J. Clin. Endocrinol. Metab. 2002 87: 3941-3946, doi: 10.1210/jc.87.8.3941  

G. Jaffe and A. Fusco B. J. Collins, G. Chiappetta, A. B. Schneider, M. Santoro, F. Pentimalli, L. Fogelfeld, T. Gierlowski, E. Shore-Freedman,

  for Benign Conditions

RET Expression in Papillary Thyroid Cancer from Patients Irradiated in Childhood

Society please go to: http://jcem.endojournals.org//subscriptions/ or any of the other journals published by The EndocrineJournal of Clinical Endocrinology & Metabolism To subscribe to

Copyright © The Endocrine Society. All rights reserved. Print ISSN: 0021-972X. Online

RET Expression in Papillary Thyroid Cancer fromPatients Irradiated in Childhood for Benign Conditions

B. J. COLLINS, G. CHIAPPETTA, A. B. SCHNEIDER, M. SANTORO, F. PENTIMALLI,L. FOGELFELD, T. GIERLOWSKI, E. SHORE-FREEDMAN, G. JAFFE, AND A. FUSCO

Section of Endocrinology (B.J.C., A.B.S., E.S.-F.), University of Illinois at Chicago, Chicago, Illinois 60612; IstitutoNazionale dei Tumori di Napoli (G.C.), Fondazione Senatore Pascale, 80131 Naples, Italy; Michael Reese Hospital (B.J.C.,A.B.S., L.F., T.G., E.S.-F., G.J.), Chicago, Illinois 60616; and the Centro di Endocrinologia ed Oncologia Sperimentale delCNR (A.F., M.S., F.P.), Dipartimento di Biologia e Patologia Cellulare e Moleculare, Universita di Napoli “Federico II,”80131 Naples, Italy

Both external and internal exposure to radiation have beenlinked to the development of papillary thyroid cancer. Rear-rangement of the gene for RET tyrosine kinase and subse-quent expression of this protein has also been found to occurin many papillary thyroid cancers, and with increased fre-quency in radiation-related cancers following the Chernobylaccident. However, little has been reported on the frequencyof RET rearrangements in cancers after exposure to externalradiation. We here report on RET protein immunoreactivityin paraffin-embedded thyroid samples from 30 patients withpapillary thyroid cancer who received radiation treatmentduring childhood for benign conditions at Michael Reese Hos-pital in Chicago, and in 34 patients identified from the tumorregistry as having papillary thyroid cancer with no history oftherapeutic radiation. The subjects were characterized bysex, age at surgery, and the following attributes of tumor pa-thology: size, number of lobes involved, number of foci, lymph

node metastases, and soft tissue invasion. Representative tis-sue samples were reacted with an antibody against the RETtyrosine kinase domain whose expression has been shown tocorrelate highly with RET/PTC rearrangements. A greaterpercentage of cancers positive for RET immunoreactivity wasfound in the radiation-exposed group (86.7% vs. 52.9%, P �0.006). Although the mean age at surgery of the exposed groupwas lower than the control group, there was no correlation ofpositive RET immunoreactivity with the age at surgery. Nocharacteristics of the tumors were associated with positiveRET immunoreactivity. In summary, the greater incidence ofRET-immunopositives in the irradiated group indicates thatthe expression of RET immunoreactivity is strongly associ-ated with radiation exposure, but the prognostic significanceof this is not yet clear. (J Clin Endocrinol Metab 87: 3941–3946,2002)

THE RELATIONSHIP BETWEEN external radiation andthyroid cancer was well established many years ago

(1–3). More recently, a similar relationship was establishedfor internal radiation from isotopes of iodine, as the accidentat the Chernobyl nuclear power plant has resulted in anunprecedented number of childhood thyroid cancers (4, 5).Almost all thyroid cancers arising after either external orinternal radiation exposure occur after childhood exposureand are of the papillary type (2, 6–9).

Following its description in 1990 (10), the activation of theRET gene by somatic rearrangement (to form the RET/PTConcogene) has been found to be a frequent event in papillarythyroid cancers (11, 12). As measured by immunohistochem-ical or RT-PCR methods and as reported from many geo-graphical areas, the prevalence of RET expression in spon-taneous papillary thyroid cancers varies widely. Forexample, prevalences of 3, 8, and 11% have been reported inSaudi Arabia, Germany, and France, whereas 70 and 85%have been reported in New Caledonia and Australia (12–15).Whether this wide range is due to methodological, geograph-ical, or other variables is not fully known. The reportedfrequency of RET-positive papillary thyroid cases in theChernobyl population is 51–76%, higher than that for almostall groups of spontaneous thyroid cancers (16–21).

