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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 5 2318-2326
Copyright © 2003 by The Endocrine Society

RAS Point Mutations and PAX8-PPAR{gamma} Rearrangement in Thyroid Tumors: Evidence for Distinct Molecular Pathways in Thyroid Follicular Carcinoma

Marina N. Nikiforova, Roy A. Lynch, Paul W. Biddinger, Erik K. Alexander, Gerald W. Dorn, II, Giovanni Tallini, Todd G. Kroll and Yuri E. Nikiforov

Department of Pathology and Laboratory Medicine (M.N.N., P.W.B., Y.E.N.) and Division of Cardiology (R.A.L., G.W.D.), University of Cincinnati, Cincinnati, Ohio 45267-0529; Department of Medicine (E.K.A.), Brigham and Women’s Hospital, Boston, Massachusetts 02115; Department of Pathology (G.T.), Yale University School of Medicine, New Haven, Connecticut 06510; and Department of Pathology and Laboratory Medicine (T.G.K.), Emory University School of Medicine, Winship Cancer Institute, Atlanta, Georgia 30322

Address all correspondence and requests for reprints to: Dr. Yuri Nikiforov, Department of Pathology, University of Cincinnati, 231 Albert Sabin Way, P.O. Box 670529, Cincinnati, Ohio 45267-0529. E-mail: yuri.nikiforov{at}uc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A series of 88 conventional follicular and Hürthle cell thyroid tumors were analyzed for RAS mutations and PAX8-PPAR{gamma} rearrangements using molecular methods and for galectin-3 and HBME-1 expression by immunohistochemistry. A novel LightCycler technology-based method was developed to detect point mutations in codons 12/13 and 61 of the H-RAS, K-RAS, and N-RAS genes. Forty-nine percent of conventional follicular carcinomas had RAS mutations, 36% had PAX8-PPAR{gamma} rearrangement, and only one (3%) had both. In follicular adenomas, 48% had RAS mutations, 4% had PAX8-PPAR{gamma} rearrangement, and 48% had neither. Follicular carcinomas with PAX8-PPAR{gamma} typically showed immunoreactivity for galectin-3 but not for HBME-1, tended to present at a younger patient age and be smaller size, and were almost always overtly invasive. In contrast, follicular carcinomas with RAS mutations most often displayed an HBME-1-positive/galectin-3-negative immunophenotype and were either minimally or overtly invasive. Hürthle cell tumors infrequently had PAX8-PPAR{gamma} rearrangement or RAS mutations. These results suggest that conventional follicular thyroid carcinomas develop through at least two distinct and virtually nonoverlapping molecular pathways initiated by either RAS point mutation or PAX8-PPAR{gamma} rearrangement.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FOLLICULAR THYROID TUMORS include conventional follicular carcinoma, Hürthle cell (oncocytic) carcinoma, and their benign counterparts, follicular adenoma and Hürthle cell (oncocytic) adenoma. Follicular cancers account for approximately 15% of all thyroid malignancies, with about two thirds being conventional and one third Hürthle cell types (1). Conventional follicular carcinomas virtually never involve regional lymph nodes and have distant metastases, most commonly to the lungs and bones (10–20% of cases). Hürthle cell carcinomas develop blood-borne metastases but also spread to regional lymph nodes.

Preoperative diagnosis of follicular tumors is difficult because adenomas and carcinomas share similar cytologic features. Only two reliable histologic criteria, capsular and vascular invasion, are known to detect malignancy before local invasion or metastases, but these can be evaluated only after surgical resection. In addition, some cellular hyperplastic nodules and follicular variants of papillary carcinoma may mimic follicular tumors in fine-needle aspiration (FNA) specimens. As a result, many follicular lesions diagnosed as indeterminate or suspicious by FNA cytology are referred for surgery when only a small fraction (8–17%) prove to be malignant (2).

