help button home button Endocrine Society JCEM JCEM Call for Nominations for EIC
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2006-1006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
91/9/3603    most recent
Author Manuscript (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhu, Z.
Right arrow Articles by Nikiforov, Y. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhu, Z.
Right arrow Articles by Nikiforov, Y. E.
Related Collections
Right arrow Endocrine Oncology
Right arrow Thyroid
The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 9 3603-3610
Copyright © 2006 by The Endocrine Society

Prevalence of RET/PTC Rearrangements in Thyroid Papillary Carcinomas: Effects of the Detection Methods and Genetic Heterogeneity

Zhaowen Zhu, Raffaele Ciampi, Marina N. Nikiforova, Manoj Gandhi and Yuri E. Nikiforov

Department of Pathology and Laboratory Medicine (Z.Z., R.C., M.G., Y.E.N.), University of Cincinnati, Cincinnati, Ohio 45267; and Division of Pathology (M.N.N.), Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45229

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
 
Context: RET/PTC rearrangements have been reported in papillary thyroid carcinomas with variable frequency in studies that used different detection methods.

Objective: Our objective was to determine the role of different detection methods and tumor genetic heterogeneity on RET/PTC detection.

Design: Sixty-five papillary carcinomas were analyzed for RET/PTC1 and RET/PTC3 using five detection methods: standard-sensitivity RT-PCR, high-sensitivity RT-PCR, real-time LightCycler RT-PCR, Southern blot analysis, and fluorescence in situ hybridization.

Results: RET/PTC rearrangements were detected by standard-sensitivity RT-PCR in 14 tumors. High-sensitivity RT-PCR detected RET/PTC in all of these and in 12 additional cases, where the levels of expression corresponded to one to five positive cells. Real-time LightCycler RT-PCR detected RET/PTC in 12 and Southern blot analysis in 11 tumors. By fluorescence in situ hybridization, 14 tumors were positive, including nine cases with 50–86% positive cells and five cases with 17–35% positive cells. Overall, nine (14%) tumors harbored clonal rearrangements, which were present in the majority of tumor cells and detected by all five methods. Five (8%) cases had subclonal rearrangements present in a smaller portion of tumor cells and detected by most methods. Twelve (18%) tumors had nonclonal RET/PTC that were detected only by high-sensitivity RT-PCR. No other mutations were found in tumors harboring clonal RET/PTC, whereas 60% of tumors with subclonal and 42% of tumors with nonclonal RET/PTC harbored additional mutations.

Conclusions: Our data suggest that broad variability in the reported prevalence of RET/PTC rearrangement is at least in part a result of the use of different detection methods and tumor genetic heterogeneity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CHROMOSOMAL REARRANGEMENT involving the RET receptor tyrosine kinase gene is a characteristic feature of papillary thyroid carcinoma. The rearrangement, named RET/PTC, fuses the 3'-terminal portion of RET coding for tyrosine kinase domain with the 5'-terminal sequence of different unrelated genes, leading to constitutive activation of the RET tyrosine kinase (1, 2). The two most common rearrangement types, RET/PTC1 and RET/PTC3, account for the vast majority of all rearrangements found in the general population. RET/PTC1 is formed by fusion with the H4 (D10S170) gene (2), and RET/PTC3 by fusion with the NCOA4 (ELE1, RFG or ARA70) gene (3, 4).

A longstanding controversy exists with respect to the prevalence of RET/PTC in papillary thyroid carcinomas and its specificity for this tumor type. The reported frequency of RET/PTC in papillary carcinomas in different studies varies from 0–87% (reviewed in Refs. 5 and 6). In part, it is likely because of geographic variability and a well-established higher incidence of this rearrangement in tumors associated with radiation exposure (7, 8, 9, 10, 11, 12). However, this cannot serve as the only explanation, because a striking variability in the frequency has been reported in the same geographical regions [8 and 85% in Australia (13, 14), 5 and 77% in Canada (15, 16)]. In the United States, the five largest series reported the frequency of RET/PTC ranging from 11–43% (17, 18, 19, 20, 21).

A variety of methods have been used for the detection of RET/PTC, but the sensitivity of the detection and quantitation of the rearrangement have rarely been performed. Most studies reporting a high incidence of the rearrangement used sensitive RT-PCR, as opposed to less sensitive but more reliable techniques such as Southern blot analysis (SB) and fluorescence in situ hybridization (FISH). Additional variability is likely to originate from applying the same RNA-based detection techniques for the analysis of snap-frozen and paraffin-embedded tissue, despite profound degradation of RNA in the latter. In addition, the quantitative analysis has suggested that levels of the RET/PTC mRNA may vary significantly between individual tumors (22). RET/PTC rearrangement may also have a patchy distribution within the tumor nodule and be present only in a portion of tumor cells, as it was shown for radiation-associated papillary carcinomas (23). Thus, it is conceivable that the discrepancy between the reported prevalence of RET/PTC in papillary carcinomas may be a result of different sensitivities of the detection methods, particularly if the rearrangement is present only in a small portion of tumor cells or if cells express the RET/PTC transcripts at low levels.

Similar factors could be responsible for a controversy surrounding the RET/PTC specificity for papillary carcinomas. Indeed, the rearrangement is generally believed to be restricted to the papillary type of thyroid carcinoma (17, 21, 24). The original study of 281 thyroid tumors using SB detected RET rearrangement in 19% of papillary carcinomas but not in any other malignant and benign thyroid tumors (21). However, the specificity of RET/PTC for papillary carcinomas, reported in those and several other studies, has been later challenged by several observations that found RET/PTC in thyroid follicular adenomas, oncocytic tumors, and even in nonneoplastic thyroid lesions (11, 25, 26, 27, 28, 29, 30).

