bu cy ologically indis inguishable om in asi e MTC (20, 21). One pa ien (case CCH-6) did no show RET mu a ion and was Abb e ia ions: AR, And ogen ecep o ; CCH, C cell hype plasia; HPF, high-powe ield; ISEL, in si u end labeling; LOH, loss o he - e ozygosi y; MEN 2, mul iple endoc ine neoplasia ype 2; MTC, med- ulla y hy oid ca cinoma; NF1, neu o ib oma osis 1; TP53, umo p o ein p53; RB1, e inoblas oma; SNP, single nucleo ide polymo phism; TSG, umo supp esso genes; WT1, Wilms’ umo 1; XCI, X-ch omosome inac i a ion. 0013-7227/01/$03.00/0 The Jou nal o Clinical Endoc inology & Me abolism 86(8):3948–3957 P in ed in U.S.A. Copy igh © 2001 by The Endoc ine Socie y 3948 Downloaded om h ps://academic.oup.com/jcem/a icle/86/8/3948/2849067 by Uni e sidad de Se illa use on 06 Feb ua y 2023 excluded om u he analyses. The same a eas in consecu i e sec ions we e used in each s udy, and hei cellula composi ion was con i med in adjacen hema oxylin-eosin-s ained sec ions. PCR analysis o clonali y and TSG mic osa elli es One CCH ocus con aining a leas 100 C cells (0.5 mm; Re . 2) was mic odissec ed om each hy oid lobe ( wo 20- ␮ m uns ained sec ions/ ocus). App op ia e con ols ( ollicula cells, lymph node, pe iphe al ne es, and hy oidal so issue) we e ca ied ou . DNA was ex ac ed using a modi ied phenol-chlo o o m p o ocol, (22) and diges ed wi h HhaI (New England Biolabs, Inc., Be e ly, MA). Hal o each sample unde wen enzyma ic diges ion (0.8 U/ ␮ l), whe eas he emaining hal was kep as undiges ed con ol. Bo h samples we e equally p ocessed, bu excluding HhaI in he undiges ed ones (10, 23, 24). A mimicke (0.3 ␮ g o double-s anded and XhoI-linea ized ␾ X174-RII phage) (Li e Technologies, Inc., Gai he sbu g, MD) was included in each eac ion o diges ion es ing. Comple e diges ion was checked by gel elec opho esis; incomple ely diges ed samples we e phenol chlo o o m pu i ied and ediges ed wi h highe HhaI concen a ion. HhaI was hen inac i a ed by phenol-chlo o o m ex ac ion (22). DNA was p ecipi a ed wi h ice-cold absolu e e hanol in he p esence o 0.3 mol/li e sodium ace a e (pH 5.2) and esuspended in 10 ␮ l o 10 mmol/li e T is-HCl (pH 8.4), 50 mmol/li e KCl, 1.5 mmol/li e MgCl2, and 100 ␮ g/ml BSA. The CAG epea in he i s exon o he human and ogen ecep o (AR) gene was ampli ied using bo h diges ed and undiges ed DNA empla es (13, 23, 25, 26). The undiges ed DNA was also used o am- pli ica ion o in on mic osa elli es a TSG (TP53,RB1,WT1, and NF1) loci, acco ding o he op imized condi ions shown in Table 1 (25, 27). The es s we e un in a Pe kin-Elme he mal cycle model 480 (Pe kin-Elme Co p., No walk, CT). The whole PCR olume (10 ␮ l) was elec opho- esed in o 0.75-mm hick 8% polyac ylamide gels, nondena u ing o he AR p oduc and 20–80% dena u ing g adien ( om op o bo om) o TSG p oduc s (13, 25, 26). The gels we e un a 5 V/cm un il xylene cyanol band was wi hin he bo om inch o he gel. The gels we e hen ixed wi h 7% ace ic acid (5 min), d ied unde acuum (40 min, 80 C), and pu inside a de eloping casse e con aining one in ensi ying sc een and p e lashed ilms (Kodak XAR; Kodak, Roches e , NY) acing he in ensi ying sc een (16–48 h, ⫺70 C). The au o adiog ams we e de el- oped using an au oma ed p ocesso Kodak-Oma 100 (Kodak). Only in o ma i e cases (balanced allele a ios in undiges ed and diges ed con ols) we e included in he inal analysis (10, 11, 13, 25, 26, 28). Allelic imbalance was densi ome ically e alua ed (EC model 910 op ical densi ome e ; EC Appa a us Co., S Pe e sbu g, FL), conside ing only allele a ios g ea e han 4:1 in he no malized diges ed lanes e idence o monoclonali y. Lanes we e no malized wi h hei co e- sponding undiges ed sample and con ols. Only allele a ios g ea e han 4:1 in any TSG was equally conside ed e idence o LOH; o he wise, e en ion o cons i u ional he e ozygosi y was assigned. Addi