How do the results on RET activation from Chernobyl,

where radiation exposure was primarily internal throughingestion of iodine isotopes, compare to subjects receivingexternal ionizing radiation? Two studies have reported find-ings from patients receiving external ionizing radiation treat-ment (22, 23). However, in one of these studies, less than halfwere children receiving treatment for benign conditions andin the other the doses and reasons for treatment were notgiven. The “Chicago cohort” is a large group of patients whoreceived radiation for benign conditions during childhoodwith doses comparable to those received as a result of theChernobyl accident. Of these subjects, over 12% have devel-oped thyroid cancer (24). In the present study, we report onthe prevalence of RET protein immunoreactivity in thyroidcancers in this group, comparing it to a control group ofpapillary thyroid cancers in patients with no known historyof radiation.

Subjects and MethodsStudy subjects

At Michael Reese Hospital (Chicago, IL), 4296 children less than 16yr of age received external beam radiation therapy for benign conditionsbetween 1939 and 1962 (25, 26). As of January 2001, 3083 of these patientshave been located. In this group, 357 have developed thyroid cancer, 121of whom were operated on at Michael Reese Hospital. Paraffin blocksfor 63 of these cases were located, of which 30 included usable papillarythyroid cancer samples. A control group of 34 cases of papillary thyroidcancer cases was compiled from 157 cases in the Tumor Registry atMichael Reese Hospital whose medical records indicated no history ofAbbreviation: ABC, Avidin biotin complex.

0013-7227/02/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 87(8):3941–3946Printed in U.S.A. Copyright © 2002 by The Endocrine Society

3941

radiation treatment. An attempt was made to match the control cases tothe radiation-exposed group by age and sex, but the limited availabilityof paraffin blocks from subjects in either group precluded a completematch. The study protocol was approved by the Michael Reese Hospitaland University of Illinois at Chicago Institutional Review Boards.

Study parameters

The demographic characteristics of the study subjects included in theanalyses were age at radiation, radiation dose to the thyroid, sex, andthe age at thyroid cancer surgery. The characteristics of the thyroidcancers for each subject were derived from operative and surgical pa-thology reports. The largest dimension of the largest malignant nodulewas used as a measure of size. The number of thyroid lobes involved (1or 2), the number of tumor foci (single or multiple), lymph node me-tastases (present or absent), and the presence of tumor invasion into softtissue adjacent to the thyroid (present or absent) were recorded.

Immunohistochemistry

Paraffin blocks had been stored for a mean of 25.7 yr for the radiation-exposed cases and 11.0 yr for the unexposed cases. Sections were cutfrom those blocks where the likelihood of papillary tumor samples wasgreatest, based on the descriptions in the surgical pathology reports.Sections of 5-�m thickness were mounted on Fisher Scientific (Pitts-burgh, PA) Superfrost slides, deparaffinized with xylene, and quenchedfor endogenous peroxidase by reaction in a solution of 0.3% hydrogenperoxide in absolute methanol for 30 min. After washing in distilled H2Oand phosphate-buffered saline, the slides were treated with dilutedblocking serum for 20 min before incubating overnight at 4 C with anaffinity-purified rabbit polyclonal antibody against a glutathione S-transferase fusion protein containing 116 amino acids of the RET cyto-plasmic tyrosine kinase domain (27). The antibody reacts with bothfull-length and rearranged RET, and does not stain normal thyrocytes.Previous results obtained by immunohistochemistry have been shownto correlate highly with the molecular results obtained by RT-PCR forRET gene rearrangements in papillary thyroid cancers (18, 28–33), al-though recent reports indicate that the antibody may also recognize onimmunoblots activated RET protein from papillary thyroid cancers inthe absence of RET/PTC rearrangements (34). After incubation with theprimary antibody, the slides were washed and incubated with biotin-ylated goat antirabbit IgG [Vectastain avidin biotin complex (ABC) kit,Vector Laboratories, Burlingame, CA] for 20 min and reacted with theABC in the ABC kit before visualizing the reaction product with 0.06 mmdiaminobenzidine (DAKO Corp., Carpinteria, CA) and 2 mm hydrogenperoxide in 0.05% phosphate-buffered saline for 5 min. The slides werewashed, counterstained with hematoxylin, dehydrated through gradedethanol and xylene, and mounted in Permount (Fisher Scientific). Ex-pression of the RET product was scored by independent observers,based on the number of cells reacting to the antibody, not the intensityof the reaction. The specimen was scored as “3�” if more than 60% ofthe cells stained positive, “2�” if 40–60% of the cells were positive, and“1�” if only 20–40% of the cells were positive. Visible staining of lessthan 20% was scored as “trace” but was considered negative for theanalysis of the data.

Statistical analysis

Binary variables were analyzed by logistic regression and the Fisherexact test and continuous variables were analyzed by multiple regres-sion, ANOVA, and t tests.

