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Original Studies |
Thyroid Carcinogenesis Group, University of Cambridge, Strangeways Research Laboratory (G.A.T., H.B., H.A.C., E.D.W.), Cambridge, United Kingdom CB1 8RN; the Institute of Pathology, Minsk State Medical Institute (A.N., E.D.C.), Minsk 220600, Belarus; the Institute of Endocrinology and Metabolism (N.D.T., T.I.B.), Kiev 254114, Ukraine; the Istituto Nazionale dei Tumori di Napoli, Fondazione Senatore Pascale (G.C., G.V., F.P.), Naples 80131, Italy; Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli Federico II (G.S., M.S., G.V.), Naples 80131, Italy; and the Dipartimento di Medicina Sperimentale e Clinica, Università di Catanzaro (A.F.), Catanzaro 88100, Italy
Address all correspondence and requests for reprints to: Dr. Massimo Santoro, Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Universita’ degli Studi di Napoli, Via S. Pansini 5, 80131 Naples, Italy. E-mail: masantor{at}unina.it
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Gene rearrangements generating the chimeric RET/PTC
oncogenes are found in thyroid papillary carcinomas (13, 14, 15, 16, 17, 18, 19). These
rearrangements cause the fusion of RET to heterologous
genes. RET encodes the tyrosine kinase receptor for growth
factors of the GDNF family (20). The oncogene resulting from the fusion
of RET to the H4 gene has been designated
RET/PTC1 (14). RET/PTC2 and RET/PTC3
are generated by the fusion between RET and the gene for the
RI
subunit of protein kinase A or the RFG (also named ELE1) gene
(21, 22, 23), respectively. Finally, RET/PTC5 is the fusion
between RET and the RFG5 gene (24). A paracentric inversion
of the long arm of chromosome 10 is responsible for the generation of
RET/PTC1 and RET/PTC3, whereas
RET/PTC2 is generated by a reciprocal balanced translocation
(25). RET/PTC rearrangements are restricted to thyroid
carcinomas of the papillary histotype (15, 18). They are found with a
high frequency in clinically silent, small papillary carcinomas; this
suggests that they can be early events in the tumorigenesis process
(18, 19, 26). Mice transgenic for RET/PTC oncogenes develop
papillary thyroid carcinomas (27, 28, 29). Moreover, both
RET/PTC1 and RET/PTC3 (30) (Melillo, R. M.,
et al., manuscript in preparation) oncogenes have
transforming effects for thyroid cells in culture.
In 1994, Ito et al. reported the frequent (57%) presence of RET rearrangements in 7 post-Chernobyl papillary carcinomas (31). Subsequently, similar results were obtained by Fugazzola et al. (32) and Klugbauer et al. (33) on 2 other small series of Belarussian samples (6 and 12 samples, respectively). Nikiforov et al. analyzed 38 post-Chernobyl Belarussian papillary carcinomas and found that 58% of the cases were positive for RET/PTC3, 16% were positive for RET/PTC1, and 3% for RET/PTC2 (34); intriguingly, 79% of solid variant tumors had RET/PTC3, whereas only 7% had RET/PTC1 (34).
Although a strong increase in the incidence of childhood thyroid papillary carcinomas was observed in Ukraine after the nuclear accident (35), RET/PTC activation has not been previously studied in Ukrainian samples. We have analyzed post-Chernobyl pediatric papillary carcinomas from Ukraine and Belarus for RET/PTC activation. The results indicate that a high frequency of RET/PTC activation is a feature of both Belarussian and Ukrainian cases. Moreover, in both series a strong correlation between the solid-follicular carcinoma and activation of the RET/PTC3 oncogene was observed.