ional al- lele bands in he CCH samples no p esen in he co esponding con ol we e conside ed e idence o SNP in dena u ing g adien gels. Nuclea DNA quan i ica ion by slide cy ome y Feulgen-s ained sec ions we e used o densi ome ic e alua ion o DNA con en (29). CAS model 200 and he Quan i a i e DNA Analysis so wa e (Bec on Dickinson and Co., F anklin Lakes, NJ) we e used o ha pu pose, measu ing a leas 200 nuclei (o he whole lesion i smalle ) in e e y CCH ocus in he mos cellula a ea un il comple ion in consecu i e mic oscope high powe ields (HPFs; ⫻400). Only com- ple e, nono e lapping and ocused nuclei we e quan i ied in each HPF. In e nal con ols (bo h lymphocy es and his ologically no mal ollic- ula cells om he same sec ion) we e i s no malized wi h comple e a hepa ocy es (ex e nal con ols, one slide/s aining holde ) (Bec on Dick- inson and Co.). They we e hen used o se ing he diploid con ols and calcula ing he DNA index o G 0 /G 1 cells (ⱖ10% o measu ed cells wi h e idence o G 2 ⫹M cells) (30). The p oli e a ion a e was calcula ed om he DNA his og am by sub ac ing he numbe o cells wi hin G 0 /G 1 limi s om he o al numbe o measu ed cells and exp essed as pe - cen age (29, 31). Bo h mean nuclea a ea and nuclea a ea o DNA index a io o G 0 /G 1 cells we e eco ded. The la e ep esen s a mo phome ic pa ame e o apop osis when coupled wi h in si u end labeling (ISEL) (13, 30). Age- and sex-ma ched no mal hy oids om 10 au opsies we e selec ed o nuclea a ea and DNA index analysis. A leas 1000 C cells we e e alua ed as con ols o his pu pose. ISEL o agmen ed DNA Ex ensi e DNA agmen a ion associa ed wi h apop osis was de- ec ed by ISEL as p e iously epo ed (31–33). Sec ions we e incuba ed in 2⫻SSC (20 min, 80 C) and diges ed wi h p o einase K [100 ␮ g/ml in T is-HCl (pH 7.6), 30 min] a oom empe a u e in mois chambe . DNA agmen s we e digoxigenin-labeled on 5⬘-p o uding e mini using he Klenow agmen o Esche ichia coli DNA polyme ase I (1 h a 37 C), de ec ed using an idigoxigenin Fab agmen s labeled wi h alkaline phospha ase, and de eloped wi h ni oblue e azolium-X phospha e (31–34). App op ia e con ols we e un, including posi i e ( eac i e lymph node), nega i e (omi ing DNA polyme ase I), and enzyma ic (DNase I diges ion be o e end labeling). The enzyma ic con ols allowed es ablishing a eliable posi i i y h eshold in each sample. The ISEL index was exp essed as pe cen age o posi i e nuclei compa ed wi h he o al numbe o C cells in he same HPF (35–37). The whole lesion was sc eened. TABLE 1. P ime sequences and PCR cycling condi ions o he ampli ica ion o polymo phic DNA egions P ime s P ime sequences Tandem epea /PCR p oduc AR-a a 5⬘-CCGAGGAGCTTTCCAGAATC-3⬘CAG epea / AR-b a 5⬘-TACGATGGGCTTGGGGAGAA-3⬘215–300 bp p53(1)-a b 5⬘-AGGGATACTATTCAGCCC-3⬘CA epea / p53(1)-b b 5⬘-ACTGCCACTCCTTGCCCCATTC-3⬘103–135 bp p53(2)-a b 5⬘-GAATCCGGGAGGAGGTTG-3⬘AAAAT epea / p53(2)-b b 5⬘-AACAGCTCCTTTAATGGCAG-3⬘140–175 bp RB1-a b 5⬘-CTCCTCCCTACTTACTTGT-3⬘CTTT(T) epea / RB1-b b 5⬘-AATTAACAAGGTGTGGTGGTACACG-3⬘266–306 bp WT1-a b 5⬘-AATGAGACTTACTGGGTGAGG-3⬘CA epea / WT1-b b 5⬘-TTACACAGTAATTTCAAGCAACGG-3⬘⬃144 bp NF1-a b 5⬘-CAGAGCAAGACCCTGTCT-3⬘CA epea / NF1-b b 5⬘-CTCCTAACATTTATTAACCTTA-3⬘171–187 bp All eac ions we e un in duplica e using 1.5 mMMgCl 2 and 1 ␮ l empla e. A long dena u a ion (4 min) and expansion (90 sec) we e used in he i s h ee cycles o each se o p ime s. a The AR alleles we e ampli ied using 0.3 ␮ Mo each p ime and 200 ␮ Mo each dNTP (including 7-deaza-dGTP ins ead o dGTP) (Boeh inge -Mannheim, Indianapolis, IN). The amplicon was in e nally labeled wi h 0.3 ␮ Ci ␣ [ 32 P]-dTTP (800 Ci/mmol, 10 mCi/ml) (New England Nucleo ide, Bos on, MA). A “ho s a ”p o ocol was also used, comple ing 28 cycles wi h an annealing empe a u e o 55°C. b The polymo phic egions o TSG we e ampli ied using 0.25 ␮ Mo each p ime , 50 ␮ Mo