Understanding the nature of these controversies and finding a reliable and biologically relevant strategy for RET/PTC detection has important clinical and diagnostic implications. Indeed, detection of RET/PTC has been offered as a diagnostic tool for papillary thyroid carcinoma in the surgical and preoperative cytological material (31, 32). Moreover, the emergence of drugs that selectively inhibit RET kinase activity (33) dictates the necessity of a better understanding of RET/PTC distribution within the tumor volume and of standardization of the detection methods for this rearrangement in tumor samples.

In this study, we compare the efficiency of RET/PTC detection by the five most common methods in snap-frozen tissue from a large series of thyroid papillary carcinomas. We demonstrate that different methods have variable sensitivities of the detection and define three subsets of papillary carcinomas based on the distribution of RET/PTC rearrangement within the tumor volume.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tumor samples and nucleic acid extraction

We analyzed 65 snap-frozen samples from papillary thyroid carcinomas collected at the Department of Pathology, University of Cincinnati, or obtained through the Cooperative Human Tissue Network. The study protocol was approved by the University of Cincinnati Institutional Review Board. Sections from the frozen tissue samples were examined microscopically to ensure that a pure population of tumor cells was present in the areas used for further analysis. Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA), and genomic DNA was isolated using phenol/chloroform extraction as previously described (20).

Standard-sensitivity (SS) RT-PCR

Detection of RET/PTC1 and RET/PTC3 by this method involved conventional two-step RT-PCR followed by agarose gel electrophoresis. Briefly, 3 µg total RNA was reverse transcribed in a volume of 20 µl with random hexamer primers and Superscript II RT (Invitrogen) according to the manufacturer’s protocol. Two microliters of cDNA samples were analyzed for RET/PTC1 and RET/PTC3 using 35 cycles of PCR amplification as previously reported (34). All samples were assessed for the adequacy of RNA by amplification of the 3-phosphoglycerate kinase (PGK) housekeeping gene. The PCR products were electrophoresed on a 1.5% agarose gel and visualized by ethidium bromide staining.

High-sensitivity (HS) semiquantitative RT-PCR

For this method, 5 µl of each PCR product obtained by conventional PCR during the SS RT-PCR detection were electrophoresed in a 1.5% agarose gel and blotted onto a nylon membrane. Each filter was then hybridized with internal oligonucleotide probe specific for RET/PTC1 or RET/PTC3. The probes were labeled with [{gamma}-32P]ATP by T4 kinase (Promega, Madison, WI). The filters were washed, exposed to Phosphor Screen, and analyzed using STORM840 PhosphorImager (Molecular Dynamics, Sunnyvale, CA). For quantitation, a positive control RNA harboring RET/PTC1 or RET/PTC3 rearrangement was 10-fold serially diluted, amplified in the same PCR, and used for generation of standard curves. The positive control for RET/PTC1 was obtained from the TPC-1 cell line (35) and for RET/PTC3 from a primary cell culture from the tumor positive for this rearrangement. Band intensities were measured using ImageQuant 5.2 software (Molecular Dynamics).

Real-time LightCycler (LC) RT-PCR

Detection of RET/PTC1 and RET/PTC3 by real-time PCR was performed on a LightCycler (Roche, Indianapolis, IN). Amplification was achieved in a 20-µl volume containing 2 µl cDNA, 2 µM of each primer, 0.1 µM of each probe, and LightCycler FastStart DNA Master HybProbe mixture according to the manufacturer’s protocol. The reaction mixture was subjected to 40 cycles of amplification followed by post-PCR fluorescence melting curve analysis (FMCA). The forward primer for RET/PTC1 was 5'-CAA GAG AAC AAG GTG CTG AAG-3' and for RET/PTC3 was 5'-CGG TAT TGT AGC TGT CCC TTT C-3', and the common reverse primer was 5'-GCA GGT CTC AAG CTC ACT C-3'. The probes for RET/PTC1 were 5'-CCC ACT TTG GAT CCT CGA TGG TAA C-fluorescein-3' and 5'-LC Red705-CTG GCT TTG CGC AGG TCG CG-phosphate-3' and for RET/PTC3 5'-CCC ACT TTG GAT CCT CCT GAC TGT TC-fluorescein-3' and 5'-LC Red640-CCA AGG TCT GCT TTT GCG TAA GCC A-phosphate-3'.

SB

From each tumor, 10 µg genomic DNA was digested with one of the four enzymes (EcoRI, HindIII, BamHI, and BglII) (Invitrogen). The digests were electrophoresed on 0.8% Tris-acetate-EDTA agarose gel and transferred to nylon filters (Osmonics, Minnetonka, MN). Hybridization was performed with a 1.0 kb BamHI-BglII RET-specific probe corresponding to the RET gene intron 11 (gift of Dr. M. Santoro) labeled with [32P]dCTP using a random oligonucleotide primer kit (Amersham Biosciences, Piscataway, NJ). After washing, the filters were analyzed using STORM840 PhosphorImager (Molecular Dynamics).