Recently, galectin-3 and HBME-1 immunostains have been suggested to be helpful for preoperative diagnosis of follicular carcinomas. Galectin-3, a member of the ß-galactosidase-binding lectin family, is reportedly up-regulated in thyroid malignant tumors and undetectable in normal thyroid tissue or benign lesions (3, 4, 5, 6). HBME-1 antibody, generated against a suspension of malignant mesothelial cells, is reported to be a sensitive and specific marker of thyroid malignancy as well (7, 8, 9). Each of these markers is imperfect in separating benign and malignant thyroid lesions, and their specificity and sensitivity vary greatly in different series (10, 11, 12).

Follicular tumors have been known to harbor activating point mutations of the RAS genes. Three RAS genes, H-RAS, K-RAS, and N-RAS, encode highly related 21-kDa proteins located at the inner surface of the cell membrane and play a central role in the transduction of signals arising from tyrosine kinase and G protein-coupled receptors. RAS point mutations in thyroid and other human neoplasms occur typically in codons 12, 13, or 61, leading to constitutive activation of downstream signaling pathways. Somatic missense mutations in codons 12/13 and 61 of one of the three RAS genes have been found in 18–52% of follicular carcinomas (13, 14, 15, 16, 17, 18) and 24–53% of follicular adenomas (13, 14, 15, 16, 19). A much lower incidence has been reported in Hürthle cell tumors (15–25% of carcinomas and 0–4% of adenomas) (15, 20, 21).

Recently a PAX8-PPAR{gamma} gene fusion was identified in a significant portion of follicular carcinomas, some with a cytogenetically detectable translocation t(2;3)(q13;p25) (22). The t(2;3) rearrangement leads to an in-frame fusion of the PAX8 gene, which encodes a paired domain transcription factor, with the peroxisome proliferator-activated receptor (PPAR) {gamma} gene. In the original report, PAX8-PPAR{gamma} fusion was detected in 63% of follicular carcinomas but not in follicular adenomas, papillary carcinomas, or nodular hyperplasias, suggesting that it may represent a specific molecular marker of follicular carcinomas (22). In recent follow-up series, PAX8-PPAR{gamma} was identified by RT-PCR in 26–56% of follicular carcinomas and in 0–13% of follicular adenomas, 0–3% of Hürthle cell carcinomas, and 0–1% papillary carcinomas (23, 24, 25). Chromosomal rearrangement is likely to be a preferable mechanism of PPAR{gamma} activation in thyroid tumors because no point mutations in the PPAR{gamma} gene were found in thyroid carcinomas and cell lines (26).

To date, the correlation between PAX8-PPAR{gamma} rearrangements and RAS point mutations in thyroid follicular tumors has not been studied. In the present study, we have analyzed the association between these genetic alterations and their correlation with galectin-3 and HBME-1 immunoreactivity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Sample selection and tumor classification

We studied 88 thyroid tumors including 33 conventional follicular carcinomas, 23 conventional follicular adenomas, 19 Hürthle cell carcinomas, and 13 Hürthle cell adenomas. The tumor samples were collected from the Cincinnati University Hospital (48 samples), Brigham and Women’s Hospital (16 samples), Yale New Haven Hospital (10 samples), or obtained through the Cooperative Human Tissue Network (CHTN) (14 samples). In 52 cases, snap-frozen tissue and in 36 cases paraffin-embedded tissue was available. The study was approved by the University of Cincinnati Institutional Review Board.

Tumors were classified according to widely accepted histologic criteria (27, 28). Briefly, tumors were diagnosed as Hürthle cell (oncocytic) neoplasms when more than 75% of cells had oncocytic features. Follicular and Hürthle cell carcinomas were distinguished from adenomas on the basis of invasion of the entire thickness of the tumor capsule and/or invasion of blood vessels within the capsule or immediately external to it. Adenomas were separated from hyperplastic nodules based on the presence of encapsulation, compression of the adjacent thyroid parenchyma, and uniform hypercellular appearance.

Histologically, follicular carcinomas are subdivided into minimally invasive and widely invasive (27, 28). For the purpose of this study, we subdivided follicular carcinomas into three groups according to degree invasiveness.

Minimally invasive. This is an encapsulated neoplasm with microscopically detectable one or two microscoric foci of capsular or vascular invasion. The major differential diagnosis for such cases is follicular adenoma, and distinction from adenoma is often difficult and requires deeper sectioning of suspicious areas.