each dNTP (Boeh inge -Mannheim, Indianapolis, IN), and in e nally labeled wi h 0.3 ␮ Ci ␣ [ 32 P]-dCTP (3000 Ci/mmol, 10 mCi/ml) (New England Nucleo ide, Bos on, MA, USA). The annealing empe a u e was 55°C o all p ime se s (excep o NF1, i was 52°C), and he numbe o cycles was expe imen ally op imized o 26. Diaz-Cano e al. •Clonali y and Tumo Supp esso Genes in CCH The Jou nal o Clinical Endoc inology & Me abolism, Augus 2001, 86(8):3948–3957 3949 Downloaded om h ps://academic.oup.com/jcem/a icle/86/8/3948/2849067 by Uni e sidad de Se illa use on 06 Feb ua y 2023 Immunohis ochemical exp ession o MIB-1 and cell cycle egula o s A e quenching endogenous pe oxidase ac i i y wi h 0.5% H 2 O 2 - me hanol (10 min.), he an igens we e e ie ed by mic owa ing sec ions in 10 mmol/li e ci a e bu e (pH 6.0; 750 W, 2 cycles o 5 min) and he sec ions we e ans e ed o a mois chambe o he emaining s eps (34). The issue sec ions we e incuba ed o e nigh a 4 C wi h he co - esponding p ima y monoclonal an ibody (no mal pRB1 a 5 ␮ g/ml, abno mal p53 a 1 ␮ g/ml, and cyclin D1, p21 WAF1 , MDM-2, and MIB-1 a 2.5 ␮ g/ml) (Oncogene Science, Inc. and Calbiochem, Camb idge, MA). Nonspeci ic binding was blocked p e ea ing he sec ions wi h dilu ed ho se se um (1:100) and he signal ampli ied wi h bio inyla ed an imouse seconda y an ibody (1:200) and pe oxidase-labeled a idin- bio in complex (1:100) (Vec o Labo a o ies, Inc., Bu lingame, CA). All eac ions we e de eloped unde mic oscopic con ol wi h 3,3⬘-diamino- benzidine as ch omogen and coun e s ained wi h hema oxylin. App o- p ia e posi i e ( eac i e lymph node) and nega i e (omi ing he p ima y an ibody) con ols we e simul aneously un. The immunohis ochemical exp ession was sc eened and sco ed as desc ibed o he ISEL analysis. S a is ical analysis o quan i a i e a iables ANOVA and S uden ’s es s we e applied o assess he di e ences o mo phome ic ea u es in G 0 /G 1 cells by DNA con en and conside ed signi ican i Pless han 0.05. Resul s A he ime o hy oidec omy, he pa ien s aged 4–8y and he e was no clinical o labo a o y e idence o ad enal o pa a hy oid pa hology. All pa ien s had bila e al CCH, as- socia ed wi h MTC in wo cases (CCH-10 and CCH-11). Two cases ( ou CCH oci) we e excluded om he clonali y assay. Case CCH-6 showed no ge mline RET mu a ion and a poly- clonal seconda y CCH due o ch onic hy oidi is (Fig. 1). Case CCH-2 was nonin o ma i e due o AR locus LOH (Table 2). Monoclonal pa e ns e ealed he same inac i a ed X-ch o- FIG. 1. Allele pa e ns o and ogen e- cep o and his ologic ea u es in CCH. a, Di use and nodula g ow h pa e ns we e obse ed in CCH associa ed wi h MEN-2A and ge mline mu a ion o RET. The a ow poin s calci onin-posi i e C cells (ABC-pe oxidase, ⫻400). b, Di use CCH associa ed wi h ch onic hy oidi is and no ge mline mu a ions in codon 634 o RET highligh ed by he immunohis o- chemical demons a ion o calci onin (a ows). The lymphocy ic in il a e o ch onic hy oidi is (a owheads) is also shown (ABC-pe oxidase, ⫻100). c, Gel pa e ns in CCH and con ols. The panel shows monoclonal pa e n wi h he same XCI in bo h lobes (lanes 1–8, case CCH-1) and polyclonal pa e n in a pa ien wi h ge mline poin mu a ion in codon 634 o RET (lanes 9–18, case CCH-4) and in a pa ien wi h no ge mline mu a ion o RET (lanes 19–22, case CCH-6). Each case shows undiges ed and diges ed (D) lanes o he mic odissec ed sample and i s co esponding con ol. Lane 23 co e- sponds o he nega i e con ol wi h no empla e and lane 24 is he size ma ke (SM). C, Con ol; H, CCH. d, Th ee mono- clonal pa e ns in MEN-2A CCH (cases CCH-3, CCH-8, and CCH-11, espec i e- ly). The panel shows he same X-ch omo- some inac i a ed in bo h hy oid lobes (LL, Le lobe; RL, igh lobe) and he balanced me hyla ion o a ep esen a i e con ol. The addi ional band o he la ge allele in he diges ed lane o igh lobe (CCH-3) and le lobe (CCH-11) is ela ed wi h slippage o Taq polyme ase du ing he ampli ica ion ( he sequence co e- sponded wi h he ampli ied locus). 