Results

The demographic variables and the thyroid cancer char-acteristics in the radiation-exposed and the unexposedgroups are given in Table 1. The members of the exposedgroup were younger and had a higher percentage of malesthan did the control group. The tumors in the exposed groupwere smaller and had a greater prevalence of lymph nodemetastases and multifocal lesions.

Figure 1 shows representative sections of the reacted thy-

roid specimens from which the data for RET immunoreac-tivity were drawn. A greater percentage of the radiation-exposed group had positive RET-immunoreactive cancersthan did the unexposed control group: 86.7% vs. 52.9%, P �0.01 (Table 1 and Fig. 2). When the cases were analyzed bythe extent of immunoreactivity (0 to 3�) attributed to eachspecimen, not only were there more cases with positive RETimmunoreactivity in the exposed group, but the exposedcases accounted for more extensively stained RET-immu-nopositives than the control group. Only one of the controlcases scored above 1� (5.5%), whereas 7 of the 26 (26.9%)cases in the exposed group were scored as 2� or 3�.

No RET immunoreactivity was detected in normal tissue.There were six subjects in the exposed group and two in theunexposed group whose slides contained adenomas or ad-enomatous nodules. An additional three subjects in theradiation-exposed cohort and one subject in the controlgroup also contained adenomas but were not included in thefinal analysis because samples of their previously describedpapillary thyroid cancer were not present in the availableblocks. Of these 12 benign nodules, only one, from the ex-posed group, demonstrated any positive reactivity to theRET antibody, and this reactivity was restricted to the nucleiof the reactive cells. The accompanying sample of papillarythyroid cancer on this slide was also RET-immunopositive.In one case, an area of Hashimoto’s thyroiditis, accompanyingpapillary cancer, was positive for RET-immunoreactivity.

The RET data for thyroid cancer, plotted by age at surgery(in deciles) in Fig. 3, indicate that at any given age the per-centage of cases positive for RET immunoreactivity wasgreater in the radiation-exposed group. Controlling for ageat surgery, the difference in the proportion of positive andnegative RET-immunoreactive cases between the two groupspersisted.Althoughtheageatsurgerywaslessintheradiation-exposed group (P � 0.001), this did not explain the differencein positive RET immunoreactivity (Table 2).

The mean size of the largest malignant nodules (Table 1)in the radiation-exposed group was smaller than that inthe unexposed group (12.9 mm vs. 24.9 mm, P � 0.001).Also, when analyzed alone, there was a significant dif-ference in size by RET status (16.0 mm for positive RET

TABLE 1. Description of the members of the two study groupsand their thyroid cancers (mean � SEM)

Group

Exposed Unexposed

PatientsTotal number 30 34Age at radiation (y) 3.4 � 0.6Thyroid dosea (cGy) 72.1 � 11.1Age at surgeryb (y) 28.7 � 1.5 46.4 � 2.8Maleb 53.3% 26.5%

Cancer characteristicsRET positiveb (%) 86.7% 52.9%Sizeb (mm) 12.9 � 1.4 24.9 � 2.9Lymph node metastasesb (%) 66.7% 32.4%Soft tissue invasion (%) 26.7% 29.4%Multifocalb (%) 76.7% 52.9%Bilateral (%) 36.7% 26.4%

a Thyroid dose was available for 27 of the 30 cases.b P � 0.05.

3942 J Clin Endocrinol Metab, August 2002, 87(8):3941–3946 Collins et al. • RET Expression in Papillary Thyroid Cancer

immunoreactivity, 26.5 mm for negative RET immuno-reactivity, Table 3). However, analysis by two-way ANOVA ormultiple regression indicated a significant difference in size by

treatment (exposed vs. unexposed) group, but not by RET status(Table 3).

The 66.7% of the radiation-exposed cases having lymphnode metastases was higher than the 32.4% of the unexposedgroup with metastases (Table 1). However, the exposedgroup was younger than the unexposed group and lymphnode metastases are known to be more common in youngerpatients. To take this into account, regression analysis oflymph node metastases as a function of age at surgery andRET status was performed. Age at surgery was inverselyrelated to lymph node metastases, whereas RET status wasnot. Neither age of surgery nor treatment group (exposed vs.unexposed), when analyzed together, was independentlyrelated to lymph node metastases.

The radiation-exposed group had more multifocal cancersthan did the unexposed group (76.7% vs. 52.9%, Table 1). Thiswas significant (P � 0.01) when adjusted for age and size.There was no difference in the prevalence of multifocal can-cers between the cases with positive or negative RET im-munoreactivity (70.5% vs. 50.0%, P � 0.06, Table 3). Analyzedtogether, neither the exposure group nor RET-status inde-pendently predicted multifocal cancers. There was no dif-ference between the exposed and unexposed groups in thenumber of tumors that demonstrated invasion into the softtissue, nor was there a relationship of soft tissue invasionwith RET status.

