Ret oncogene activation in human thyroid
neoplasms is restricted to the papillary cancer
subtype.
M Santoro, … , G Della Porta, N Berger
J Clin Invest. 1992;89(5):1517-1522. https://doi.org/10.1172/JCI115743.
We have recently reported the activation of a new oncogene in human papillary thyroid carcinomas. This oncogene, papillary thyroid carcinoma (PTC), is a novel rearranged version of the ret tyrosine-kinase protooncogene. Thyroid neoplasms include a broad spectrum of malignant tumors, ranging from well-differentiated tumors to undifferentiated anaplastic carcinomas. To determine the frequency of ret oncogene activation, we analyzed 286 cases of human thyroid tumors of diverse histologic types. We found the presence of an activated form of the ret oncogene in 33 (19%) of 177 papillary carcinomas. By contrast, none of the other 109 thyroid tumors, which included 37 follicular, 15 anaplastic, and 18 medullary carcinomas, and 34 benign lesions, showed ret activation.
Research Article
Find the latest version:
Ret Oncogene
Activation in Human
Thyroid Neoplasms
Is Restricted
to
the Papillary
Cancer
Subtype
Massimo Santoro,* Francesca Carlomagno,* IanD.Hay,t Marie A.
Herrmann,*
MicheleGrieco,*Rosamarina Melillo,* Marco A. Pierotti,'Italia Bongarzone,' Giuseppe DellaPorta,*NicoleBerger,'1 Jean LouisPeix,11 ChristianPaulin,11 Nicole Fabien,11 Giancarlo Vecchio,* Robert B.Jenkins,*andAlfredo Fusco'*Centro di Endocrinologia ed Oncologia SperimentaledelCNR,%DipartimentodiBiologiaePatologiaCellulareeMolecolare, II
Facoltd' di MedicinaeChirurgia, UniversitdldiNapoli, 80131 Naples, Italy;*DivisionsofEndocrinologyand Internal Medicineand Laboratory Genetics, Mayo Clinic, Rochester, Minnesota55905;
IDivisione
diOncologia Sperimentale A,Istituto Nazionale Tumori, 20133Milan, Italy; 1Laboratoire d'Anatomie Pathologique, ServicedeChirurgie, HopitaldeL'Antiquaille,69321 LyonCedex05France;Laboratoire de Cytologie, Hopital Jules Courmont 69310, Pierre Benite, France;and'Dipartimentodi MedicinaSperimentale eClinica, Facoltd di MedicinaeChirurgiadiCatanzaro, UniversitdidegliStudi diReggio Calabria, 88100 Catanzaro, Italy
Abstract
We haverecently reported the activation ofa newoncogene in humanpapillarythyroid carcinomas. This oncogene, papillary thyroid carcinoma(PTC), is a novel rearranged version of the rettyrosine-kinase protooncogene.Thyroidneoplasms include abroad spectrum ofmalignanttumors,ranging from well-dif-ferentiated tumors toundifferentiated anaplastic carcinomas. To determine thefrequency of ret oncogeneactivation,we
ana-lyzed 286 cases of humanthyroid tumors of diversehistologic
types. We found the presence ofanactivated formoftheret
oncogene in 33(19%)of 177papillarycarcinomas.Bycontrast, none of the other 109thyroidtumors, which included 37 follicu-lar, 15anaplastic,and18medullarycarcinomas,and34 benign lesions, showed ret activation.(J.Clin. Invest. 1992.
89:1517-1522.)Key words: carcinoma * gene rearrangement *ret pro-tooncogene *tyrosinekinase * PTC
Introduction
There isgrowing evidence thattumordevelopmentand pro-gression may result from a series ofgenetic alterations that
affectthe normalmechanismscontrollingcellproliferation (1).