3950 The Jou nal o Clinical Endoc inology & Me abolism, Augus 2001, 86(8):3948–3957 Diaz-Cano e al. •Clonali y and Tumo Supp esso Genes in CCH Downloaded om h ps://academic.oup.com/jcem/a icle/86/8/3948/2849067 by Uni e sidad de Se illa use on 06 Feb ua y 2023 mosome in bo h lobes om a gi en pa ien , showing ei he he la ge allele ( wo cases, 4 CCH oci) o he smalle allele p e e en ially me hyla ed (six cases, 12 CCH oci; Fig. 1). Ano he pa ien ( wo CCH oci) displayed a polyclonal pa - e n. Assuming equal p obabili y o me hyla ion o each AR allele a he cellula le el, he p obabili y o conco dan me h- yla ion pa e ns o AR alleles will be 0.5 (Fig. 2a). Howe e , issues om in o ma i e emales can be polyclonal, mono- clonal wi h p e e en ial me hyla ion o he la ge allele, o monoclonal wi h he smalle allele p edomina ing (Fig. 2b) (38). Unde hose ci cums ances and assuming independen me hyla ion o each allele, equal a p io i p obabili y should be expec ed o each chance and issue (P⫽1/3). In issue compa isons om in o ma i e pa ien s ( wo di e en alleles p esen ), conco dan allele pa e ns would expec ed in 2(1/3) n , being 2 ⫽numbe o alleles and n ⫽numbe o lesions compa ed. The e o e, iden ical pa e ns could be andomly ound in 2(1/3) 2 ⫽0.22 when wo issue samples a e compa ed (Fig. 2b). The e o e, he p obabili y o an- domly inding iden ical allele pa e ns in issues om eigh emales would be 0.22 8 ⫽5.49 10 ⫺6 . Any issue-speci ic a i- abili y in DNA me hyla ion was excluded using mul iple ma ched con ols o he same sample size. The polyclonal case (CCH-4, Table 2) could no be accu a ely assessed o his pu pose, due o he absence o gel pa e n di e ences be ween ue polyclonal p oli e a ion and p oli e a ion o wo clones e ealing di e en X ch omosome inac i a ed (Fig. 2b). All cases showed gene ic abno mali y in a leas one TSG locus, especially TP53 LOH (Table 2). LOH/SNP analyses showed TP53 abno mali ies in 6 o 10 in o ma i e cases (12 o 20 CCH oci, 60%), RB1 al e a ions in 4 o 7 in o ma i e pa ien s (6 o 14 CCH oci, 43%), and WT1 dele ions in 2 o 10 in o ma i e pa ien s (2 o 20 CCH oci, 10%) (Table 2, Fig. 3). No al e a ions we e de ec ed in NF1 locus. TP53 abno - mali ies we e conco dan ly de ec ed in bo h lobes in all pa- ien s in a leas one polymo phic egion. Disco dan gene ic esul s we e obse ed only in wo pa ien s o RB1 (SNP) and in wo pa ien s o WT1 (LOH), and coexis en TP53 and RB1 gene ic al e a ions in wo pa ien s (Table 2). The DNA con en analysis e ealed diploid G 0 /G 1 cells in all cases, hypodiploid G 0 /G 1 cells (DNA index ⬍0.9) in 14 o 20 (70%) CCH oci, and hype diploid G 0 /G 1 cells (1.15 ⬍ DNA index ⬍1.85) in 1 o 20 (5%) CCH oci (Table 3). Hypodiploid G 0 /G 1 cells e ealed a ela i ely dec eased nu- clea a ea (35.11 ⫾4.20 ␮ m (2) and a signi ican ly highe nuclea a ea o DNA index a io [43.12 ⫾3.59 ␮ m (2), P⬍ 0.003) han con ol C cells [36.48 ⫾3.98 ␮ m (2), 37.22 ⫾4.06 ␮ m 2 , espec i ely]. Low p oli e a ion indices we e ob ained om he DNA his og am analysis (⬍5% o monoclonal oci and 7.6–8.3% o polyclonal oci). MIB-1 and cell cycle egula o immunoexp essions and ISEL e ealed a he e o- geneous ma ke dis ibu ion (Fig. 4). Bo h p oli e a ion and apop osis indices we e low, especially he ISEL index (Table 3). Rema kably, no p53 exp ession was de ec ed in CCH oci (Fig. 4). Discussion CCHs in MEN 2A pa ien s we e ound monoclonal and showed he same inac i a ed X-ch omosome in bo h lobes and mul iple TSG abno mali ies. These indings and he down- egula ion o apop osis a e consis en wi h an in a- epi helial neoplasia and sugges ha p og essional gene ic e en s occu ea ly in hy oid de elopmen . The same AR allele was p e e en ially me hyla ed in bo h lobes