Overtly invasive. These are completely or partially encapsulated tumors with multiple (at least three, typically more than five) areas of capsular and vascular invasion but still confined to the thyroid gland. Capsular invasion is typically seen as a broad-based extension through the tumor capsule, and endotheliolized tumor aggregates are usually found in several consecutive blood vessels. The diagnosis of follicular carcinoma in such cases is straightforward, although the tumor does not show extrathyroidal extension.

Widely invasive. Widely invasive neoplasm typically replaces the entire thyroid lobe, with extrathyroidal extension and invasion of perithyroidal tissues. The tumor lacks evidence of encapsulation. Diagnosis of malignancy is unequivocal, even on gross examination. Differential diagnosis includes poorly differentiated or anaplastic carcinoma.

Nucleic acid isolation and reverse transcription

DNA was extracted by the phenol-chloroform method as previously described (29). RNA isolation from snap-frozen and paraffin-embedded tissue was performed using Trizol reagent (Invitrogen, Carlsbad, CA) as previously described (30). Three micrograms total RNA extracted from frozen tissue and 5 µl RNA extracted from paraffin-embedded tissue were reverse transcribed in a volume of 20 µl, using random hexamer priming and Superscript II RT (Invitrogen) according to the manufacturer’s protocol. All cDNA samples were tested to assess the adequacy of RNA by amplifying a 247-bp control sequence of the PGK gene as reported elsewhere (31).

Detection of RAS mutations

Point mutations in codons 12/13 and 61 of the H-, K-, and N-RAS genes were detected using three different methods.

LightCycler PCR and fluorescence melting curve analysis (FMCA). LightCycler technology has been demonstrated as an efficient method for detection of N-RAS mutations in human tumors (32, 33). It is based on rapid-cycle PCR amplification of the locus containing a mutational hot spot on the LightCycler (Roche Molecular Biochemicals, Manheim, Germany) using a hybridization probe format, followed by FMCA. This format requires a pair of primers and two fluorescently labeled oligonucleotide probes and relies on fluorescence resonance transfer interaction between two probes that hybridize adjacent to one another, so that one fluorophore functions as the donor and another as the acceptor, the latter emitting a light of specific wavelength (34). For the detection of a point mutation, one probe is designed to span the mutation site, and the products are distinguished based on their distinct melting temperatures (Tm), which reflect the thermodynamic stability of the perfectly complementary and mismatched probe-target duplexes.

For each mutational hot spot, a pair of primers and two internal oligonucleotide probes were obtained (Table 1Go). To detect N-RAS 12/13 and N-RAS 61 mutations, primers and probes were used as described by Elenitoba-Johnson et al. (32) with some modifications. Primers and probes for the other four hot spots were designed with the help of Olfert Landt, TIB Molbiol (Berlin, Germany). All primers and probes were obtained from TIB Molbiol. Amplification was performed in a glass capillary using 50–100 ng DNA in a 20-µl volume containing 2 µl of 10x LightCycler DNA master hybridization probes [containing PCR buffer, deoxynucleotide triphosphates, 10 mM MgCl2, and Taq polymerase (Roche), 1.6 µl of 25 mM MgCl2, 40 pmol of each primer, and 2 pmol of each hybridization probe]. The reaction mixture was subjected to 40 cycles of rapid PCR consisting of denaturation at 94 C for 1 sec, annealing at 50–54 C for 20 sec, and extension at 72 C for 10 sec. Postamplification FMCA was performed by gradual heating of samples at a rate of 0.2 C/sec from 45 C to 95 C. Fluorescence melting peaks were built by plotting of the negative derivative of fluorescent signal corresponding to the temperature (-dF/dT).


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  		Table 1. Primers and probes used for LightCycler amplification and FMCA

 
Normal placental DNA was used as a negative control and DNA from cell lines carrying one of the RAS mutations served as a positive control (29). All PCR products that showed deviation from the wild-type (placental DNA) melting peak were sequenced after purification through Microcon PCR kit (Millipore Corp., Billerica, MA) to verify the presence of RAS mutation and detect the exact nucleotide change.