The data for the radiation group alone were analyzed for

FIG. 1. Immunohistochemical analysis of RET. Paraffin sections of normal thyroid tissue and thyroid carcinomas were analyzed by immu-nohistochemistry using antibodies against the RET protein. A, Immunostaining of normal thyroid tissue (�200). B, Negative control byimmunostaining of a carcinoma sample (�200) using antibodies against the RET protein preincubated with the peptide against which theantibodies were raised. No immunoreactivity was observed. C and D, Immunostaining of carcinoma samples (�400) using antibodies againstthe RET protein. Strong cytoplasmic staining is observed.

FIG. 2. RET immunoreactivity as a function of radiation exposure.The number of cases in the exposed (left) and unexposed (right) groupswere tabulated by RET score. Absence of RET immunoreactivity orfewer than 20% of cells scoring positive was regarded as negative,represented as a shaded bar on the left side of each group. The casesscoring positive for RET are displayed in the bar on the right side ofeach group. The bar is subdivided by the RET score as described inSubjects and Methods. The percentage of each group scoring negativeor positive is given at the top of each bar. The number of RET-positivecases in the exposed group was significantly greater than the numberof RET-positive cases in the unexposed controls (P � 0.003, Fisherexact test).

Collins et al. • RET Expression in Papillary Thyroid Cancer J Clin Endocrinol Metab, August 2002, 87(8):3941–3946 3943

possible correlations between RET status and the dose ofradiation, age at treatment or latency (time between radiationand surgery). No associations were found for any of thevariables (Table 2). Also, none of these factors was related tothe characteristics of the thyroid cancers in the exposedgroup.

Discussion

Most of the data related to the expression of the RETproto-oncogene in papillary thyroid cancers have been eitherfrom sporadic cases or from cases related to radiation expo-sure after the Chernobyl accident. In this paper, we report

results of RET protein expression in thyroid cancers in apopulation exposed to external radiation during childhoodfor the treatment of benign conditions, a group that has beensubject to much less analysis of RET expression. Our datashow that there is an increased expression of positive RETimmunoreactivity in the thyroid cancers of the radiation-exposed group, with 86.7% of the radiation group positive forRET immunoreactivity vs. 52.9% of the control group. Thefrequency of RET protein expression in our control group ishigher than the frequency of RET expression reported inmany, but not all studies of spontaneous papillary thyroidcancers (15, 35) and is similar to what has been observed inthe Chernobyl population (56–76%) (16, 19, 20, 23, 36). Thefrequency of RET expression we observed by immunohis-tochemistry in the radiation group is comparable to thatreported by Bounacer et al. (22) for RT-PCR data, who found84% RET positives in 19 externally irradiated subjects. How-ever, they included irradiated adults and patients who re-ceived high dose radiation therapy in their population. It isalso comparable to the RT-PCR data recently reported byElisei et al. (23) who observed 76% (19 of 25) RET-positivethyroid cancers removed from adults who had been exposedto external irradiation in childhood. Because they did notreport the dose of treatment and whether it was administeredfor benign or malignant conditions, the comparison to ourstudy and to Chernobyl cases is uncertain.

As the exposed and unexposed groups were not balancedin demographic characteristics, we performed multivariateanalyses. The subjects in our radiation-exposed group weresignificantly younger than the control group (Table 1). How-ever, we found no association of positive RET immunore-activity with age at surgery. Similarly, no relationship be-tween the frequency of RET rearrangements and age wasfound for nonradiation related cases in Japan, England, andItaly (23, 37, 38). Thus, the age difference in the two groupsdoes not explain the higher frequency of positive RET im-munoreactivity in irradiated cases.

The fact that the radiation-exposed group contained morelymph node-positive cases might be attributed to the lowerage of this group and the well-known association of youngage with lymph node metastases. In fact, an age differencebetween lymph node positivity and negativity existed onlyin the unexposed group, suggesting that the association of

FIG. 3. The number of cases of RET immunopositive and immunon-egative tumors by age at surgery (in deciles) in the exposed andunexposed groups. Each bar represents the number of cases whosesurgery occurred in the age decile bracketed by the age at either sideof the bar. The RET-positive cases are represented by the shadedportion of the bar and the RET-negative cases by the open bar. Thepercentage of the positive cases in each decile is indicated at the topof the bar.