Moleculareventsthat have beenidentified includeinactivation
oftumor-suppressor genes(2),aswellasactivatingmutations
ofcellular protooncogenes (3). In somecases, specific
onco-genes arealtered inspecifictumors. Thus,theN-mycand
c-erb-B2/neu
oncogenesareamplifiedin neuroblastomas(4)and in breast carcinomas(5),respectively. Similarly,c-myc andc-ablareinvolved in the chromosomaltranslocationswhich
char-acterize Burkitt'slymphoma and chronicmyelogenous leuke-mia (3),respectively.Moreover, there is someevidence suggest-ing a specificity for activation ofparticular ras gene family
members in someforms ofcancer:e.g., K-ras inlung, colon, and pancreascarcinomas (6-8), and N-ras in acute myeloid leukemia (9).
Address correspondence and reprint requests to Dr. Massimo Santoro, CentrodiEndocrinologiaedOncologia,Sperimentale del CNR, Uni-versita di Napoli, Via S. Pansini 5, 80131 Naples, Italy.
Receivedfor publication9September1991andinrevisedform 11
December1991.
Thethyroid provides an attractive model to study the steps that are involved in the neoplastic process. Most thyroid neo-plasmsoriginate from a single cell type, the thyroid follicular cell. However,theycomprisea broad spectrumoftumors with differentphenotypic characteristics and variable biological and clinical behavior: i.e., from the benign colloid adenomas, through the slowly progressive differentiated papillary and fol-licular carcinomas, to the invariably fatal anaplastic carci-nomas (10).Papillary and follicular carcinomas are considered to betwodifferent biological entities. Typically, papillary carci-nomaismultifocal. It generally metastasizes to regional lymph nodes, and has been associated with previous radiation expo-sure.Conversely, follicular carcinoma is solitary and encapsu-lated. Itinvades blood vessels and spreads often to bones; its
highest incidence has been reported from endemic goitrous areas(10). It seems probable that the differences observed in
biologicalbehavior could be explained by the involvement of
differentgenetic events in the pathogenesis of these two
differ-ing follicularcell-derived malignancies.
We have recently demonstrated the frequent activation of a newoncogene in human papillary thyroid carcinomas(1 1-13). Wehave shown that this oncogene, named papillary thyroid
carcinoma
(PTC),I
isthe productoftherecombinationofthetyrosine-kinasedomain ofthe ret protooncogenewithathus faruncharacterizedgenethat we have named H4 (13). In
addi-tion, we have observed that an inversion (l0)(ql1.2-q2 1) is present in asignificantnumberofpapillary thyroid carcinomas
(14).The ret and H4 genes are both localized on the long arm of thechromosome 10(15-17),and we have demonstrated that at least infourcases, theinversion on chromosome 10 determines
their fusion(17a).
Toconfirmthefrequency of activation of ret in papillary tumors in awider collection of samples, and to ascertain what role retactivationmay play in the pathogenesis of nonpapillary
thyroid tumors, we have analyzed 286 human thyroid neo-plasms collected at the Mayo Clinic, Rochester, Minnesota, at theHospitals ofLyon, France, at the Istituto Nazionale Tu-moriof Milan, and at the II Medical School of Naples, Italy. In this study we demonstrate that ret oncogene is frequently acti-vated only in the papillary histotype of thyroid carcinomas.
Fromthesefindings, we would suggest that ret activation may representaspecific genetic event that may be of value in the differentialdiagnosis of papillary thyroid cancer in man.
1.Abbreviationsusedinthis paper:PCR,polymerasechainreaction;
PTC,papillary thyroidcarcinoma. J.Clin. Invest.
© The AmericanSocietyfor ClinicalInvestigation,Inc. 0021-9738/92/05/1517/06 $2.00
Figure 1. Schematic ; + + __,,representation of the ge-2 e 10 3 nomic restriction maps
'____ ,I, oftheH4andprotoret
R RR R RR R R R genes. Closed boxes
rep-resent theapproximate
positions ofthecoding
12 7 sequences. Thearrows
RET mark the positions of
R R R R the previouslydescribed
Kb R = Eco~i breakpointsin the two
3 Kb R=EcoRI
genes
(13).