FISH

Tumor touch imprints were prepared from snap-frozen tissue. A 207-kb BAC clone (RPC11-351D16, CHORI) was used to generate a probe for the RET gene. BAC DNA was extracted and directly labeled with Green-dUTP using a nick translation kit (Vysis, Downers Grove, IL). The labeled probe was coprecipitated in the presence of the 10-fold excess of Cot1 DNA and 100-fold excess of salmon sperm DNA (Life Technologies, Inc., Gaithersburg, MD) and resuspended in the hybridization buffer (14.3% dextran sulfate, 78.6% formamide, 2.9x standard saline citrate, pH 7.0). For dual-color FISH, either the H4 or NCOA4 probes were labeled with Orange-dUTP (Vysis) and coprecipitated together with the RET probe. After co-denaturation of the nuclei and the probe at 73 C for 3 min, hybridization was performed overnight at 37 C in a Hybrite hybridization chamber (Vysis). Nuclei were counterstained with 4',6'-diamidino-2-phenylindole. Microscopy was performed with a Leica TCS 4D confocal microscope. In every preparation, 200 nuclei were scored. To establish the cutoff level to consider a tumor positive for RET/PTC, the baseline frequency of the finding of three RET signals (which indicates a split of one probe) was analyzed in normal thyroid cells and tumors negative for RET/PTC by PCR. A mean value (x) and SD were calculated, and a value of 3x was adopted as the cutoff level (23).

Detection of BRAF and RAS mutations

Detection of V600E BRAF mutation was performed using real-time PCR and FMCA on a LightCycler as previously reported (36). Point mutations in the RAS genes that are most commonly found in thyroid cancer, N-RAS codon 61, H-RAS codon 61, and K-RAS codon 12/13, were detected using PCR and FMCA on a LightCycler as previously reported (37).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SS RT-PCR

By SS RT-PCR, RET/PTC rearrangements were detected in 14 of 65 papillary carcinomas. Of them, nine tumors revealed RET/PTC1 and five RET/PTC3. The intensity of bands amplified using this standard PCR procedure and resolved by agarose gel electrophoresis and staining with ethidium bromide was strong or moderate in nine cases and weak, but clearly recognizable, in another five cases (Fig. 1Go). All other samples had sufficient quality and quantity of RNA, as confirmed by amplification of the PGK gene, but were negative for RET/PTC.


Figure 1
View larger version (77K):
[in this window]
[in a new window]
  		FIG. 1. Detection of RET/PTC1 and RET/PTC3 rearrangements by SS RT-PCR. Some tumor samples showed strong to moderate (for example, tumors 19, 16, 18, and 51) or weak (22 and 57) amplification bands. L, 123-bp marker; NC, negative control; PC, positive control.

 
HS RT-PCR

To achieve a higher sensitivity of the detection, the PCR products obtained by SS RT-PCR were blotted onto a nylon membrane and hybridized with radioactively labeled oligonucleotide probes. Sensitivity of this method was established using serial dilution of the positive controls. Both RET/PTC1 and RET/PTC3 were detected at dilution 1:10,000 but not 1:100,000, which corresponded to the detection limit between 5 and 0.5 cells positive for each type of the rearrangement (Fig. 2AGo). Using this method, RET/PTC was detected in all 14 samples that were positive by SS RT-PCR and in 12 additional tumors that were negative by SS RT-PCR. The latter samples revealed a faint amplification band corresponding to RET/PTC1 in nine samples and to RET/PTC3 in two samples, with one more sample being positive for both rearrangement types (Fig. 2BGo). Relative quantitation of cells positive for RET/PTC was performed assuming that the levels of expression of RET/PTC were similar between the positive controls and tumor samples. For the 14 samples positive by SS RT-PCR, there was overall a good correlation between the intensity of bands seen in the agarose gel and quantitation of signals after hybridization. The intensity of bands corresponded to the presence of 22,500–50,000 (45–100%) RET/PTC-positive cells among nine cases with strong or moderate band intensity in the agarose gel and to the presence of 200–13,000 (4–26%) cells with the rearrangement among five samples showing a weak band by SS RT-PCR. In the 12 additional samples scored positive only by HS RT-PCR, the intensity of amplification bands corresponded to the presence of 1.5–5 (0.003–0.01%) positive cells.


Figure 2
View larger version (52K):
[in this window]
[in a new window]
  		FIG. 2. Detection and quantitation of RET/PTC1 and RET/PTC3 expression by HS RT-PCR. A, Serial dilutions of the RET/PTC1 and RET/PTC3 positive controls starting from 50,000 positive cells (1:1 dilution) to 0.5 positive cells (1:100,000 dilution). The detection limit of both rearrangement types was between 5 and 0.5 cells. B, RET/PTC was detected in tumor samples previously found to be positive by SS RT-PCR (for example, 65 and 52) and in those that were negative by SS RT-PCR (28, 26, 37, 54, and 55). NC, Negative control; PC, positive control.

 
LC RT-PCR

Using real-time LC RT-PCR, RET/PTC was detected in 12 of 65 tumor samples. Among them, nine tumors were positive for RET/PTC1 and three tumors for RET/PTC3 (Fig. 3Go). All of the samples positive by LC RT-PCR were also positive by SS RT-PCR and by HS RT-PCR, with identical RET/PTC type identified in all experiments. However, LC RT-PCR failed to detect RET/PTC in two additional cases that were tested positive by SS RT-PCR and HS RT-PCR and in an additional 12 samples tested positive only by HS RT-PCR.