TABLE 2. Description of the study groups by RET status

Group

RET � RET �

No. of patientsa 44 20Exposed 26 4Unexposed 18 16

Age at radiationExposed 3.65 � 0.62 1.39 � 0.48

Thyroid dose (cGy)Exposed 69.1 � 12.2 89.3 � 29.0

Age at surgery 36.9 � 2.3 40.8 � 3.7Exposed 29.2 � 1.7 25.4 � 3.6Unexposed 48.0 � 3.9 44.7 � 4.0

Male 19 of 44 (43.2%) 6 of 20 (30.0%)Exposed 15 of 26 (57.7%) 1 of 4 (25.0%)Unexposed 4 of 18 (22.2%) 5 of 16 (31.3%)

a P � 0.05.

TABLE 3. Characteristics of the thyroid cancer by RET status

Group

RET � RET �

Size (mm)a 16.0 � 2.1 26.5 � 3.1Exposed 13.0 � 1.6 12.3 � 0.6Unexposed 20.3 � 4.4 30.1 � 3.4

Lymph node metastases 23 of 44 (52.3%) 8 of 20 (40.0%)Exposed 16 of 26 (61.5%) 4 of 4 (100.0%)Unexposed 7 of 18 (38.9%) 4 of 16 (25.0%)

Soft tissue invasion 13 of 44 (29.5%) 5 of 20 (25.0%)Exposed 8 of 26 (30.8%) 0 of 4 (0.0%)Unexposed 5 of 18 (27.8%) 5 of 16 (31.3%)

Multifocal 31 of 44 (70.5%) 10 of 20 (50.0%)Exposed 21 of 26 (80.8%) 2 of 4 (50.0%)Unexposed 10 of 18 (55.5%) 8 of 16 (50.0%)

a P � 0.05.

3944 J Clin Endocrinol Metab, August 2002, 87(8):3941–3946 Collins et al. • RET Expression in Papillary Thyroid Cancer

young age and lymph node metastases may be stronger inthe absence of radiation treatment.

As there were differences in the presenting features of thecancers in the exposed and unexposed groups, we analyzedthe data to see if RET status was related to any pathologicalcharacteristic. The smaller size of the cancers in the radiationgroup is due, in large part, to the effect of surveillance of thispopulation, which has been screened on a regular basis since1975 for the presence of thyroid nodules. The control group,on the other hand, represents spontaneous cancers that likelyhad gone undetected until they reached a larger size. Al-though treatment (exposed vs. unexposed) and RET statuswere independently related to size, when combined, onlyradiation exposure status was significant.

Attributes of the thyroid cancers were analyzed for a re-lationship to RET status. None were found for lymph nodemetastases, multifocality, and local invasion. However, thenumber of cases was too small to determine whether RETstatus has prognostic significance in general or in radiation-related cases. A recent study (32) similarly found no prog-nostic impact of RET protein expression on the long-termoutcome of papillary thyroid cancers.

The specificity of RET/PTC rearrangements to papillarycarcinomas has been questioned by findings in “benign”thyroid neoplasms in patients who were exposed to radia-tion, either for therapeutic purposes or after the Chernobylaccident. Bounacer et al. (22) found RET rearrangements in45% of radiation-related follicular adenomas. Elisei et al. (23)found rearrangements in 52.4% “benign thyroid neoplasms”following exposure from Chernobyl, in 37.5% (3 of 8) casesfollowing external irradiation, and in 13.9% of sporadic cases.In contrast, we observed positive RET immunoreactivity inonly one of nine (11%) external radiation-related benign nod-ules. The reason for this discrepancy, possibly due to thelimited number of cases studied or the difference in meth-odologies, and the potential role of RET activation in radi-ation-related benign nodules requires further investigation.

It has recently been shown that, contrary to previous as-sumptions, the RET gene may not be completely silent innormal thyroid follicular cells and that the expression ofwild-type RET may be increased above low basal levels inmany papillary thyroid cancers (34, 39). Bunone et al. (39)found RET/ELE1 transcripts, the reciprocal form of the on-cogenic ELE1/RET translocation, in thyroid carcinomas, con-firming the activity of the RET promoter in thyroid cells.Fluge et al. (34) confirmed the presence of low levels ofwild-type c-ret mRNA in normal thyroid and overexpressionof these wild-type transcripts in papillary thyroid carcino-mas by RT-PCR, although the latter also harbored translo-cations. They also demonstrated RET immunoreactivity byWestern blotting of a large amount (250 �g) of protein, usingthe same antibody against RET which we have used in thisstudy and two other antibodies (Santa Cruz Biotechnology,Inc., Santa Cruz, CA).