DNA heteroduplex analysis by HPLC (WAVE HPLC). To detect RAS mutations using this method, 200 ng DNA from each sample were amplified using conventional PCR with primers flanking codons 12/13 and 61 of the three RAS genes. The PCR products (ranging in size from 182–400 bp) were then characterized by temperature-dependent denaturing HPLC on WAVE apparatus (Transgenomic, Inc., Omaha, NE) and eluted using a linear gradient (typically 10–20%) of acetonitrile in 0.1 M triethyl ammonium acetate over 7–10 min, as described previously (35). Optimal melting temperature and elution gradient acetonitrile programs for each PCR product were initially estimated using WAVEMaker 4.0 software (Transgenomic). Negative and positive controls were as described above. All samples that displayed a deviation from normal (placental DNA) elution profiles were directly sequenced.

Direct DNA sequencing. Two hundred nanograms of DNA were PCR amplified using primers and conditions reported previously (29) and direct sequenced using an automated ABI model 377 sequencer (Applied Biosystems, Foster City, CA). Both sense and antisense primers were used for sequencing to assure the detection of heterozygous mutations in the RAS genes.

Detection of PAX8-PPAR{gamma} rearrangement

Most of the cases employed in this study were previously analyzed for PAX8-PPAR{gamma} rearrangement by RT-PCR (58 patients) (23) and PPAR{gamma} with 3p25 probe by fluorescence in situ hybridization (16 patients) (22, 25). Fourteen additional tumors were studied for PAX8-PPAR{gamma} fusion by RT-PCR as previously described (23). PCR products were resolved by electrophoresis in a 1.5% agarose gel and in selected cases sequenced using an automated ABI model 377 sequencer.

Immunohistochemical analysis

Immunostaining was performed on paraffin-embedded sections using avidine-streptavidin immunoperoxidase method with monoclonal antibodies against galectin-3 (Vector Laboratories Ltd., Burlingame, CA) at a dilution of 1:100 and HBME-1 (DAKO Corp., Carpinteria, CA) at a dilution of 1:80 on automated Benchmark system (Ventana Medical Systems, Inc., Tuscon, AZ). Antigen retrieval was achieved by incubation at mild degree in cell condition 1 solution (Ventana). Paraffin sections from a papillary thyroid carcinoma were used as a positive control. For galectin-3, staining was considered specific when cytoplasmic and/or nuclear reactivity was noted. For HBME-1, only membranous and apical/colloidal immunoreactivity was considered specific (9). For both antibodies, tumors were scored positive when more than 10% of cells were immunoreactive.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RAS point mutations

First, DNA from 47 tumors, including 20 samples isolated from frozen tissue and 27 from paraffin-embedded tissue, and six cell lines with known RAS mutations were analyzed using LightCycler FMCA and WAVE HPLC. The detection of RAS mutations by both methods was compared with that by direct DNA sequencing as the gold standard. Nine thyroid tumors had N-RAS codon 61 mutations (7 CAA->CGA and 2 CAA->AAA) and another two cases had H-RAS codon 61 CAG->AAG mutation by sequencing. LightCycler FMCA identified RAS mutations in all 11 tumors and in all six cell lines with known RAS mutations (Fig. 1Go).



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  		Figure 1. LightCycler FMCA detection of RAS point mutations based on distinct Tm of duplexes formed between the wild-type probe and either wild-type (wt) or mutant sequences. For N-RAS 12/13 locus, the wt sequence Tm was 60.6 C and the GGT->TGT mutation Tm was 55.2 C. For N-RAS 61, the wt Tm was 62.1 C, the CAA->CGA mutation Tm was 58.8 C, and the CAA->AAA mutation Tm was 54.7 C. For H-RAS 12/13, the wt Tm was 66.6 C and the GGC->GTC mutation Tm was 59 C. For H-RAS 61, the wt Tm was 66.4 C, the CAG->CGG mutation Tm was 61.7 C, and the CAG->AAG mutation Tm was 58.8 C. For K-RAS 12/13, the wt Tm was 69.4 C and the GGT->GTT mutation Tm was 62.8 C. For K-RAS 61, the wt Tm was 59.5 C and the CAA->CAT mutation Tm was 53.6 C. All mutations shown were heterozygous because the products also demonstrated a wt peak, except for the homozygous H-RAS 12 GGC->GTC mutation in the T24 cell line.