in monoclonal CCHs (eigh o nine in o ma i e pa- ien s, 89%; Table 2), sugges ing ha a common p ogeni o con ibu ed o hose lesions (25, 39, 40), and suppo a neo- plas ic na u e. These conco dan pa e ns also suppo a clonal expansion o C-cell p ecu so s be ween he andom XCI and he p ecu so mig a ion in o he hy oid anlage (⬃15 TABLE 2. Analysis o polymo phic DNA egions o AR gene and TSG in CCH oci om pa ien s wi h MEN-2A and poin mu a ion in codon 634 Sample Me hyla ion o AR alleles TP53 (1) TP53 (2) RB1 WT1 NF1 CCH1-LL Unbalanced LOH-S ROH ROH ROH ROH CCH1-RL Unbalanced LOH-S ROH ROH ROH ROH CCH2-LL a NI-LOH ROH ROH NI LOH-L ROH CCH2-RL a NI-LOH ROH ROH NI ROH ROH CCH3-LL Unbalanced NI ROH LOH-L ROH ROH CCH3-RL Unbalanced NI ROH LOH-L ROH ROH CCH4-LL a Balanced LOH-S NI ROH/SNP-SL ROH ROH CCH4-RL a Balanced LOH-S NI ROH ROH ROH CCH5-LL Unbalanced LOH-S ROH ROH ROH ROH CCH5-RL Unbalanced LOH-S ROH ROH ROH ROH CCH7-LL a Unbalanced ROH ROH NI ROH ROH CCH7-RL a Unbalanced ROH ROH NI LOH-S ROH CCH8-LL a Unbalanced LOH-S ROH ROH/SNP-L ROH ROH CCH8-RL a Unbalanced LOH-S ROH ROH ROH ROH CCH9-LL Unbalanced NI ROH LOH-S ROH ROH CCH9-RL Unbalanced NI ROH LOH-S ROH ROH CCH10-LL b Unbalanced LOH-S NI NI ROH NI CCH10-RL b Unbalanced LOH-S NI NI ROH NI CCH11-LL b Unbalanced LOH-L NI ROH ROH NI CCH11-RL b Unbalanced LOH-L NI ROH ROH NI ROH, Re en ion o he e ozygosi y; LL, le lobe; RL, igh lobe; L, la ge allele; S, smalle allele; NI, nonin o ma i e. a Cases CCH-2, -4, -7, and -8 e ealed gene ic he e ogenei y in le and igh lobes. b Cases CCH-10 and CCH-11 e ealed coexis en medulla y hy oid ca cinoma. Case CCH-6 showed no RET poin mu a ions in codon 634. Diaz-Cano e al. •Clonali y and Tumo Supp esso Genes in CCH The Jou nal o Clinical Endoc inology & Me abolism, Augus 2001, 86(8):3948–3957 3951 Downloaded om h ps://academic.oup.com/jcem/a icle/86/8/3948/2849067 by Uni e sidad de Se illa use on 06 Feb ua y 2023 FIG. 2. Compa ison o XCI pa e ns. Only he ac i e and nonme hyla ed and ogen ecep o allele is exp essed in a gi en cell, and can be dis inguished by he leng h o he polymo phic ma ke in he exon 1 in in o ma i e pa ien s. Howe e , compa a i e analyses gi e di e en possibili ies a he cellula and issue le els. a, The cell-cell compa ison shows ou po en ial possibili ies, wo e ealing di e en X-ch omosomes inac i a ed and sugges ing di e en p ogeni o s (50%), and o he wo displaying he same X-ch omosome inac i a ed (ei he he la ge allele o he smalle allele, 50%), consis en wi h common p ogeni o . b, Th ee possibili ies can be ound a he issue le el, depending on he inac i a ion o only one allele in monoclonal issues ( he la ge o he smalle one) o bo h alleles in polyclonal issues. The compa ison o wo gi en issues (1 and 2) would hen esul in nine heo e ical op ions (each one ep esen ed in di e en gels); only wo o hem a e consis en wi h a common p ogeni o con ibu ing he cells, bo h issues exp essing ei he he la ge o he smalle allele (pink). I andom inac i a ion o bo h alleles is p esen (g een), no clonali y assessmen can be achie ed because he p ogeni o cell can be ei he common o di e en . The emaining cases (black) ep esen issue de elopmen om di e en p ogeni o s. 3952 The Jou nal o Clinical Endoc inology & Me abolism, Augus 2001, 86(8):3948–3957 Diaz-Cano e al. •Clonali y and Tumo Supp esso Genes in CCH Downloaded om h ps://academic.oup.com/jcem/a icle/86/8/3948/2849067 by Uni e sidad de Se illa use on 06 Feb ua y 2023 wk, Fig. 5) (11, 41). Suppo ing his poin , pa ien s o hese kind ed also e ealed conco dan monoclonal pa e ns in 90% nodules om ad enal medulla y hype plasias and mi- c osa elli e abno mali ies in a leas one TSG in 75% pheo- ch omocy omas (26, 42). Ea ly clonal expansions in neu al c es de i a i es could explain hose indings, bu o he al- e na i e explana ions o he monoclonal pa e n mus be excluded. The