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Materials and Methods |
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Twenty-eight thyroid papillary carcinomas from Ukraine and 39 from Belarus that occurred in patients living in areas contaminated by the Chernobyl nuclear accident and that were operated between January 1995 and July 1997 were studied. Seven follicular carcinomas and 9 follicular adenomas from Ukraine were also studied. The age of the patients ranged between 6–18 yr; 18 patients were male, and 49 were female. The morphological and epidemiological features of these tumors will be described elsewhere (Thomas, G., et al., manuscript in preparation). Histological slides stained by hematoxylin-eosin were reviewed by at least two pathologists from Cambridge and either Minsk or Kiev. Overall classification was performed according to the WHO recommendations (1, 36).
Immunohistochemistry
Five- to 6-µm paraffin sections were deparaffinized, placed in a solution of absolute methanol and 0.3% hydrogen peroxide for 30 min, and then treated with blocking serum for 20 min. The slides were incubated with affinity-purified polyclonal antibodies specific for the RET tyrosine kinase domain (18, 26) (1:100) and with biotinylated anti-rabbit IgG (Vectostain ABC kits, Vector Laboratories, Inc., Burlingame, CA) and with premixed reagent ABC (Vector Laboratories, Inc.). Immunostaining was performed with a diaminobenzidine (DAKO Corp., Carpinteria, CA) solution (0.06 mmol/L diaminobenzidine and 2 mmol/L hydrogen peroxide). The slides were counterstained with hematoxylin.
RT-PCR for RET/PTC expression
Ribonucleic acid (RNA) extraction, RT, and subsequent PCR amplification were performed as previously reported (26). Positive controls were represented by tumor samples harboring RET/PTC1, RET/PTC2, or RET/PTC3 rearrangements (18). Forward primers were: RET/PTC1, 5'-ATTGTCATCTCGCCGTTC-3' (nucleotides 196–214) (14); RET/PTC2, 5'-TATCGCAGGAGAGACTGTGAT-3' (nucleotides 483–503) (21); and RET/PTC3, 5'-AAGCAAACCTGCCAGTGG-3' (nucleotides 697–714) (22). Another RFG primer (5'-AACTGTCCTGCTCTTTGA-3', nucleotides 481–498) was used to detect alternative types of RET/PTC3 (RET/PTC3r2) rearrangements (37). The primer 5'-TACTAGAATACTGCAATC-3' was used to detect the RFG5-RET (RET/PTC5) rearrangement (24). The sequence of the common reverse primer (on the RET tyrosine kinase sequence) was 5'-TGCTTCAGGACGTTGAAC-3' (nucleotides 543–561) (14). Five hundred nanograms of RNA were reverse transcribed and subjected to 40 cycles of PCR (Perkin-Elmer Corp., Norwalk, CT; 94 C for 30 sec, 55 C for 2 min, and 72 C for 2 min). The product was analyzed on a 2% agarose gel and hybridized with a RET probe covering the tyrosine kinase domain. The human hypoxanthine phosphoribosyltransferase-specific primers were 5'-CCTGCTGGATTACATCAAAGCACTG-3' (nucleotides 316–340) and 5'-CCTGAAGTATTCATTATAGTCTCAAGG-3' (nucleotides 685–661) (38). Nine of the RET/PTC1-positive and eight of the RET/PTC3-positive amplified products were completely sequenced to confirm the rearrangement (Sequenase, U.S. Biochemical Corp., Cleveland, OH).
Statistical analysis
The association between the RET/PTC3 rearrangement
and the solid-follicular variant was analyzed by the
2 test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and shown in Fig. 1 demonstrated a high prevalence of
RET rearrangements. The prevalence of
RET/PTC-positive cases was slightly higher in the Ukrainian
(60.7%) than in the Belarussian (51.3%) series. No case of
RET/PTC2 rearrangement was observed; 23 samples were
positive for RET/PTC3, and 14 were positive for
RET/PTC1. Sixteen follicular neoplasms from Ukraine scored
negative (Table 1).
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shows that a correlation
(P < 0.01) between the solid-follicular histotype and
the type of RET/PTC rearrangement was observed; 41% of the
solid-follicular carcinomas were positive for RET/PTC3,
whereas only 10% of them were RET/PTC1 positive.