These findings show that wild-type RET mRNA is ex-pressed at a low level in normal thyroid tissue, but theprotein from normal cells was not detectable on Westernblots (34) and therefore is not likely to be detectable on tissuesections. In our study and in other studies using this anti-body, positive RET immunoreactivity has not been detected

in normal thyroid. However, the correlation reported be-tween histologically detectable RET immunoreactivity usingthe antibody employed here and the finding of a somatic RETrearrangement is very high (18, 28–33), although some dis-cordance between immunohistochemical and RT-PCR datahas been observed (18, 28). Absence of RET protein expres-sion in the presence of a RET/PTC translocation may arisebecause RT-PCR is a more sensitive technique than immu-nohistochemistry. Conversely, expression of the RET proteinin the absence of a detectable translocation may have beendue to the presence of a translocation that was missed by theRT-PCR primers used, or in accordance with Fluge’s work,may represent a splice variant or some alternative mecha-nism of activation. We cannot, therefore, be sure that everypositive RET immunoreactive case in our study represents acorresponding gene rearrangement. However, the immuno-reactivity recognized by the antibody in this study is cyto-plasmic, indicating overproduction of RET protein that haslost its transmembrane localization due to transposition ofthe tyrosine kinase domain with the N terminus of a cyto-plasmic protein. The wild-type and splice variants observedby Fluge et al. (34) do not have altered transmembrane andtyrosine kinase domains, indicating that the protein productshould still be targeted to the plasma membrane. Furtherwork is necessary to confirm the cellular localization anddetectability of the reported splice variants and wild-typeRET under different fixation and detection protocols.

In conclusion, positive RET immunoreactivity in radia-tion-related thyroid cancers is more common than in cancersfrom patients without a history of radiation exposure. Thefrequency of activation reported in cases arising from inter-nal, Chernobyl-related cases is similar to the frequency re-ported here. The cases reported here were exposed to radi-ation at about the same ages and with similar doses. Thefindings predict that as cases continue to occur, as a result ofeither external or internal radiation, they will be associatedwith a high frequency of RET activation. How this will affecttheir clinical behavior, if at all, remains to be seen. This isimportant because of the well-recognized poorer prognosisof thyroid cancer with advancing age.

Acknowledgments

Received February 13, 2002. Accepted May 3, 2002.Address all correspondence and requests for reprints to: Barbara J.

Collins, Ph.D., University of Illinois at Chicago, Section of Endocrinol-ogy and Metabolism (MC 640), 1819 West Polk Street, Chicago, Illinois60612. E-mail: bcollins@uic.edu.

This work was supported in part by Grant R01-CA-21518 from theNational Cancer Institute.

References

1. Duffy BJ, Fitzgerald P 1950 Thyroid cancer in childhood and adolescence: areport on twenty-eight cases. Cancer 10:1018–1032

2. Ron E, Lubin JH, Shore RE, Mabuchi K, Modan B, Pottern LM, SchneiderAB, Tucker MA, Boice Jr JD 1995 Thyroid cancer after exposure to externalradiation: a pooled analysis of seven studies. Radiat Res 141:259–277

3. Schneider AB, Ron E 2000 Thyroid diseases: tumors: carcinoma of follicularepithelium. In: Braverman LE, Utiger RD, eds. Werner Ingbar’s the thyroid.Philadelphia: Lippincott Williams and Wilkins; 875–886

4. Robbins J, Schneider AB 2000 Thyroid cancer following exposure to radio-active iodine. Rev Endocrinol Metab Dis 1:197–203

5. Tuttle RM, Becker DV 2000 The Chernobyl accident and its consequences:update at the millennium. Semin Nucl Med 30:133–140

Collins et al. • RET Expression in Papillary Thyroid Cancer J Clin Endocrinol Metab, August 2002, 87(8):3941–3946 3945

6. Nikiforov Y, Gnepp DR 1994 Pediatric thyroid cancer after the Chernobyldisaster—pathomorphologic study of 84 cases (1991–1992) from the Republicof Belarus. Cancer 74:748–766

7. Astakhova LN, Anspaugh LR, Beebe GW, Bouville A, Drozdovitch VV,Garber V, Gavrilin YI, Khrouch VT, Kuvshinnikov AV, Kuzmenekov YN,Minenko VP, Moschik SI, Nalivko AS, Robbins J, Shemiankina EV,Shinkarev S, Tochitskaya SI, Waclawiw MA 1998 Chernobyl-related thyroidcancer in children of Belarus: a case-control study. Radiat Res 150:349–356

8. Tronko MD, Bogdanova TI, Komissarenko IV, Epstein OV, Oliynyk V,Kovalenko A, Likhtarev IA, Kairo I, Peters SB, Livolsi VA 1999 Thyroidcarcinoma in children and adolescents in Ukraine after the Chernobyl nuclearaccident—statistical data and clinicomorphologic characteristics. Cancer 86:149–156