Above the maps areillustratedthe DNAprobes used in thisstudy. They corre-spond to theprobes indicatedinFig. 1 of reference 13. Probe 12 is a1.8-kbpBamHIrestrictionfragment.Therestriction sites shown are R: EcoRI.Methods
DNAextractionand Southern blot analysis. The tumor samples were frozeninliquid nitrogenand stored frozen until DNA extractions were
performed.Thespecimens obtained fromtheMayoClinicwere evalu-atedfor theircontent in tumoral cells. A certaindegreeof
contamina-tionby normal cells was observed. However, DNAextractionswere
performedfrom thosesectionscontainingnotless than 70% of neoplas-ticcells. High molecular weight DNA extraction fromtumorsand Southern blotanalyseswereperformed, accordingtostandard
proce-dures(18).Briefly, 10,ggofDNAweredigested, asrecommendedby
the enzymemanufacturer(Amersham Corp., Arlington Heights, IL;
Promega Biotec., Madison, WI), electrophoresed through0.8% aga-rose,transferredtonylonfilters(Hybond-N;AmershamCorp.),and
hybridizedto32P-labeledprobes bythe randomoligonucleotideprimer
kit (AmershamCorp.). Hybridization andwashingswerecarriedout
9 10 N 14 9 14 N 9 14 N
understringent conditions, as previously described (13). Autoradiogra-phy wasperformed by using Kodak XAR films at -70°C for 1-7 d with
intensifyingscreens.
Transfection assay. The DNAsextracted from tumor specimens weretransfectedontoNIH/3T3cells by the calcium phosphate copre-cipitation technique, following a modified version of the protocol of Chen and Okayama, as previously described (12). Foci of transformed cells wereevaluatedafter2-3 wk.
RNAanalysis and polymerase chain reaction (PCR). RNA extrac-tionwasperformed,aspreviously described(19). The RNA
amplifica-tionwasdonefollowingthemethoddescribedby E. S.Kawasaki(20),
starting from5,goftotal RNA. The sequencesofthe sense
H44-196-213)and theantisenseRET(544-561)-relatedprimers were as follows: H4: 5'-ATTGTCATCTCGCCGTTC-3'; RET: 5'-CTGCTTCAG-GACGTTGAA-3'. Thebreak-pointis at nucleotides 351-352. The ex-pectedamplified product of the PTC mRNAis 363 bp long.
Results
Southern blotanalysis. Theretprotooncogeneencodesa
pro-teinstructurally relatedtotransmembranereceptorswitha
cy-toplasmic tyrosine-kinase domain(21-23). We have demon-stratedthatthe truncation of theNH2-terminal region leadsto
its activationinthyroid papillary carcinomas. This
rearrange-ment occurs in anintronicsequencethat lies between the TK and thetransmembrane-encoding domain of theret
protoon-cogene,and it ispossibletodetectthisrearrangementby South-ern blotanalysisof the tumor DNA(13). Fig. 1 showsa sche-maticrepresentationofthegenomicrestriction maps ofretand
H4, andofthe rearrangements between thetwogenes previ-ously reported (13).
10 10 10
6.3
Kbp-BamH I Hind m
42 N 53 55 42 N 53 55
_~
O r- E IL) *- m
-Ib m
-9.3 Kbp
-6.3Kbp
EcoRI
R R
B B Bgl]IB
R
BB B
R
-9.3 Kbp
-3.7 Kbp
Figure 2.Examples ofret
rearrange-mentinpapillary thyroid
carci-nomascollectedatthe Hospitals of Lyon. Southern blot analysis of DNAextracted from the neoplastic specimens.10 gofDNAwere di-gested with EcoRI, BamHI,or
HindIIl restrictionenzymes
(Amer-shamCorp.), transferredtonylon filters(Hybond-N; Amersham Corp.)andhybridizedtoa 1.0-kbp BamHI-BglII ret-specific probe.The
arrowsindicate the sizeof the
nor-malfragments. (Lanes 9, 10, 14, 42, 53, and55) DNAs extractedfrom
different tumoralsamples. (Lane N)
DNAextracted fromnormal human tissue. Apartialrestrictionmapof theregionof theretgeneanalyzed and theprobeused(shaded area)
areshown.