Conversely, a large portion (41%) of the carcinomas of the classic
type were RET/PTC1 positive. Interestingly, in the case of
one Ukrainian solid-follicular carcinoma (not included in the tables),
both RET/PTC1 and RET/PTC3 rearrangements were
detected (Fig. 1
, sample 2); this suggests that more than one
independent neoplastic clone was present in the same gland.
Diffuse-sclerosing carcinomas (four cases) were present only in the
Ukraine series. Intriguingly, a high proportion of them (three of four)
were RET/PTC positive (two RET/PTC1 and one
RET/PTC3; Table 2). The analysis of a higher number of
samples will be required to assess whether this papillary carcinoma
subtype is associated with such a high frequency of RET
rearrangements. Normal thyroid tissue samples were available in the
case of seven RET/PTC-positive and three
RET/PTC-negative Ukrainian samples; all were negative for
RET/PTC activation (Table 3
and Fig. 2). Lymph node metastasis
specimens were available for eight Ukrainian patients; the primary
tumor scored positive for RET rearrangement in five and
negative in three. In all cases in which the primary tumor was
positive, the metastasis also scored positive and showed the same type
of RET/PTC rearrangement; two representative examples are
shown in Fig. 2. In two cases, two independent metastases were
available from the same patient, and both showed the presence of the
same rearrangement (Table 3). Interestingly, one of the three
metastatic lesions derived from patients whose primary tumor was
negative for RET activation showed the presence of a
RET/PTC3 rearrangement; the patient had a solid-follicular
carcinoma (Fig. 2, lane 4, and Table 3). It is possible that the
rearrangement occurred during the metastatic dissemination or,
alternatively, that it was already present, but in only a minority of
the cells of the primary tumor.
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To confirm the presence of a RET rearrangement and to
demonstrate the expression of the rearranged RET product,
the 17 Ukrainian samples that were RET/PTC positive in the
RT-PCR and the 19 negative Ukrainian samples (4 classic and 6
solid-follicular papillary carcinomas and 9 adenomas) were analyzed by
immunohistochemistry with anti-RET antibodies.
RET expression is not normally found in follicular cells
(41); after the rearrangements, RET transcriptional
regulatory sequences are replaced by those belonging to the fused
genes, and this causes RET/PTC expression (18, 26). A good
correlation was found between RET/PTC rearrangement detected
by RT-PCR and RET protein expression. All 17 positive
samples showed an intense immunostaining with the anti-RET
antibodies. Representative examples of immunostaining of classic and
solid-follicular papillary carcinomas are shown in Fig. 3. When the primary tumor was positive
for RET/PTC protein expression, the metastatic lesion (5
cases) was positive also (1 representative example is shown in Fig. 4B). In some cases the
anti-RET antibody was able to detect the presence of a
neoplastic embolus in the infiltrated vessels of the thyroid tumor
(Fig. 4A). The 9 follicular adenomas were negative on
immunohistochemistry. As a negative control, 10 samples of normal
thyroid tissue were analyzed and invariably scored negative. One of the
10 papillary carcinomas that were negative at the RT-PCR showed a focal
positivity on immunohistochemistry (not shown); it is possible that
only a few neoplastic cells carried the rearrangement in that case
and/or that RET rearrangements other than those detectable
with the primer pairs used were present.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Together, these studies show that post-Chernobyl tumors have a high
frequency of RET activation; this frequency has continued
from the early years after the accident up to the present. The
frequency of RET/PTC activation in nonexposed adult
populations has been reported to vary from 2.5–40% in different
series (13, 14, 15, 16, 17, 18, 19). These differences have been ascribed at least in part
to the geographic origin of the patients studied. In one recent report
the age of the patients has also been demonstrated to be an important
factor, as thyroid carcinomas in patients under 30 yr of age, lacking
evidence of exposure to radiation, showed a higher frequency of
RET/PTC activation than older patients (42). Thus, a matched
control (age and region) would be necessary to ascertain whether the
exposure to radioactive isotopes has really caused an increase in the
prevalence of RET rearrangements. For a number of reasons,
including the extreme rarity of thyroid carcinomas in children in the
Chernobyl area before the accident (0.5 cases/million·yr) (12), such
a control is very difficult to obtain. However, several considerations
support the idea of the important role played by RET
rearrangements in radiation-associated thyroid papillary carcinomas.