9. Jacob P, Kenigsberg Y, Zvonova I, Goulko G, Buglova E, Heidenreich WF,Golovneva A, Bratilova AA, Drozdovitch V, Kruk J, Pochtennaja GT, Ba-lonov M, Demidchik EP, Paretzke HG 1999 Childhood exposure due to theChernobyl accident and thyroid cancer risk in contaminated areas of Belarusand Russia. Brit J Cancer 80:1461–1469

10. Grieco M, Santoro M, Berlingieri MT, Melillo RM, Donghi R, BongarzoneI, Pierotti M 1990 PTC is a novel rearranged form of the ret proto-oncogeneand is frequently detected in vivo in human thyroid papillary carcinomas. Cell60:557–563

11. Jhiang SM, Caruso DR, Gilmore E, Ishizaka Y, Tahira T, Nagao M, Chiu JM,Mazzaferri EL 1992 Detection of the PTC/retTPC oncogene in human thyroidcancers. Oncogene 7:1331–1337

12. Santoro M, Carlomagno F, Hay ID, Herrmann MA, Grieco M, Melillo R,Pierotti MA 1992 Ret oncogene activation in human thyroid neoplasms isrestricted to the papillary cancer subtype. J Clin Invest 89:1517–1522

13. Zou MJ, Shi YF, Farid NR 1994 Low rate of ret proto-oncogene activation(PTC/retTPC) in papillary thyroid carcinomas from Saudi Arabia. Cancer 73:176–180

14. Mayr B, Potter E, Goretzki P, Ruschoff J, Dietmaier W, Hoang-Vu C, DralleH, Brabant G 1998 Expression of Ret/PTC1, -2, -3, -�3 and -4 in Germanpapillary thyroid carcinoma. Brit J Cancer 77:903–906

15. Chua EL, Wu WM, Tran KT, McCarthy SW, Lauer CS, Dubourdieu D,Packham N, O’Brien CJ, Turtle JR, Dong QH 2000 Prevalence and distributionof ret/ptc 1, 2, and 3 in papillary thyroid carcinoma in New Caledonia andAustralia. J Clin Endocrinol Metab 85:2733–2739

16. Fugazzola L, Pilotti S, Pinchera A, Vorontsova TV, Mondellini P, Bongar-zone I, Greco A, Astakhova L, Butti MG, Demidchik EP, Pacini F, PierottiMA 1995 Oncogenic rearrangements of the RET proto-oncogene in papillarythyroid carcinomas from children exposed to the Chernobyl nuclear accident.Cancer Res 55:5617–5620

17. Klugbauer S, Lengfelder E, Demidchik EP, Rabes HM 1995 High prevalenceof RET rearrangement in thyroid tumors of children from Belarus after theChernobyl reactor accident. Oncogene 11:2459–2467

18. Thomas GA, Bunnell H, Cook HA, Williams ED, Nerovnya A, Cherstvoy ED,Tronko ND, Bogdanova TI, Chiappetta G, Viglietto G, Pentimalli F, Salva-tore G, Fusco A, Santoro M, Vecchio G 1999 High prevalence of RET/PTCrearrangements in Ukrainian and Belarussian post-Chernobyl thyroid papil-lary carcinomas: a strong correlation between RET/PTC3 and the solid-fol-licular variant. J Clin Endocrinol Metab 84:4232–4238

19. Ito T, Seyama T, Iwamoto KS, Mizuno T, Tronko ND, Komissarenko IV,Cherstvoy ED, Satow Y, Takeichi N, Dohi K, Akiyama M 1994 Activated REToncogene in thyroid cancers of children from areas contaminated by Chernobylaccident. Lancet 344:259

20. Nikiforov YE, Rowland JM, Bove KE, Monforte-Munoz H, Fagin JA 1997Distinct pattern of ret oncogene rearrangements in morphological variants ofradiation-induced and sporadic thyroid papillary carcinomas in children. Can-cer Res 57:1690–1694

21. Rabes HM, Demidchik EP, Sidorow JD, Lengfelder E, Beimfohr C, HoelzelD, Klugbauer S 2000 Pattern of radiation-induced RET and NTRK1 rear-rangements in 191 post-Chernobyl papillary thyroid carcinomas: biological,phenotypic, and clinical implications. Clin Cancer Res 6:1093–1103

22. Bounacer A, Wicker R, Caillou B, Cailleux AF, Sarasin A, Schlumberger M,Suarez HG 1997 High prevalence of activating ret proto-oncogene rearrange-ments, in thyroid tumors from patients who had received external radiation.Oncogene 15:1263–1273

23. Elisei R, Romei C, Vorontsova T, Cosci B, Veremeychik V, Kuchinskaya E,Basolo F, Demidchik EP, Miccoli P, Pinchera A, Pacini F 2001 RET/PTCrearrangements in thyroid nodules: studies in irradiated and not irradiated,malignant and benign thyroid lesions in children and adults. J Clin EndocrinolMetab 86:3211–3216