-6.3Kbp
EcoR
Here, we report the analysis of 286 neoplastic thyroid sam-ples for ret activation. This analysis has been performed by probing Southern blots with a 1-kbp BglII-BamHI DNA frag-ment. This fragment is able to identify the region within the ret gene whererearrangement occurs (Probe 7of Fig. 1)(13). This probe hybridizes to a 6.3-kbp EcoRI, a 3.7-kbpBamHI,and a 9.3-kbp HindIII restriction fragment in normal human DNA (see map on Fig. 2). We found that 33 (11.5%) out of 286 samples showed adjunctive rearranged bands, and this result was demonstrable with at least three different restriction en-zymes (examples are in Figs. 2 and 3). In some cases, the rearranged band had a decreased intensity with respecttothe normal one(forexample, lane 10, Fig. 2). This is most likely due to contamination of the tumoral specimen by normal surrounding tissue. The possibility that the rearrangement might involve only a fraction of the neoplastic cells is currently underinvestigation by using the in situ RNA hybridization technique.
Therearrangement was specific for the papillary histotype; it was present in 11 outof 65 papillary samples (16.9%) col-lected at the MayoClinic, 8outof70(11.4%) from Lyon, and 14 out of 42 (33.3%) from Milan. We did not find any rear-rangement in the 37 follicular, 15 anaplastic, and 18 medullary
carcinomas,nor inthe 34benignlesions that we have analyzed (TableI).
Transfectionassay. We evaluated thetransformingactivity ofthe 14samplescollectedattheIstituto Nazionale Tumori of
Milanwhich already scoredpositivefor ret rearrangement. All
TableI. RETAcitvation in Human ThyroidTumors
Tumor type Positive/analyzed
USA France Italy Total
Papillary 11/65 8/70 14/42 33/177 Follicular 0/11 0/13 0/13 0/37
Anaplastic 0/2 0/5 0/8 0/15 Medullary 0/0 0/3 0/15 0/18 Adenoma 0/0 0/18 0/16 0/34
*Other 0/0 0/1 0/4 0/5
Total 78 110 98 286
*Squamouscellcarcinoma, sarcomatoidcarcinoma.
thesamples,testedby the standardfocus formationassay on NIH/3T3 cells, gave rise totransformedfoci in 2-3 wk (Table
II). Also, the available metastases ofthe ret-positivetumors scoredpositive (TableII).Hybridization oftheDNAextracted from theprimaryfociof transformedNIH/3T3 cells with
ret-specificprobesallowedus to conclude that thetransferred on-cogene was actually an activated version of ret (data not
shown). We alsotransfectedtheDNAextracted from nine
fol-liculartumors;nonegaverisetotransformed foci
(Table
II). Further characterizationof
gene rearrangement. We hadpreviously reported that when tumorscarryingan activated version of ret were probed with a NH2-terminal
protoret-speci-H I L M
-3.7Kbp
Bam H I
U X R I I
l-I
IFigure 3. Examples of ret rearrangement in papillary thyroid
carci-nomascollectedatthe Mayo Clinic. (Lanes B, C, D, E, F, G, H, L,
andM)DNAs extractedfromdifferent tumoral samples. (LanesA
and I)DNAextractedfrom normal human tissue.
TableII. TransformingActivityofthe ActivatedRETOncogene
fromPapillary and Follicular ThyroidCarcinomas
Patient Transforming H4
No. Histologicaltype Specimen* activityt positivity
I PapillaryCa. T 0.05 yes
1 M 0.06 yes
2 T 1.10 yes
2 M 0.03 yes
3 T 0.10 no
3 M 0.30 no
4 T 0.20 yes
4 M 0.03 yes
5 T 0.07 no
5 M 0.10 no
6 M 0.05 no
7 T 0.35 no
8 T 0.15 yes
9 T 0.63 no
9 M 0.85 no
10 T 0.31 yes
11 M 0.65 yes
12 T 0.08 no
12 M 0.75 no
13 M 0.07 yes
14 T 0.15 no
15-24 Follicular Ca. T <0.003 no
*T,
Primarytumor,M,Lymphnodalmetastasis. tNIH/3T3Transformedfoci/AgofDNAtransfected.9 14 42 53 55 N 9 14 42 53 55
9.3 Kb
Hind III EcoRI
R
M N L N B C E D G H
w...o'.I
Eco RI
R R
l
process
l
I I
i
I
B B B B B B
Bgl II
Figure4.Rearrangement of the ret
5'terminalregioninpapillary thy-roidcarcinomas. Southernblot
analysis ofDNAextractedfromthe neoplastic specimens. 10ugof DNA werehybridized to a 1.8-kbp DNA probespecific forthe 5' terminal
re-gionoftheretprotooncogene. The 6.3 Kb arrowsindicatethesize ofthe nor-malfragment.Apartial restriction
mapoftheregion oftheret gene
analyzedand theprobe used (shaded area)areshown.(Left)Samples
col-lected at theHospitals ofLyon.
