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. San o o M, Ca lomagno F, Romano A, e al. 1995 Ac i a ion o RET as a
dominan ans o ming gene by ge mline mu a ions o MEN2A and MEN 2B.
Science 267:381–383
2. Mulligan LM, Kwok JB, Healey CS, e al. 1993 Ge m-line mu a ions o he RET
p o o-oncogene in mul iple endoc ine neoplasia ype 2A. Na u e 363:458–460
3. Lips CJ, Lands a e RM, Hoppene JW, e al. 1994 Clinical sc eening as
compa ed wi h DNA analysis in amilies wi h mul iple endoc ine neoplasia
ype 2A. N Engl J Med 331:828–835
4. Nowell PC 1976 The clonal e olu ion o umo cell popula ions. Science 194:
23–28
5. Fialkow PJ 1976 Clonal o igin o human umo s. Biochim Biophys Ac a 458:
283–321
6. Kapple JW 1971 The 5-me hylcy osine con en o DNA: issue speci ici y.
J Cell Physiol 78:33–36
7. Lai d PW, Jaenish R 1994 DNA me hyla ion and cance . Hum Mol Gene
3:1487–1495
8. Lyon MF 1992 Some miles ones in he his o y o X-ch omosome inac i a ion.
Annu Re Gene 26:17–28
9. S e nlich M, Mi ell C, Sa a ians S, Ba sky SH 1994 A no el s a egy o he
in es iga ion o clonali y in p ecance ous disease s a es and ea ly s ages o
umo p og ession. Biochem Biophys Res Commun 199:511–518
10. Diaz-Cano SJ, Blanes A, Wol e HJ 2001 PCR echniques o clonali y assays.
Diagn Mol Pa hol 10:24–33
11. Diaz-Cano SJ 1998 Clonali y s udies in he analysis o ad enal medulla y
p oli e a ions: Applica ion p inciples and limi a ions. Endoc Pa hol 9:301–316
12. Salomon RN, Diaz-Cano S 1995 In oduc ion o apop osis. Diagn Mol Pa hol
4:235–238
13. Diaz-Cano SJ, de Miguel M, Blanes A, Tashjian R, Gale a H, Wol e HJ 2000
Clonali y as exp ession o dis inc i e cell kine ics pa e ns in nodula hype -
plasias and adenomas o he ad enal co ex. Am J Pa hol 156:311–319
14. G aze K, Spile IJ, Tashjian AH, J , e al. 1978 Na u al his o y o amilial
medulla y hy oid ca cinoma: e ec o a p og am o ea ly diagnosis. N Engl
J Med 299:980–985
15. Gagel RF, Tashjian J AH, Cummings T, e al. 1988 The clinical ou come o
p ospec i e sc eening o mul iple endoc ine neoplasia ype 2 a: an 18 yea
expe ience. N Engl J Med 318:478–485
16. Gagel RF, Co e GJ, Ma ins Bugalho MJ, e al. 1995 Clinical use o molecula
in o ma ion in he managemen o mul iple endoc ine neoplasia ype 2A.
J In e n Med 238:333–341
17. Eng C, Clay on D, Schu enecke I, e al. 1996 The ela ionship be ween
speci ic RET p o o-oncogene mu a ions and disease pheno ype in mul iple
endoc ine neoplasia ype 2. In e na ional RET mu a ion conso ium analysis.
JAMA 276:1575–1579
18. Wol e HJ, Mel in KE, Ce i-Skinne SJ, e al. 1973 C-cell hype plasia p e-
ceding medulla y hy oid ca cinoma. N Engl J Med 289:437–441
19. Biddinge PW, Ray M 1993 Dis ibu ion o C cells in he no mal and diseased
hy oid gland. Pa hol Annu 28 :205–229
20. Pe y A, Molbe g K, Albo es-Saa ed a J 1996 Physiologic e sus neoplas ic
C-cell hype plasia o he hy oid: sepa a ion o dis inc his ologic and biologic
en i ies. Cance 77:750–756
21. McDe mo MB, Swanson PE, Wick MR 1995 Immunos ains o collagen ype
IV disc imina e be ween C-cell hype plasia and mic oscopic medulla y ca -
cinoma in mul iple endoc ine neoplasia, ype 2a. Hum Pa hol 26:1308–1312
22. Diaz-Cano SJ, B ady SP 1997 DNA ex ac ion om o malin- ixed pa a in-
embedded issues: p o ein diges ion as a limi ing s ep o e ie al o high
quali y DNA. Diagn Mol Pa hol 6:342–346
23. Mu e GL, Chapono ML, Fle che JA 1995 A polyme ase chain ea ion assay
o non- andom X ch omosome iden i ies monoclonal endome ial cance and
p ecance s. Am J Pa hol 146:501–508
24. Mu e GL, Boyn on KA 1995 PCR bias in ampli ica ion o AR alleles, a
inucleo ide epea ma ke used in clonali y s udies. Nucleic Acids Res 23:
1411–1418
25. Diaz-Cano SJ, Blanes A, Rubio J, Ma illa A, Wol e HJ 2000 Molecula e o-
lu ion and in a umo he e ogenei y by opog aphic compa men s in muscle-
in asi e ansi ional cell ca cinoma o he u ina y bladde . Lab In es 80:
279–289
26. Diaz-Cano SJ, de Miguel M, Blanes A, Tashjian R, Gale a H, Wol e HJ 192
2000 Clonal pa e ns in phaech omocy omas and MEN-2A ad enal medulla y
hype plasias: his ologic and kine ic co ela es. J Pa hol 192:221–228
27. Cawkwell L, Bell SM, Lewis FA, Dixon MF, Taylo GR, Qui ke P 1993 Rapid
de ec ion o allele loss in colo ec al umou s using mic osa elli es and luo-
escen DNA echnology. B J Cance 67:1262–1267
28. Diaz-Cano SJ 2000 Designing a molecula analysis o clonali y in umou s.
J Pa hol 191:343–344
29. Bibbo M, Ba els PH, Dy ch HE, Wied GL 1991 Cell image analysis. In: Bibbo
M, ed. Comp ehensi e cy opa hology. Philadelphia: W.B. Saunde s Co.;
965–983
30. She wood SW, Schimke RT 1995 Cell cycle analysis o apop osis using low
cy ome y. In: Schwa z LM, Osbo ne BA, eds. Cell dea h. San Diego: Aca-
demic P ess; 77–97
31. Koch M, de Miguel M, Ho¨ le H, Diaz-Cano SJ 2000 Kine ic p o iles o
in aepi helial and in asi e p os a ic neoplasias: he key ole o down- egu-
la ed apop osis in umo p og ession. Vi chows A ch 436:413–420
32. Wijsman JH, Jonke RR, Keijze R, an de Velde CJH, Co nelisse CJ, an
Die endonck JH 1993 A new me hod o de ec apop osis in pa a in sec ions:
In si u end-labeling o agmen ed DNA. J His ochem Cy ochem 41:7–12
33. Diaz-Cano SJ, Ga cia-Moline M, Ca ney W, Wol e HJ 1997 Bcl-2 exp ession
and DNA agmen a ion in b eas ca cinoma, pa hologic and s e oid ho mone
ecep o s co ela es. Diagn Mol Pa hol 6:199–208
34. Taylo CR, Co e RJ 1994 Immunomic oscopy: a diagnos ic ool o he su gical
pa hologis s. Philadelphia: W.B. Saunde s Co
35. Simpson JF, Du PL, Page DL 1992 Exp ession o mi osis pe housand cells
and cell densi y in b eas ca cinomas. A p oposal. Hum Pa hol 23:608–611
36. Diaz-Cano SJ, Leon MM, de Miguel M, Gale a-Da idson H, Wol e HJ 1996
Mi o ic index quan i ica ion: di e en app oaches and hei alue in ad enal
co ical p oli e a i e lesions. Lab In es 74:170A
37. Ha jacek M, Diaz-Cano S, Alman BA, e al. 2000 P ominen exp ession o
mRNA o p oin lamma o y cy okines in syno ium in pa ien s wi h ju enile
heuma oid a h i is o ch onic lyme a h i is. J Rheuma ol 27:497–503
38. Diaz-Cano SJ, Wol e HJ 1997 Clonali y in Kaposi’s sa coma [Le e ; commen ].
N Engl J Med 337:571–572
39. Quade BJ, McLachlin CM, So o-W igh V, Zucke man J, Mu e GL, Mo on
CC 1997 Dissemina ed pe i oneal leiomyoma osis. Clonali y analysis by X
ch omosome inac i a ion and cy ogene ics o a clinically benign smoo h mus-
cle p oli e a ion. Am J Pa hol 150:2153–2166
40. Alman BA, Paje ski ME, Diaz-Cano S, Co boy K, Wol e HJ 1997 Agg essi e
ib oma osis (desmoid umo ) is a monoclonal diso de . Diagn Mol Pa hol
6:98–101
41. Sugiyama S 1969 Emb yonic de elopmen o human hy oid gland and ul-
imob anchial body. Ac a Endoc 179(Suppl 138):179
42. Diaz-Cano SJ, Blanes A 1999 In luence o in a umou he e ogenei y in he
in e p e a ion o ma ke esul s in phaeoch omocy omas. J Pa hol 189:627–628
43. Gale RE, Wheadon H, Boulos P, Linch DC 1994 Tissue speci ici y o X-ch o-
mosome inac i a ion pa e ns. Blood 83:2899–2905
44. B ady SP, Mag o CM, Diaz-Cano SJ, Wol e HJ 1999 Analysis o clonali y o
a ypical cu aneous lymphoid in il a es associa ed wi h d ug he apy by PCR/
DGGE. Hum Pa hol 30:130–136
45. Fe a is AM, Mange ini R, Gae ani GF, Romei C, Pinche a A, Pacini F 1997
Polyclonal o igin o medulla y ca cinoma o he hy oid in mul iple endoc ine
neoplasia ype 2. 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