24. Schneider AB, Ron E, Lubin J, Stovall M, Gierlowski TC 1993 Dose-responserelationships for radiation-induced thyroid cancer and thyroid nodules: evi-dence for the prolonged effects of radiation on the thyroid. J Clin EndocrinolMetab 77:362–369

25. Favus M, Schneider A, Stachura M, Arnold J, Ryo UY, Pinsky S, Colman M,Arnold M, Frohman L 1976 Thyroid cancer occurring as a late consequence ofhead-and-neck irradiation. N Engl J Med 294:1019–1025

26. Schneider AB, Shore-Freedman E, Ryo UY, Bekerman C, Favus M, PinskyS 1985 Radiation-induced tumors of the head and neck following childhoodirradiation: prospective studies. Medicine 64:1–15

27. Santoro M, Wong WT, Aroca P, Santos E, Matoskova B, Grieco M, Fusco A,di Fiore PP 1994 An epidermal growth factor receptor/ret chimera generatesmitogenic and transforming signals: evidence for a ret-specific signaling path-way. Mol Cell Biol 14:663–675

28. Tallini G, Santoro M, Helie M, Carlomagno F, Salvatore G, Chiappetta G,Carcangiu ML, Fusco A 1998 RET/PTC oncogene activation defines a subsetof papillary thyroid carcinomas lacking evidence of progression to poorlydifferentiated or undifferentiated tumor phenotypes. Clin Cancer Res 4:287–294

29. Viglietto G, Chiappetta G, Martinez-Tello FJ, Fukunaga FH, Tallini G, Rigo-poulou D, Visconti R, Mastro A, Santoro M, Fusco A 1995 RET/PTC oncogeneactivation is an early event in thyroid carcinogenesis. Oncogene 11:1207–1210

30. Giannini R, Salvatore G, Monaco C, Sferratore F, Pollina L, Pacini F, BasoloF, Fusco A, Santoro M 2000 Identification of a novel subtype of H4-RETrearrangement in a thyroid papillary carcinoma and lymph node metastasis.Int J Oncol 16:485–489

31. Papotti M, Volante M, Giuliano A, Fassina A, Fusco A, Bussolati G, SantoroM, Chiappetta G 2000 RET/PTC activation in hyalinizing trabecular tumorsof the thyroid. Am J Surg Pathol 24:1615–1621

32. Basolo F, Molinaro E, Agate L, Pinchera A, Pollina L, Chiappetta G, MonacoC, Santoro M, Fusco A, Miccoli P, Elisei R, Capezzone M, Pacini F 2001 RETprotein expression has no prognostic impact on the long-term outcome ofpapillary thyroid carcinoma. Eur J Endocrinol 145:599–604

33. Basolo F, Giannini R, Monaco C, Melillo RM, Carlomagno F, Pancrazi M,Salvatore G, Chiappetta G, Pacini F, Elisei R, Miccoli P, Pinchera A, FuscoA, Santoro M 2002 Potent mitogenicity of the RET/PTC3 oncogene correlateswith its prevalence in tall-cell variant of papillary thyroid carcinoma. Am JPathol 160:247–254

34. Fluge O, Haugen DRF, Akslen LA, Marstad A, Santoro M, Fusco A, VarhaugJE, Lillehaug JR 2001 Expression and alternative splicing of c-ret RNA inpapillary thyroid carcinomas. Oncogene 20:885–892

35. Rabes HM, Klugbauer S 1998 Molecular genetics of childhood papillarythyroid carcinomas after irradiation: high prevalence of RET rearrangement.Rec Results Cancer Res 154:248–264

36. Klugbauer S, Lengfelder E, Demidchik EP, Rabes HM 1996 A new form ofRET rearrangement in thyroid carcinomas of children after the Chernobylreactor accident. Oncogene 13:1099–1102

37. Williams GH, Rooney S, Thomas GA, Cummins G, Williams ED 1996 RETactivation in adult and childhood papillary thyroid carcinoma using a reversetranscriptase-n-polymerase chain reaction approach on archival-nested ma-terial. Br J Cancer 74:585–589

38. Motomura T, Nikiforov YE, Namba H, Ashizawa K, Nagataki S, YamashitaS, Fagin JA 1998 ret rearrangements in Japanese pediatric and adult papillarythyroid cancers. Thyroid 8:485–489

39. Bunone G, Uggeri M, Mondellini P, Pierotti MA, Bongarzone I 2000 RETreceptor expression in thyroid follicular epithelial cell-derived tumors. CancerRes 60:2845–2849

3946 J Clin Endocrinol Metab, August 2002, 87(8):3941–3946 Collins et al. • RET Expression in Papillary Thyroid Cancer