i s issue o conside is he pa ch size concep o con iguous cellula egions o he same lineage (10, 11, 13, 25). The mosaic pa ch size could be he cause o monoclonal pa e ns in small samples, bu i would no explain he con- co dan pa e n in bo h lobes, especially wi h samples in- cluding a leas 100 cells (10, 38, 43). In addi ion, echnical aspec s such as PCR bias agains he la ge allele could con- ibu e o p e e en ial ampli ica ion o he smalle allele. Ou DNA ex ac ion p o ocol included a long p o ein diges ion and e ie ed DNA o ⬃1 kb (da a no shown), excluding deg aded DNA as cause (13, 22, 25, 28, 44). Ou PCR design also included long dena u a ion and ex ension in he i s h ee cycles and 7-deaza-dGTP in he ampli ica ion mix u e o a oid de ec i e ampli ica ion o CG- ich sequences (Table 1) (10, 13, 24, 25, 40). These echnical conside a ions could easonably exclude he PCR bias as cause o conco dan monoclonal pa e ns, as e ealed by he wo cases showing he la ge allele me hyla ed (Fig. 1). The polyclonal pa e n in pa ien s wi h ge mline RET mu- a ion (CCH-4) needs some conside a ions. XCI assays can esul in polyclonal pa e ns i he es ic ion endonuclease diges ion is incomple e o he a ge DNA is hype me hyl- a ed (10, 11, 13, 25, 28). Subop imal enzyma ic diges ion may change he clonali y pa e n o monoclonal issues (10, 28), bu ha possibili y was excluded keeping in e nal con ols o endonuclease diges ion. Al hough some DNA dena u a ion mus be expec ed du ing embedding and ex ac ion, bo h he long diges ion (16 h) and he HhaI ac i i y on single-s anded DNA assu e comple e diges ion. Repea ed polyclonal pa - e ns we e ob ained using nonboiled empla es. We a e cu - en ly es ing me hyla ion in hese lesions. Likewise, any signi ican con amina ion can explain polyclonal esul s as hose epo ed in MEN 2 MTC (45), opposed o he clonal o igin p e iously epo ed in inhe i ed MTC and pheoch o- mocy omas (46, 47). Con amina ion was excluded by ca e ul mic odissec ion unde mic oscopic con ol and mul iple sampling (10, 11). Finally, he coexis ence o TP53 and RB1 abno mali ies (Table 2) suppo a neoplas ic na u e and ag ees wi h he high MTC incidence (60%) in double he - e ozygous animals o TP53 and RB1 (48). In his scena io, he polyclonal pa e n could be he esul o ei he C-cell p e- cu so expansions be o e he andom XCI o clonal p oli - e a ion o cells showing di e en inac i a ed X-ch omosome (Fig. 5) (11, 45). The inhe i ed gene ic de ec can de e mine an accele a ed p ocess o soma ic al e a ions and DNA dele ions (hypodip- loid G 0 /G 1 cells and TSG-LOH) (11, 13), a o ing cellula he e ogenei y and molecula p og ession (11, 25). Ou esul s sugges ha p og ession in CCHs in ol es mul iple genes, especially TP53 and RB1 as epo ed in MTC (48, 49). Al- hough he numbe o cases is no su icien o exclude chance, he conco dan TP53 dele ion ound in h ee pa ien s (Table 2) sugges s ha TP53 dele ions ha e occu ed a a poin in emb yonic de elopmen be o e di e gence o he C-cell p ecu so s o each hy oid lobe in his pa ien subse . The e o e, hese mu a ions should occu ea lie han p e i- ously hough and co ela e wi h he clinical de ec ion o MTC in pa ien s younge han 5 y . Con e sely, he he e - ogenei y o RB1 and WT1 al e a ions and in agene ic poly- mo phism o TP53 mu a ions suppo hei soma ic na u e and a so o “ho spo ” o such mu a ions. WT1 and RET ha e al eady shown collabo a i e e ec s, as e ealed by he p esence o geni ou ina y abno mali ies in RET o GDNF knockou mice (50, 51). In con as , no pa ien in his se ies showed NF1 al e a ions, al hough his locus has e ealed FIG. 3. Mic osa elli e analysis o umo supp esso genes (1, TP53 (1); 2, TP53 (2); 3, RB1; 4, WT1; 5, NF1) in CCH. LOH o SNP was de ec ed in polymo phic DNA egions o umo supp esso genes. The a ows poin o he he e ogeneous gene ic lesions in TP53 (LOH; a and b) and