Figure 3
View larger version (28K):
[in this window]
[in a new window]
  		FIG. 3. Detection of RET/PTC1 (A) and RET/PTC3 (B) rearrangement by amplification using real-time LC RT-PCR. NC, Negative control; PC, positive control.

 
The results obtained by these three methods, all of which used the PCR-based technique and therefore were directly related to the levels of expression of RET/PTC transcripts, suggested two possibilities: 1) RET/PTC is indeed present in various proportions of tumor cells, or 2) the rearrangement is present in most cells within the tumor but is expressed at highly variable levels. These possibilities were tested using SB and FISH.

SB

SB was conducted by digesting the genomic DNA from each tumor with EcoRI, HindIII, BamHI, or BglII. These restriction enzymes were chosen because of their cutting position close to the intron 11 of RET, which is a breakpoint cluster region in all types of RET/PTC rearrangement. By this method, RET/PTC was detected in 11 (17%) tumors. Among those, eight positive samples were detected after HindIII, seven cases after EcoRI, two after BglII digestion, and one after BamHI digestion (Fig. 4Go). All tumors tested positive by SB were also positive by SS RT-PCR, HS RT-PCR, and LC RT-PCR.


Figure 4
View larger version (81K):
[in this window]
[in a new window]
  		FIG. 4. SB of DNA digested with EcoRI, HindIII, BamHI, and BglII. A 1.0-kb RET probe recognizes the 6.3-kb EcoRI fragment of the wild-type RET, 9.3-kb fragment for HindIII, 3.7-kb fragment for BamHI, and 7.8-kb fragment for BglII. The asterisks indicate cases that showed additional hybridization bands and therefore were diagnosed as positive for RET rearrangement.

 
FISH

RET/PTC detection by FISH was performed in two steps. First, hybridization of a 207-kb probe spanning the entire RET gene was used to identify all possible types of RET rearrangement, which is expected to show a split of one of the hybridization signals, resulting in the presence of three RET signals in the cell. Cases that appeared positive by this screening technique were subjected to the two-color FISH using the RET and either H4 or NCOA4 probes to determine the exact type of rearrangement. Before applying this method, its resolution was tested by hybridizing the RET probe to five normal thyroid tissues and 10 papillary carcinomas tested negative by all three RT-PCR-based methods, including HS RT-PCR. Two hundred consecutive cells were counted in each case, and 0.5–6% of cells were scored as having three RET signals. The resulting mean value was 2.9 ± 2.1, and therefore a 9% cutoff was accepted as the limit of resolution of this method. As a result, only those tumors that revealed 9% or more of cells with three hybridization signals were considered positive for RET/PTC rearrangement by FISH.

Using FISH with the RET probe, 14 tumors revealed 17–86% of cells with three hybridization signals and were considered positive for RET/PTC (Fig. 5Go). Using two-color FISH, nine of these tumors were found to have RET/PTC1 and five tumors RET/PTC3 rearrangement. The 14 cases positive for RET/PTC by FISH were all detected by SS RT-PCR with total concordance with respect to the rearrangement types. Moreover, nine tumors that showed moderate or strong band intensity in the agarose gel on SS RT-PCR were found to contain 50–86% of cells with the rearrangement and five tumors with a weak band on SS RT-PCR had 17–35% of cells with RET/PTC. None of the cases positive for RET/PTC by HS RT-PCR contained more than 7% of cells with three RET hybridization signals and therefore were considered negative by FISH.


Figure 5
View larger version (30K):
[in this window]
[in a new window]
  		FIG. 5. FISH. A, Hybridization with the RET probe showing four cells, three of which revealed three signals from a split of one probe and therefore were considered positive for the rearrangement; B, cohybridization with RET and H4 probes reveals RET/PTC1 rearrangement; C, cohybridization with RET and NCOA4 probes reveals RET/PTC3 rearrangement.

 
Comparison between the detection methods, RET/PTC quantitation, and tumor grouping

The efficiency of RET/PTC detection in individual tumor cases by the five methods is summarized in Table 1Go. Overall, RET/PTC was identified by HS RT-PCR in 40% of tumors, by SS RT-PCR and FISH in 22%, by LC RT-PCR in 19%, and by SB in 17% of tumors. Of those, 11 tumors (17%) were found positive for RET/PTC by all five methods, one tumor (2%) by four methods, two tumors (3%) by three methods, and 12 tumors (19%) by a single method, HS RT-PCR.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Detection of RET/PTC in thyroid papillary carcinomas by different methods

 
The quantitation of cells within the tumor that harbor RET/PTC by FISH revealed that nine tumors had 50–86% of cells with the rearrangement. Because the number of positive cells is likely to be even slightly underestimated by FISH, we can conclude that in those cases RET/PTC is present in the majority of tumor cells and is therefore a clonal event. The rearrangement in tumors from this group was detected by all five methods. In another five cases, FISH revealed 17–35% of cells positive for RET/PTC. It appears that in these tumors a significant proportion of cells, but not the majority of them, harbor RET/PTC, and therefore the rearrangement is subclonal. In this group, RET/PTC was identified by at least three different methods. Finally, the third group included 12 cases where RET/PTC rearrangement was detected only by HS RT-PCR. By FISH, the fraction of positive cells in these tumors was below the cutoff level of 9%. The intensity levels of the hybridization bands detected by HS RT-PCR corresponded to the presence of 0.003–0.01% positive cells or 1.5–5 cells with RET/PTC. Therefore, in these tumors, the rearrangement is nonclonal (Table 2Go).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Tumor grouping based on the fraction of cells with RET/PTC rearrangement