(Lanes 9, 10, 14, 42, 53,and55)
DNAsextractedfrom different tu-moral samples. Lane N: DNA ex-tractedfromnormal human tissue.
(Right) Samplescollected at the MayoClinic. (Lanes B, C, D, E, G, H,L,and M) DNAs extracted from
different tumoral samples. (Lane N)
DNAextractedfromnormal human
tissue.
fic sequence, a rearranged bandwasalsodetectable. This sug-gests that the chromosomal event leading to ret activation shouldnotinvolvedeletion ofitsNH2-terminal encoding
do-main (13). Subsequently,wehavedemonstrated that an inver-sion (10) (q 11.2-21)determinedthe H4-retfusion inatleast fourcasesofpapillarythyroidcarcinoma(1 7a).In thepresent
study,wefoundthatmanyofthesamplesalso showed a similar
mechanism. In fact, 10/11 samples from Mayo, 5/8 from
Lyon, and 9/9 from Milan showed rearranged bands when
probed witha 5'-terminal 1.8-kbp ret-specific BamHI DNA
fragment (probe 12ofFig. 1) (Examplesare showninFig. 4). The PTC oncogene thatwehadisolated frompapillary
thy-roid carcinomas bymeansofthetransfectionassay was shown
tobe theproduct ofafusionbetweenatruncatedretgene anda
novel sequence thatwenamed H4(13).Wedemonstrated that
the truncation ofthe H4 gene couldoccur indifferentpoints
over asequenceof several thousand basepairs (Fig. 1) (13). Thus,theanalysis ofH4 involvement has beenperformed by usingdifferentprobes (showninFig. 1)from Alu-freeregions ofthe H4 gene(probes 2, 8, 9, 10, 11, 3,ofFig. 1) (13). Fig. 5
shows the H4 gene rearrangementinthesample 14fromLyon (probe2ofFig. 1) (13).We have shown H4 rearrangement also in one othersamplethatwascollectedattheMayoClinic
(sam-pleDofFig. 2)and
cytogenetically
showedachromosome lOqinversion (14, 1 7a). Moreover, 7outofthe 14samples which scoredpositivein the transfection assay showed H4 involve-ment,sincein these cases theprimary NIH/3T3foci contained
H4-specificsequences (Table II).
RNAandPCRanalysis.Wehavepreviouslyshownthat ret
rearrangement leadingto PTC formation gives riseto
trun-catedtranscripts.TheH4promoter, which fusestoret, is able
to drive its expression in the thyroidtumorthat carries the
rearrangement. To increase thepossibilityofdetectingthe PTC formation,and toverifyH4involvement in the cases inwhich
apparently by the Southern blot analysis H4 was not
rearranged, we performed a PCR analysis on the RNA
ex-tracted-from five
ret-rearranged
tumors. We copied 5 Mg oftumoralRNAby usingreversetranscriptase and an antisense 18-bases long 3' ret-specific oligonucleotide primer. We
ampli-fiedtheresultant cDNAwiththesame 3'primer and a sense
H4-specific 18-basesoligomer. An amplified 363-bp long frag-mentindicatedthe presence of a fused H4-ret transcript (i.e.,
activatedPTC). The correct sized fragment was found in sam-ples10, 14, and 53 from Lyon, but not in samples 9 (Fig. 6) and 55(notshown).Thus, in samples 10, 14, and 53, ret was fused
toH4;converselyin the samples 9 and 55, the rearrangement
ofthe ret protooncogene probably involved a gene distinct
fromH4.