RB1 (SNP; b) obse ed. Diaz-Cano e al. •Clonali y and Tumo Supp esso Genes in CCH The Jou nal o Clinical Endoc inology & Me abolism, Augus 2001, 86(8):3948–3957 3953 Downloaded om h ps://academic.oup.com/jcem/a icle/86/8/3948/2849067 by Uni e sidad de Se illa use on 06 Feb ua y 2023 al e a ions in neu al c es umo s (especially pheoch omo- cy omas) (52, 53). Ea ly clonal expansions in neu al c es de i a i es accumula ing gene ic al e a ions would be he exp ession o mul is ep umo igenesis and is also suppo ed by he epo ed abno mali ies o loci on ch omosomes 1 and 3 (54, 55). The p esence o addi ional soma ic RET mu a ions has been epo ed in MTC and should be conside ed exp es- sion o gene ic p og ession and umo cell selec ion a he han e idence agains he clonali y o hose lesions (54). An- o he aspec o conside is he o igin o all pa ien s om a single amily and he in luence o amilial e ec s on he esul s. Howe e , he amilial gene ic backg ound canno explain he associa ion o un ela ed aspec s such as conco - dan pa e ns o clonali y and he p esence o mul iple TSG abno mali ies. ATP53- and RB1-knockou model e ealed MTC wi hin he i s 9–15 mon hs o mouse li e (60–83%) wi h he highes incidence in double he e ozygous animals (mu a ed RB1 and TABLE 3. Resul s o DNA-Ploidy, MIB-1, and cell cycle egula o immunoexp ession, and ISEL in CCH om MEN-2A pa ien s wi h poin mu a ion in codon 634 a Case no. DNA-Ploidy MIB-1 (%) MDM2 (%) pRB (%) p21 WAF1 (%) ISEL (%) CCH-1 Hypodiploid 3.1 4.2 4.8 1.4 0.03 CCH-2 Hypodiploid 2.5 6.2 4.0 2.1 0.05 CCH-3 Hypodiploid 2.1 4.3 4.3 1.7 0.03 CCH-4 Hypodiploid 3.7 5.5 5.1 1.7 0.04 CCH-5 Diploid 6.8 5.6 4.6 1.6 0.02 CCH-7 Diploid 5.9 3.1 7.0 1.4 0.03 CCH-8 Hypodiploid 4.3 3.2 8.7 1.2 0.01 CCH-9 Hypodiploid 4.6 3.0 8.1 1.2 0.02 CCH-10 b Hypodiploid 4.3 4.6 5.9 1.5 0.03 CCH-11 b Hypodiploid 4.5 4.4 6.6 1.5 0.02 A ⫾SD 4.18 ⫾1.44 4.41 ⫾1.11 5.91 ⫾1.64 1.53 ⫾0.27 0.03 ⫾0.01 a These sco es show he combined esul s CCH a eas om bo h lobes in each pa ien because o he small size ha a oid o sc een 50 HPF/CCH ocus. b Cases CCH-10 and CCH-11 showed coexis en MTC oci (excluded in he sc eening). Case CCH-6 did no show RET poin mu a ions in codon 634. FIG. 4. Immunohis ochemis y and ISEL in CCHs. C cells show cy oplasmic MIB-1 s aining (b) along wi h ela i ely low nuclea p21 WAF1 labeling (e) and ISEL index (d). c, No abno mal p53 immunos aining was de ec ed. (a, H&E, ⫻400; b, c, and e, ABC-pe oxidase and 3,3⬘- diaminobenzidine, ⫻100; d, digoxigenin end labeling de eloped wi h alkaline phospha ase and NBT-X phospha e, ⫻100). The a ows poin o posi i e signal. 3954 The Jou nal o Clinical Endoc inology & Me abolism, Augus 2001, 86(8):3948–3957 Diaz-Cano e al. •Clonali y and Tumo Supp esso Genes in CCH Downloaded om h ps://academic.oup.com/jcem/a icle/86/8/3948/2849067 by Uni e sidad de Se illa use on 06 Feb ua y 2023 TP53) (48). The absence o no mal TP53 unc ion would p o- ec umo cells om apop osis (56) and con ibu es o an inc eased RB1 mu a ion a e in ad anced umo s (48). Re- ma kably low ISEL indices and low- o-no mal p oli e a ion indices we e obse ed in his CCH se ies. The educed cel- lula loss associa ed wi h longe su i al o ini ia ed/ mu a ed cells would p omo e p og ession om p ein asi e s ages o e an ex ended pe iod o ime (11, 13, 57), and gene ic ins abili y (49). T ans o ming mu a ions ha escape cell epai sys ems will accumula e and p omo e ea ly p e- neoplas ic oci only a e inhibi ing apop osis (11, 13, 58). The pos mi o ic epai ing sys ems a e less e ec i e in low- p oli e a ion lesions (59), explaining he p esence o he e - ogeneous and non andomly dis ibu ed mu a ions (60). In con as , high p oli e a ion and mu a ion a es de e mine homogeneous DNA damage and ac i a ion o apop osis (57, 60, 61). No mal pRB1 and low p21 WAF1 exp essions ound in his se ies a e consis en wi h low p