 
For WAVE HPLC, all 20 DNA samples extracted from fresh tissue were efficiently amplified by PCR and amendable to the analysis, whereas of 27 cases from paraffin-embedded tissue, 9–18 samples failed to amplify various RAS regions, probably because of a limited DNA quality and larger size of amplicons (182–400 bp). Nine informative cases revealed abnormal elution profiles, five of which corresponded to specific RAS mutations with another four having a codon 27 H-RAS polymorphism, as detected by sequencing. Among six informative cases with RAS mutations identified by direct sequencing, five (83%) were detected by WAVE HPLC (data not shown).

Taking into account that LightCycler FMCA was as sensitive and specific as direct sequencing and no new mutations were detected by WAVE HPLC, remaining tumor samples were analyzed for RAS mutations by LightCycler FMCA and sequencing of PCR products showing abnormal melting curves. Overall, RAS mutations were detected in 17 (52%) follicular carcinomas, 11 (48%) follicular adenomas, 2 (11%) Hürthle cell carcinomas, and 1 (8%) Hürthle cell adenoma (Table 2Go). In follicular carcinomas and adenomas, the CAA->CGA mutation of N-RAS codon 61 was the most common. In Hürthle cell tumors, mutations were observed only in H-RAS codon 61 and K-RAS codon 12. The overall spectrum of RAS mutation detected in these cases is shown in Table 3Go.


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  		Table 2. Prevalence of RAS point mutations and PPAR{gamma} rearrangement in conventional follicular and Hürthle cell tumors

 

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  		Table 3. Spectrum of RAS mutations identified in conventional follicular and Hürthle cell tumors

 
PPAR{gamma} rearrangement

Seventy-two tumors were studied for PAX8-PPAR{gamma} rearrangement by RT-PCR. The fusion was found in 5 of 17 follicular carcinomas, in 1 of 23 follicular adenomas, but not in 32 Hürthle cell tumors. Five tumors (four follicular carcinomas and one follicular adenoma) with PAX8-PPAR{gamma} rearrangement showed products consistent with fusion between exon 9 of PAX8 and exon 1 of PPAR{gamma}. One other follicular carcinoma showed fusion between exon 7 of PAX8 and exon 1 of PPAR{gamma}. Sixteen additional follicular carcinomas were selected for study based on rearrangement status by fluorescence in situ hybridization with 3p25 probes flanking the PPAR{gamma} gene. Eight follicular carcinomas were rearrangement positive, eight cases with rearrangement negative. The overall prevalence of PPAR{gamma} rearrangement in these tumors is summarized in Table 2Go.

Galectin-3 and HBME-1 immunohistochemistry

Thirty-three follicular carcinomas and 23 follicular adenomas were analyzed for galectin-3 and HBME-1 expression by immunohistochemistry (Fig. 2Go). Overall, 70% of follicular carcinomas and 30% of follicular adenomas showed immunoreactivity with at least one of the antibodies (Table 4Go). In general, immunoreactivity with both antibodies in the follicular tumors was more often focal than diffuse and of weaker intensity in comparison with those we typically observe in papillary carcinomas.



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  		Figure 2. Immunohistochemical analysis for galectin-3 and HBME-1. Gelactin-3 immunoreactivity was typically both cytoplasmic and nuclear, whereas specific HBME-1 immunoreactivity was membranous and apical/colloidal. Follicular carcinomas with the PPAR{gamma}+/RAS- genotype were commonly positive for galectin-3 and negative for HBME-1 (top), whereas tumors with the RAS+/PPAR{gamma}- genotype were typically negative for galectin-3 and positive for HBME-1 (bottom).