Discussion
The identification ofthenature of the genetic mutations in
thyroid neoplasms, and the assessment of their prevalence in thevarious tumor phenotypes, iscriticalto an understanding
A
9
3Kbp- 10
10 14 42 53 55
B
9 10 14 42 53 55
t
Hind m EcoRI
Figure 5. RearrangementofH4gene in one papillary thyroid carci-noma.Southern blotanalysisof the DNAs extracted from the samples collected at theHospitalsof Lyon. TheH4-specificDNAprobe used is the probe 2 of reference 13. The arrows indicate the sizes of the normalfragments.
WI:
B
A. 1 2 3 4
-363bp
-Figure6.Expressioninpapillary thyroidcarcinomasofafused H4-ret
transcript.PCRanalysisontheRNAextracted from fourpapillary
carcinomas collectedattheHospitalsofLyon.5tig of totalRNAwere reversetranscribedusinganantisenseret-specific oligonucleotide,
andamplified usingthe sameoligo togetherwithanH4-specific
am-plimer. 1/10of theproductofthe amplificationwassubjectedto
electrophoreticseparationon a2%agarose gel. (Lanes 2, 3,and4)
RNA extracted from thepatients 10, 14,and 53 ofFig. 1, respectively. (Lane 1)RNAextracted frompatient9.(A)Ethidium bromide
stain-ingof theagarose gel. (B) Hybridizationoftheamplified productto
aret-specificcDNAprobe (I18).
of their pathogenesis. Different geneticevents may occurto determine the different phenotypic characteristics ofthyroid neoplasms, suchasthe abilitytoinvade thelymphatics (papil-lary) orthe blood vessels(follicular), or thepropensityto
be-comededifferentiated(anaplastic).
Wepreviously demonstrated theactivation of theret onco-gene inpapillary carcinomas (12, 13). To date,the
ret-trans-forminggenehas been found activatedinvivoonlyinthyroid carcinomas(1 1-13) andin apapillarythyroid carcinoma cell
line(24). Previouslydescribedrearrangementsof theret
onco-genewerefoundtohave occurred in vitroduringthe
transfec-tionprocedure(25-27). Moreover,wedid notfindany
exam-ple ofret rearrangementby screening about 150nonthyroid tumors (data notshown), and Ishizaka etal. have reporteda
similar result in a study of more than 100 nonthyroid
tu-mors(28).
These published observations suggestthatretactivation is
specifically involved onlyinthyroidtumorigenesis,buttheydo
notdetermineifaparticular subtypeofthyroidtumorhasret
rearrangements. Therefore, we collected 286 human thyroid neoplastic samples and screenedthembySouthern blot analy-sis forret rearrangement. We foundret activatedonlyin the papillary subtype of these tumors: specifically, in 11 tumors
collected at the Mayo Clinic, 8 collected at the Hospitals of
Lyon, and 14collected in Italyat the Istituto Nazionale
Tu-mori of Milan andatthe IIMedical School ofNaples.Our data
stronglysupport thehypothesisthatretactivation isspecificfor papillary carcinoma. In fact, noneof either the 37 follicular
carcinomasorthe 72othernonpapillarythyroidtumorsscored positively.
Recently,Ishizakaetal. (29)publishedthedetection ofan
activatedret(PTC) in1 outof11thyroid papillarycarcinomas, but also in4 outof19follicular adenomas andinoneoftwo
adenomatous goiters. In the adenomas, however, the activa-tion ofret was detectedonly insomeregions of thetumors. Theydonot report anyanalysis of nonpapillary malignant tu-mors.Since the prevalence of occult thyroid carcinomas in the Japanesepopulation is high,it ispossiblethat thePTC-positive
casesoffollicular adenomas and adenomatous goiter could
re-flectaminor populationofcancerouscells,asthe authors sug-gestintheir discussion.