 
Correlation between the distribution of RET/PTC and prevalence of BRAF and RAS mutations

The prevalence of BRAF and RAS mutations, also known to occur in papillary thyroid carcinomas, was tested in all three tumor groups. In tumors with clonal RET/PTC, no BRAF or RAS mutations were found. Among five tumors containing subclonal RET/PTC, three tumors (60%) also revealed T1799A BRAF mutation. Among 12 tumors containing nonclonal RET/PTC, five (42%) revealed additional mutations, including three cases of N-RAS codon 61 and two cases of BRAF mutations (Table 1Go). In three tumor cases (one with subclonal and two with nonclonal RET/PTC) that were also positive for BRAF (two cases) or N-RAS codon 61 (one case) mutations, three to six separate frozen tissue samples harvested from different areas within the tumor nodule were also available. They were studied for RET/PTC, BRAF, and N-RAS codon 61 mutations. No RET/PTC was detected using HS RT-PCR, whereas all additional samples within each tumor were positive for either BRAF or RAS mutations. This indicated that in these tumors, BRAF or RAS mutations were present in the majority of tumor cells, whereas RET/PTC involved only an isolated group of cells in one area of the tumor nodule.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of our study obtained using five common detection methods indicate that the broad variability in the reported prevalence of RET/PTC is at least in part caused by the use of different detection techniques and genetic heterogeneity of thyroid papillary carcinomas. We show that distribution of RET/PTC rearrangement within each tumor can vary from involving almost all neoplastic cells to only a small fraction, probably just a few cells. Depending on the fraction of involved tumor cells, we propose to subclassify the rearrangement as clonal, subclonal, or nonclonal. Although nonclonal RET/PTC can be detected using highly sensitive methods, these tumors commonly have other and more dominant genetic alterations, arguing against the biological and diagnostic importance of the low-level RET/PTC detection in these tumor samples.

All methods used in this study accurately detected RET/PTC and its type in 17% of papillary cancers. Based on the FISH analysis, these tumors contained 35–86% of cells harboring the rearrangement. This indicates that all of these methods have a comparable accuracy of detection when the rearrangement is present in a significant portion of tumor cells. Three other cases with 17–24% of cells positive for RET/PTC by FISH were detected by several, but not all, methods. They were all tested positive by SS RT-PCR. This indicates that a standard detection procedure using conventional RT-PCR with 35 cycles of amplification is sufficient to identify RET/PTC in all tumors where at least 17% of cells harbor the rearrangement. The use of more sensitive techniques, such as HS RT-PCR, will detect additional tumor cases where only a small fraction of cells is positive for RET/PTC. In this study, this fraction was definitely less than 9%, as judged by the FISH data, and appeared to correspond to one to six positive cells based on the intensity of amplification of RET/PTC transcripts. Of note, a high sensitivity of RT-PCR can be achieved not only by post-PCR hybridization with the radioactive probe but also by using two rounds of amplification with nested primers. The latter technique not only increases dramatically the sensitivity but is also prone to generation of nonspecific amplicons and requires particular caution with result interpretation.

This study revealed a substantial number of papillary carcinomas where most of the tumor cells harbored RET/PTC as detected by FISH. FISH, depending on the probe design, may underestimate or overestimate the prevalence of RET/PTC because RET is located close to its rearrangement partners in a significant portion of interphase thyroid cell nuclei (38). In this study, the quantitation by FISH revealed nine tumors (14%) containing more than 50% of cells carrying the rearrangement. Taking into account that some positive cells would still be missed because of the limitations of hybridization and two-dimensional microscopy, we can conclude that in these 14% of tumors, the vast majority if not all tumor cells carried the rearrangement. This contrasts with the previous finding by Unger et al. (23), who examined by FISH radiation-induced papillary carcinomas from children and detected RET/PTC in a minority of tumor cells, with no tumors containing more than 46% of positive cells. The discrepancy may reflect different tumor origins, age variation of patients, different specimen preparations, or other technical variations between the two studies.

Our findings provide a rationale for subclassification of papillary carcinoma into three groups based on the proportion of tumor cells with RET/PTC. In one group of tumors, the majority of cells were positive for the rearrangement. These tumors had clonal RET/PTC that could be detected by all methods and did not reveal other mutations, suggesting that RET/PTC was a dominant genetic event. These tumors are expected to respond to RET-receptor-targeted therapies. Another group of tumors had subclonal RET/PTC rearrangements that were identified in a minor but still significant portion of tumor cells. This finding provides evidence for intratumor genetic heterogeneity of thyroid papillary carcinomas and allows us to put forward several possible interpretations. This may reflect the multiclonal origin of the tumor or can suggest that RET/PTC is a later effect in thyroid carcinogenesis, as previously suggested by Unger et al. (23). Another possibility is that RET/PTC was the initial event that was lost during the tumor progression. Some evidence supporting this chain of events has been reported (39). It remains unclear which of these hypotheses provide a correct explanation for the occurrence of subclonal RET/PTC and whether these tumors would respond to the targeted inhibition of the RET kinase. Because other mutations were found in tumors with subclonal RET/PTC and they were present in the majority of neoplastic cells, it is more likely that RET/PTC was a late event in the progression of these malignancies. Finally, the third group of tumors had nonclonal RET/PTC rearrangements that were present in a minute fraction of cells within the tumor. The reason for such a limited presence of RET/PTC is not clear. It is likely that this is because of the late occurrence of RET/PTC in a cell within the transformed clone. The vast majority of cells within these tumors had no RET/PTC and instead contained other mutations, which are expected to be more central for their progression. Therefore, these tumors are unlikely to possess the same biological characteristics as tumors with predominant RET/PTC rearrangement and their response to RET inhibitors is questionable. It also remains unclear whether the nonclonal rearrangements are specific for papillary carcinomas or whether they can occur in other thyroid tumors. These findings point toward the necessity of calibration of the detection methods to report not only the presence of RET/PTC rearrangement but also the extent of its distribution within the tumor, particularly when testing for RET/PTC is used for tumor diagnosis or justification for RET-receptor-targeted therapy.