 

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  		Table 4. Immunoreactivity for galectin-3 and HBME-1 in thyroid tumors

 
Correlation between RAS mutations and PPAR{gamma} rearrangement and galectin-3 and HBME-1 immunohistochemistry

Of 88 total tumors, 34% had only RAS mutations, 15% had only PPAR{gamma} rearrangement, and 1% had both. Among conventional follicular carcinomas, 49% had a RAS+/PPAR{gamma}- genotype and 36% had a PPAR{gamma}+/RAS- genotype. One follicular carcinoma (3%) harbored both mutations (Table 2Go). These genotypic changes were correlated with galectin-3 and HBME-1 immunohistochemistry within the same tumors. Follicular carcinomas with PPAR{gamma} rearrangement showed immunoreactivity for galectin-3 in 75% of cases and no immunoreactivity for HBME-1 in 59% of cases. In contrast, follicular carcinomas with RAS mutations were mostly negative for galectin-3 (88%) and positive for HBME-1 (62%) (Table 5Go).


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  		Table 5. Immunoprofiles and clinical-pathological features of follicular carcinomas with PPAR{gamma}+/RAS- and RAS+/PPAR{gamma}- genotypes

 
Clinical-pathological features of tumor groups are summarized in Table 5Go. Follicular carcinomas with the PPAR{gamma}+/RAS- genotype had a tendency to develop in younger patients, be of a smaller size, and almost all of them were overtly invasive at presentation (Fig. 3Go). Follicular carcinomas with RAS+/PPAR{gamma}- genotype tended to present at older age, be larger in size, and show a wide range of invasiveness. Two tumors with focal areas of poorly differentiated histology were both RAS+/PPAR{gamma}-.



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  		Figure 3. Microscopic features of overtly invasive follicular carcinoma. A, Broad-based invasion through the tumor capsule. B, Multifocal vascular invasion with tumor thrombi identifiable in four consecutive blood vessels within the tumor capsule (arrows). Note that despite the readily identifiable features of invasive growth, these tumors preserve overall encapsulation.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report here that 85% of conventional follicular thyroid carcinomas develop through two distinct and virtually nonoverlapping molecular pathways: one involving RAS point mutations and another involving PAX8-PPAR{gamma} rearrangement. The fact that only 1 of 33 follicular carcinomas had both mutations suggests at least two possibilities: These mutations involve activation of the same signaling pathway so that the occurrence of two mutations provides little additional growth advantage, or conventional follicular carcinomas consist of at least two groups of tumors developing through distinct molecular mechanisms. Our results obtained by immunohistochemistry support the second possibility because follicular carcinomas with RAS mutations were found typically to express HBME-1 and little galectin-3, whereas follicular carcinomas with PPAR{gamma} rearrangement characteristically expressed galectin-3 and less HBME-1. Potential differences were also observed in the clinical-pathological features in these two cancer groups. For example, tumors with PAX8-PPAR{gamma} tended to present mostly during the fourth decade of life and be somewhat smaller in size, whereas RAS-positive tumors presented at older age, typically during the fifth decade of life, and were larger.

There was also significant variability in histologic invasiveness between tumors harboring PAX8-PPAR{gamma} and RAS mutations (Fig. 4Go). For example, 12 of 13 tumors with PPAR{gamma} rearrangement were follicular carcinomas, but only one was a follicular adenoma. Eleven of the 12 follicular carcinomas with PAX8-PPAR{gamma} were overtly invasive and demonstrated multifocal capsular and vascular invasion. The follicular adenoma had a thick capsule and displayed strong and diffuse HBME-1 and galectin-3 (carcinoma-like) immunoreactivity, raising the possibility it might be a preinvasive or in situ follicular carcinoma. Thus, it is likely that PAX8-PPAR{gamma} rearrangement confers invasive potential at the earliest tumor stages such that a benign follicular adenoma precursor lesion does not exist. High frequency of vascular invasion in these tumors correlates with the possibility of advanced disease, and distant metastases have been observed in follicular carcinomas with PAX8-PPAR{gamma} (36) (Dwight, T., and C. Larsson, personal communication; Kroll, T. G., unpublished data).



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  		Figure 4. Spectrum of invasiveness of follicular tumors with PPAR{gamma} rearrangement and RAS mutations.