oli e a ion and apop o ic indices. Only highe p oli e a ion a es and inc eased p21 WAF1 would inac i a e pRB1 esul ing in highe apop osis (59). The combined deli e y o wo coope a i e genes, like p21 WAF1 and TP53, would lead he cyclin D/CDK-pRB1 pa h- way owa d g ow h a es and apop osis (62, 63). Some clinically ele an opic s ill emains unknown. RET encodes a ecep o y osine kinase ha is exp essed in neu al c es de i a i es and hei co esponding umo s, MTC, and pheoch omocy oma (64). RET mu a ions o codon 634 cause ecep o dime iza ion and cons i u i ely ac i a e he y osine kinase (65), he eby mimicking he e ec caused by he bind- ing o a ligand o he ecep o . In gene al, a single mu a ion causing a neoplasm is unlikely, and o he gene ic al e a ions (TP53 and RB1 in his se ies) would be equi ed (11). Al- hough no mal issues a e he e ogeneous and show andom LOH and DNA dele ions in 4–20% (66–68), ha le el o FIG. 5. a, Rela i e iming o clonal expansion o C cells and he loca ion o C-cell p ecu so s in he hy oid anlage Clonal Signal and expansion o C cells a e loca ing in he hy oid would make unlikely cells showing he same allele me hyla ed (le ). The opposi e scena io should be conside ed o consis en ly conco dan me hyla ion pa e n o and ogen ecep o alleles (MePARA): clonal signal and expansions should happen be o e he loca ion o C-cell p ecu so s in he hy oid ( igh ). b and c, Rela i e iming o clonal expansion o C cells and XCI. b, Monoclonal p oli e a ion equi es an al eady es ablished inac i a ion o he X ch omosome be o e he clonal expansion o esul in monoclonal gel pa e n. c, I he clonal signal and expansion an ecedes he XCI, selec ed cells can po en ially inac i a e any o he alleles and show a “pseudopolyclonal” gel pa e n. Diaz-Cano e al. •Clonali y and Tumo Supp esso Genes in CCH The Jou nal o Clinical Endoc inology & Me abolism, Augus 2001, 86(8):3948–3957 3955 Downloaded om h ps://academic.oup.com/jcem/a icle/86/8/3948/2849067 by Uni e sidad de Se illa use on 06 Feb ua y 2023 soma ic gene ic al e a ions canno explain his se ies inci- dence o TSG abno mali ies, especially o CCH oci wi h al e a ions in wo loci (CCH-4 and CCH-8). Simila ly, so- ma ic missense mu a ions a codon 918 o RET ha e been epo ed in 20% MTC and in a sample o CCH om MEN 2 pa ien s (54). These ac o s and he p eexis ing ge mline mu- a ion o RET may con ibu e o C-cell umo igenesis. Those indings sugges ha CCHs in MEN 2A a e in aepi helial neoplasias. In conclusion, gene ic (monoclonal p oli e a ion wi h mul- iple TSG abno mali ies) and kine ic (down- egula ed apo- p osis) e idences sugges ha MEN 2A CCHs a e in aepi- helial neoplasias. Conco dan XCI pa ens in bo h lobes suppo ea ly clone expansions p eceding di e gence o C- cell p ecu so s o each hy oid lobe; his may ep esen a pa adigm o o he ge mline mu a ions du ing emb yo- genesis. Acknowledgmen s Recei ed Ap il 12, 2000. Accep ed Ap il 2, 2001. Add ess all co espondence and eques s o ep in s o: Sal ado J. Diaz-Cano, M.D., Ph.D., Depa men o His opa hology and Mo bid Ana omy, The Royal London Hospi al, Whi echapel, London E1 1BB, Uni ed Kingdom. E-mail: [email p o ec ed]. This wo k was p esen ed in pa as abs ac o ms a he Uni ed S a es and Canadian Academy o Pa hology (USCAP) Mee ings in O lando, Flo ida (Ma ch 1997), and Bos on, Massachuse s (Feb ua y 1998). This wo k was pe o med a he Depa men s o Pa hology, Tu s Uni e si y– New England Medical Cen e (Bos on, MA) and S . Ba holomew’s and he London NHS T us (London, UK). Re e ences 1. 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Hum Gene 99:202–205 3956 The Jou nal o Clinical Endoc inology & Me abolism, Augus 2001, 86(8):3948–3957 Diaz-Cano e al. •Clonali y and Tumo Supp esso Genes in CCH Downloaded om h ps://academic.oup.com/jcem/a icle/86/8/3948/2849067 by Uni e sidad de Se illa use on 06 Feb ua y 2023