Wealso notedadifference in thefrequency ofretactivation
indifferent geographicareas,rangingfrom 11% in the French samplesto33% in the Italian samples. The U. S.tumorshadan
intermediate frequency of17%.Thismay suggestthe
involve-ment ofdifferent genetic or environmental factors in these threeareas. The recent findingofhighratesofrascodon 61 mutation in thyroid tumors in aniodide-deficient area (30), and the differencesdetected in the patterns ofras mutation
betweenradiation-associatedandspontaneoushumanthyroid
carcinomas (31)seem tosupportsuchsuggestion.Itis
notewor-thy thatonlyoneofthe
patients positive
forretactivation inthisstudy, hada
prior
documentedradiationexposure.We havealso been ableto demonstrate that the H4 gene was involvedin retactivation in 11 outof 33 retrearranged samples. Wedemonstrated this involvement in two casesby Southern blotanalysis (samples 14and D), in three casesby PCR analysis (samples 10, 53, and again, 14), and in seven cases (Milan samples) by the transfection assay. We cannot
exclude, however, thatH4 maybeinvolved in theremaining
casesbecausewedidnothaveenoughRNAandDNA to
per-form PCR ortransfection assays, respectively, in the
appar-entlynegativesamples. In atleast 5ofthese 22cases wehave
conclusivelydemonstrated thatretisnotfusedtoH4, and still otheruncharacterizedgenes maybeinvolved in the rearrange-ment.Interestingly, cytogenetic studies of papillary carcinoma have demonstrated that the region where ret is mapped
(10q11.2)is also involved in translocations with other
chromo-somes(14).
In24of33 ret-rearrangedcases,the5'terminal region ofthe
retgene (extracellular and transmembrane encoding region)
was notdeleted,but was present ratherinarearrangedstatus,
similartopreviously describedcases(13).
There still remain majorunansweredquestions about the
mannerin which thyroidtumors evolve, and the molecular
eventsthatdetermine whetheratumorbehaves eitheras a
dif-ferentiatedpapillary/follicularor as adedifferentiated anaplas-tic one. Other genetic alterations are frequently detected in
thyroid tumors and include mutations involving ras genes.
However, thepublishedstudieson ras oncogeneinvolvement
(30-36)indicateasignificantheterogeneityamongtheresults obtainedby differentgroups.Whereasraspointmutationsare presentwithanaboutequivalent frequencyin bothbenignand
malignant thyroidtumors, the sameconclusion isnotreadily established when the follicular and papillary types are
com-pared. We have previously demonstrated that the oncogene
trk, belongingtothetyrosinekinase family, is alsofrequently
(about 10%) activated in papillary carcinomas (12), and
re-cently, Suarezetal. (37) observedgspmutations in - 10%of
differentiated thyroid carcinomas.Inaddition,allelicloss
stud-ies ofchromosome 3 and 10 have demonstrated differences
betweenpapillary and follicular carcinomas(38). These data
A
indicatethatother somaticmutations shouldbeconsideredto
explainthediffering behaviorpatternsexhibited bydifferent thyroidtumor types.However, fromourstudieswewould
sug-gestthatretactivationrepresents animportantand apparently
specific geneticeventinthe development of human papillary thyroid carcinoma.
Acknowledgments
Weacknowledge the assistance of Dr. S. Pilotti in obtaining the tumor samples from theIstituto NazionaleTumori, and we are alsogratefulto Dr.C. S. Grant in obtaining the tumor samples from patients operated atRochesterMethodist Hospital.Wealsoacknowledge the
contribu-tion ofDr. V. N. Patelwhoperformedmanyofthe DNA extractions
fromthe Mayo Clinic material. Finally, the authors areindebtedto F. D'Agnello, F. Moscato,andM.Berardoneforthe art work.
Thiswork has been supported by the ProgettoFinalizzato "Ingeg-neriaGenetica"and the Progetto Finalizzato"Biotecnologiae
Biostru-mentazione" ofthe CNR, by the "Progetto AIDS"oftheIstituto
Su-periorediSaniti, bygrantsfromtheAssociazione Italiana Ricercasul
Cancro,andalsobyagrant from LaLiguecontrele Cancer du Rhone.
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