   Acknowledgments
 
We thank Massimo Santoro for providing the RET probe for Southern blot analysis.


   Footnotes
 
This study was supported by National Institutes of Health Grant R01 CA88041.

Disclosure statement: The authors have nothing to declare.

First Published Online June 13, 2006

Abbreviations: FISH, Fluorescence in situ hybridization; FMCA, fluorescence melting curve analysis; HS, high-sensitivity; LC, LightCycler; SB, Southern blot analysis; SS, standard-sensitivity.

Received May 9, 2006.

Accepted June 7, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Fusco A, Grieco M, Santoro M, Berlingieri MT, Pilotti S, Pierotti MA, Della Porta G, Vecchio G 1987 A new oncogene in human thyroid papillary carcinomas and their lymph-nodal metastases. Nature 328:170–172[CrossRef][Medline]
  2. Grieco M, Santoro M, Berlingieri MT, Melillo RM, Donghi R, Bongarzone I, Pierotti MA, Della Porta G, Fusco A, Vecchio G 1990 PTC is a novel rearranged form of the ret proto-oncogene and is frequently detected in vivo in human thyroid papillary carcinomas. Cell 60:557–563[CrossRef][Medline]
  3. Santoro M, Dathan NA, Berlingieri MT, Bongarzone I, Paulin C, Grieco M, Pierotti MA, Vecchio G, Fusco A 1994 Molecular characterization of RET/PTC3; a novel rearranged version of the RET proto-oncogene in a human thyroid papillary carcinoma. Oncogene 9:509–516[Medline]
  4. Bongarzone I, Butti MG, Coronelli S, Borrello MG, Santoro M, Mondellini P, Pilotti S, Fusco A, Della Porta G, Pierotti MA 1994 Frequent activation of ret protooncogene by fusion with a new activating gene in papillary thyroid carcinomas. Cancer Res 54:2979–2985[Abstract/Free Full Text]
  5. Nikiforov YE 2002 RET/PTC rearrangement in thyroid tumors. Endocr Pathol 13:3–16[CrossRef][Medline]
  6. Tallini G, Asa SL 2001 RET oncogene activation in papillary thyroid carcinoma. Adv Anat Pathol 8:345–354[CrossRef][Medline]
  7. Nikiforov YE, Rowland JM, Bove KE, Monforte-Munoz H, Fagin JA 1997 Distinct pattern of ret oncogene rearrangements in morphological variants of radiation-induced and sporadic thyroid papillary carcinomas in children. Cancer Res 57:1690–1694[Abstract/Free Full Text]
  8. Fenton CL, Lukes Y, Nicholson D, Dinauer CA, Francis GL, Tuttle RM 2000 The ret/PTC mutations are common in sporadic papillary thyroid carcinoma of children and young adults. J Clin Endocrinol Metab 85:1170–1175[Abstract/Free Full Text]
  9. Smida J, Salassidis K, Hieber L, Zitzelsberger H, Kellerer AM, Demidchik EP, Negele T, Spelsberg F, Lengfelder E, Werner M, Bauchinger M 1999 Distinct frequency of ret rearrangements in papillary thyroid carcinomas of children and adults from Belarus. Int J Cancer 80:32–38[CrossRef][Medline]
  10. Rabes HM, Demidchik EP, Sidorow JD, Lengfelder E, Beimfohr C, Hoelzel D, Klugbauer S 2000 Pattern of radiation-induced RET and NTRK1 rearrangements in 191 post-Chernobyl papillary thyroid carcinomas: biological, phenotypic, and clinical implications. Clin Cancer Res 6:1093–1103[Abstract/Free Full Text]
  11. Bounacer A, Wicker R, Caillou B, Cailleux AF, Sarasin A, Schlumberger M, Suarez HG 1997 High prevalence of activating ret proto-oncogene rearrangements, in thyroid tumors from patients who had received external radiation. Oncogene 15:1263–1273[CrossRef][Medline]
  12. Fugazzola L, Pilotti S, Pinchera A, Vorontsova TV, Mondellini P, Bongarzone I, Greco A, Astakhova L, Butti MG, Demidchik EP, Pacini F, Pierotti MA 1995 Oncogenic rearrangements of the RET proto-oncogene in papillary thyroid carcinomas from children exposed to the Chernobyl nuclear accident. Cancer Res 55:5617–5620[Abstract/Free Full Text]
  13. Chua EL, Wu WM, Tran KT, McCarthy SW, Lauer CS, Dubourdieu D, Packham N, O’Brien CJ, Turtle JR, Dong Q 2000 Prevalence and distribution of ret/ptc 1, 2, and 3 in papillary thyroid carcinoma in New Caledonia and Australia. J Clin Endocrinol Metab 85:2733–2739[Abstract/Free Full Text]
  14. Learoyd DL, Messina M, Zedenius J, Guinea AI, Delbridge LW, Robinson BG 1998 RET/PTC and RET tyrosine kinase expression in adult papillary thyroid carcinomas. J Clin Endocrinol Metab 83:3631–3635[Abstract/Free Full Text]