 
On the other hand, the RAS+/PPAR{gamma}- genotype was observed in almost equal number of follicular carcinomas and follicular adenomas (Fig. 4Go). The follicular adenomas displayed no morphologic indicators of malignancy and showed little expression of galectin-3 and some immunoreactivity for HBME-1. The follicular carcinomas demonstrated the entire spectrum of histologic invasiveness and were minimally, overtly, and widely invasive. This suggests that RAS-initiated follicular carcinomas develop through a benign follicular adenoma stage/precursor. Thus, RAS activation by itself appears insufficient to determine malignant growth but may predispose to acquisition of additional genetic or epigenetic alterations that lead to a fully transformed phenotype.

This possibility is supported by in vitro studies. Acute expression of mutant H-RAS in cultured normal human thyroid cells induces a period of rapid proliferation during which the cells exhibit a partially transformed phenotype but retain differentiation features (37, 38). Remarkably, after 15–25 population doublings, the cells stop growing and undergo senescence, despite undiminished expression of mutant RAS. This self-limited cell proliferation with the retention of differentiation properties recapitulates in many aspects the growth of human follicular adenomas. Indeed, thyroid-specific expression of mutant K-RAS in transgenic mice has shown to lead to the development of benign thyroid nodules and follicular adenomas, whereas carcinomas are rare and required additional goitrogen stimulation (39).

Hürthle cell carcinomas possess a distinct set of large-scale chromosomal abnormalities, as it has been demonstrated by comparative genomic hybridization and loss of heterozygosity studies (21, 40, 41, 42). Our data indicate that Hürthle cell adenomas and carcinomas have a very low frequency of either RAS mutations or PAX8-PPAR{gamma} rearrangement. These tumors, despite sharing some histologic features with conventional follicular tumors, apparently develop via separate molecular events and likely represent a distinct type of thyroid neoplasm.

Among unselected 17 follicular carcinomas in this study, 88% revealed either RAS (58%) or PAX8-PPAR{gamma} (30%) mutations. This suggests that molecular testing of thyroid FNA specimens may be useful to improve the accuracy of preoperative diagnosis of follicular tumors. Thus, finding of PAX8-PPAR{gamma} rearrangement in thyroid FNA samples would be a strong indicator of follicular carcinoma. As for RAS mutation, it cannot discriminate between follicular carcinoma and follicular adenoma but would allow distinction between follicular neoplasms and hyperplastic thyroid nodules. Such distinction is of considerable clinical importance because 42–77% of thyroid nodules removed surgically are nonneoplastic colloid nodules (2).

In conclusion, our data suggest that follicular thyroid carcinomas can develop through different molecular pathways (Fig. 5Go). For conventional follicular tumors, at least two distinct pathways exist, initiated by either PAX8-PPAR{gamma} rearrangement or RAS mutation. Hürthle cell tumors have a low frequency of both of these genetic alterations and apparently require a unique set of mutations for their development.



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  		Figure 5. Putative molecular pathways in thyroid conventional follicular and Hürthle cell tumors. Tumors harboring PAX8-PPAR{gamma} rearrangement directly develop as follicular carcinomas without a benign adenoma stage. Acquisition of RAS mutations leads to the development of follicular adenomas, whereas additional and still unknown genetic alterations require for malignant transformation and progression to a follicular carcinoma stage. Hürthle cell tumors lack both of these genetic alterations and require a separate set of mutations for their development. Whether Hürthle cell adenoma is a precursor lesion for Hürthle cell carcinoma remains unclear.

 


   Footnotes
 
This work was supported by NIH Grants R01-CA88041 (to Y.N.) and CA75425 and a Georgia Cancer Coalition Distinguished Clinical Scientist Award (to T.G.K.). Tissue samples were collected in part using a support by a grant from the NIH (PHS M01 RR08084, the Children’s Hospital–University of Cincinnati General Clinical Research Center Tissue Procurement Facility) and through the Cooperative Human Tissue Network (CHTN), which is funded by the National Cancer Institute.

Abbreviations: FMCA, Fluorescence melting curve analysis; FNA, fine-needle aspiration; PPAR, peroxisome proliferator-activated receptor; Tm, melting temperature.

Received December 4, 2002.

Accepted February 14, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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