The prevalence of RET/PTC activation in post-Chernobyl
carcinomas [in our study and the other published reports (31, 32, 33, 34)] is
higher than the highest frequencies reported in the literature in
nonexposed subjects. Moreover, mutations of other genes
(ras, Gs TSHR, and p53) known to be
involved in thyroid carcinogenesis have not been detected or have
occurred at a very low prevalence in post-Chernobyl tumors (43, 44)
(Santoro, M., et al., submitted). In addition, the fact that
ionizing radiation can induce RET/PTC rearrangement in
vitro (45, 46) and the high frequency of RET/PTC
rearrangements in patients exposed to external radiation (47) support
the hypothesis that these rearrangements can be a direct consequence of
radiation exposure.
One of the most striking features of post-Chernobyl papillary carcinomas is the correlation between the type of rearrangement and the morphological variant of the carcinoma. Frequently, post-Chernobyl carcinomas have a solid-follicular aspect (46 of 67 cases in the present series). This variant is characterized by solid nests of tumor cells, often with many small follicular lumina; it may also show a minor papillary component and tumor cells with ground-glass nuclei and nuclear grooves typical of papillary carcinomas (39, 48). Although we have found RET/PTC1 favored in tumors having a prevalent classic aspect (7 of 10 RET/PTC-positive samples), a prevalence of the RET/PTC3 isoform (19 of 24 RET/PTC-positive samples) was observed in papillary carcinomas of the solid-follicular type. This is intriguing, because this morphological variant is rarely found in the nonexposed population, and it is considered by some researchers as evidence of a more malignant phenotype (49); consistently, post-Chernobyl carcinomas are relatively aggressive, showing intraglandular dissemination, extension to the perithyroid tissue, and distant metastases. This correlation, described first by Nikiforov et al. (34), has been confirmed experimentally in transgenic mouse models. Although the targeted expression of RET/PTC1 to the thyroid gland caused the generation of carcinomas of the classic type (27, 28), RET/PTC3 mice develop aggressive carcinomas with a prevalent solid component, which are highly prone to metastasize to regional lymph nodes (29). It is still unknown which difference(s) between the two oncoproteins explains their different effects in vivo. The RET component of the 2 chimeric proteins is identical, and our recent findings indicate that there is no significant difference in the extent of activation of the intrinsic RET kinase function in RET/PTC1 and RET/PTC3 (Melillo, R. M., et al., unpublished observations). Thus, although the function of H4 and RFG genes (in the case of RET/PTC1 and RET/PTC3, respectively) is still unknown, it is tempting to speculate that some functional differences between the two RET fusion partners may contribute to the different neoplastic phenotypes.
We conclude that a high proportion of post-Chernobyl thyroid carcinomas in children show RET rearrangement, and that there are good reasons to believe that there is a causal link between radiation exposure and the rearrangement. We also find a strong correlation between the morphological subtype of papillary carcinoma and the type of RET rearrangement; this link is seen in both humans and transgenic animals. RET/PTC3 rearrangement is particularly prevalent in radiation-induced tumors in children, but whether this is linked primarily to the nature of the carcinogenic agent or to the age of the child remains to be determined.
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Acknowledgments |
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Footnotes |
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Received April 7, 1999.
Revised July 20, 1999.
Accepted July 22, 1999.
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gene
mutations in childhood thyroid tumors after Chernobyl in contrast to
sporadic adult thyroid neoplasia. Cancer Res. 57:2358–2361.This article has been cited by other articles:
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