  15. Sugg SL, Zheng L, Rosen IB, Freeman JL, Ezzat S, Asa SL 1996 ret/PTC-1, -2, and -3 oncogene rearrangements in human thyroid carcinomas: implications for metastatic potential? J Clin Endocrinol Metab 81:3360–3365[Abstract]
  16. Sugg SL, Ezzat S, Rosen IB, Freeman JL, Asa SL 1998 Distinct multiple RET/PTC gene rearrangements in multifocal papillary thyroid neoplasia. J Clin Endocrinol Metab 83:4116–4122[Abstract/Free Full Text]
  17. Tallini G, Santoro M, Helie M, Carlomagno F, Salvatore G, Chiappetta G, Carcangiu ML, Fusco A 1998 RET/PTC oncogene activation defines a subset of papillary thyroid carcinomas lacking evidence of progression to poorly differentiated or undifferentiated tumor phenotypes. Clin Cancer Res 4:287–294[Abstract]
  18. Jhiang SM, Caruso DR, Gilmore E, Ishizaka Y, Tahira T, Nagao M, Chiu IM, Mazzaferri EL 1992 Detection of the PTC/retTPC oncogene in human thyroid cancers. Oncogene 7:1331–1337[Medline]
  19. Lam AK, Montone KT, Nolan KA, Livolsi VA 1998 Ret oncogene activation in papillary thyroid carcinoma: prevalence and implication on the histological parameters. Hum Pathol 29:565–568[CrossRef][Medline]
  20. Nikiforova MN, Caudill CM, Biddinger P, Nikiforov YE 2002 Prevalence of RET/PTC rearrangements in Hashimoto’s thyroiditis and papillary thyroid carcinomas. Int J Surg Pathol 10:15–22[Abstract/Free Full Text]
  21. Santoro M, Carlomagno F, Hay ID, Herrmann MA, Grieco M, Melillo R, Pierotti MA, Bongarzone I, Della Porta G, Berger N, Peix JL, Paulin C, Fabien N, Vecchio G, Jenkins RB, Fusco A 1992 Ret oncogene activation in human thyroid neoplasms is restricted to the papillary cancer subtype. J Clin Invest 89:1517–1522[Medline]
  22. Rhoden KJ, Johnson C, Brandao G, Howe JG, Smith BR, Tallini G 2004 Real-time quantitative RT-PCR identifies distinct c-RET, RET/PTC1 and RET/PTC3 expression patterns in papillary thyroid carcinoma. Lab Invest 84:1557–1570[CrossRef]
  23. Unger K, Zitzelsberger H, Salvatore G, Santoro M, Bogdanova T, Braselmann H, Kastner P, Zurnadzhy L, Tronko N, Hutzler P, Thomas G 2004 Heterogeneity in the distribution of RET/PTC rearrangements within individual post-Chernobyl papillary thyroid carcinomas. J Clin Endocrinol Metab 89:4272–4279[Abstract/Free Full Text]
  24. Santoro M, Sabino N, Ishizaka Y, Ushijima T, Carlomagno F, Cerrato A, Grieco M, Battaglia C, Martelli ML, Paulin C, Fusco A 1993 Involvement of RET oncogene in human tumours: specificity of RET activation to thyroid tumours. Br J Cancer 68:460–464[Medline]
  25. Ishizaka Y, Kobayashi S, Ushijima T, Hirohashi S, Sugimura T, Nagao M 1991 Detection of retTPC/PTC transcripts in thyroid adenomas and adenomatous goiter by an RT-PCR method. Oncogene 6:1667–1672[Medline]
  26. Wirtschafter A, Schmidt R, Rosen D, Kundu N, Santoro M, Fusco A, Multhaupt H, Atkins JP, Rosen MR, Keane WM, Rothstein JL 1997 Expression of the RET/PTC fusion gene as a marker for papillary carcinoma in Hashimoto’s thyroiditis. Laryngoscope 107:95–100[CrossRef][Medline]
  27. Cinti R, Yin L, Ilc K, Berger N, Basolo F, Cuccato S, Giannini R, Torre G, Miccoli P, Amati P, Romeo G, Corvi R 2000 RET rearrangements in papillary thyroid carcinomas and adenomas detected by interphase FISH. Cytogenet Cell Genet 88:56–61[CrossRef][Medline]
  28. Sheils OM, O’Eary J J, Uhlmann V, Lattich K, Sweeney EC 2000 ret/PTC-1 activation in Hashimoto thyroiditis. Int J Surg Pathol 8:185–189[Abstract/Free Full Text]
  29. Elisei R, Romei C, Vorontsova T, Cosci B, Veremeychik V, Kuchinskaya E, Basolo F, Demidchik EP, Miccoli P, Pinchera A, Pacini F 2001 RET/PTC rearrangements in thyroid nodules: studies in irradiated and not irradiated, malignant and benign thyroid lesions in children and adults. J Clin Endocrinol Metab 86:3211–3216[Abstract/Free Full Text]
  30. Chiappetta G, Toti P, Cetta F, Giuliano A, Pentimalli F, Amendola I, Lazzi S, Monaco M, Mazzuchelli L, Tosi P, Santoro M, Fusco A 2002 The RET/PTC oncogene is frequently activated in oncocytic thyroid tumors (Hurthle cell adenomas and carcinomas), but not in oncocytic hyperplastic lesions. J Clin Endocrinol Metab 87:364–369[Abstract/Free Full Text]
  31. Salvatore G, Giannini R, Faviana P, Caleo A, Migliaccio I, Fagin JA, Nikiforov YE, Troncone G, Palombini L, Basolo F, Santoro M 2004 Analysis of BRAF point mutation and RET/PTC rearrangement refines the fine-needle aspiration diagnosis of papillary thyroid carcinoma. J Clin Endocrinol Metab 89:5175–5180[Abstract/Free Full Text]
  32. Cheung CC, Carydis B, Ezzat S, Bedard YC, Asa SL 2001 Analysis of ret/PTC gene rearrangements refines the fine needle aspiration diagnosis of thyroid cancer. J Clin Endocrinol Metab 86:2187–2190[Abstract/Free Full Text]
  33. Carlomagno F, Vitagliano D, Guida T, Ciardiello F, Tortora G, Vecchio G, Ryan AJ, Fontanini G, Fusco A, Santoro M 2002 ZD6474, an orally available inhibitor of KDR tyrosine kinase activity, efficiently blocks oncogenic RET kinases. Cancer Res 62:7284–7290[Abstract/Free Full Text]
  34. Caudill CM, Zhu Z, Ciampi R, Stringer JR, Nikiforov YE 2005 Dose-dependent generation of RET/PTC in human thyroid cells after in vitro exposure to {gamma}-radiation: a model of carcinogenic chromosomal rearrangement induced by ionizing radiation. J Clin Endocrinol Metab 90:2364–2369[Abstract/Free Full Text]
  35. Ishizaka Y, Ushijima T, Sugimura T, Nagao M 1990 cDNA cloning and characterization of ret activated in a human papillary thyroid carcinoma cell line. Biochem Biophys Res Commun 168:402–408[CrossRef][Medline]
  36. Nikiforova MN, Kimura ET, Gandhi M, Biddinger PW, Knauf JA, Basolo F, Zhu Z, Giannini R, Salvatore G, Fusco A, Santoro M, Fagin JA, Nikiforov YE 2003 BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas. J Clin Endocrinol Metab 88:5399–5404[Abstract/Free Full Text]
  37. Zhu Z, Gandhi M, Nikiforova MN, Fischer AH, Nikiforov YE 2003 Molecular profile and clinical-pathologic features of the follicular variant of papillary thyroid carcinoma. An unusually high prevalence of ras mutations. Am J Clin Pathol 120:71–77[CrossRef][Medline]
  38. Gandhi M, Medvedovic M, Stringer JR, Nikiforov YE 2006 Interphase chromosome folding determines spatial proximity of genes participating in carcinogenic RET/PTC rearrangements. Oncogene 25:2360–2366[CrossRef][Medline]
  39. Barr FG, Qualman SJ, Macris MH, Melnyk N, Lawlor ER, Strzelecki DM, Triche TJ, Bridge JA, Sorensen PH 2002 Genetic heterogeneity in the alveolar rhabdomyosarcoma subset without typical gene fusions. Cancer Res 62:4704–4710[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Endocr Relat CancerHome page
C. Romei, R. Ciampi, P. Faviana, L. Agate, E. Molinaro, V. Bottici, F. Basolo, P. Miccoli, F. Pacini, A. Pinchera, et al.
BRAFV600E mutation, but not RET/PTC rearrangements, is correlated with a lower expression of both thyroperoxidase and sodium iodide symporter genes in papillary thyroid cancer
Endocr. Relat. Cancer, June 1, 2008; 15(2): 511 - 520.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
D. V. Frau, M. L. Lai, P. Caria, T. Dettori, P. Coni, G. Faa, L. Morandi, G. Tallini, and R. Vanni
Trisomy 17 as a Marker for a Subset of Noninvasive Thyroid Nodules with Focal Features of Papillary Carcinoma: Cytogenetic and Molecular Analysis of 62 Cases and Correlation with Histological Findings
J. Clin. Endocrinol. Metab., January 1, 2008; 93(1): 177 - 181.
[Abstract] [Full Text] [PDF]

Home page
 Endocr Relat CancerHome page
G. Riesco-Eizaguirre and P. Santisteban
New insights in thyroid follicular cell biology and its impact in thyroid cancer therapy
Endocr. Relat. Cancer, December 1, 2007; 14(4): 957 - 977.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
R. Ciampi and Y. E. Nikiforov
RET/PTC Rearrangements and BRAF Mutations in Thyroid Tumorigenesis
Endocrinology, March 1, 2007; 148(3): 936 - 941.
[Abstract] [Full Text] [PDF]

Home page
 Eur J EndocrinolHome page
M. Santoro, R. M. Melillo, and A. Fusco
RET/PTC activation in papillary thyroid carcinoma: European Journal of Endocrinology Prize Lecture.
Eur. J. Endocrinol., November 1, 2006; 155(5): 645 - 653.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
91/9/3603    most recent
Author Manuscript (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhu, Z.
Right arrow Articles by Nikiforov, Y. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhu, Z.
Right arrow Articles by Nikiforov, Y. E.
Related Collections
Right arrow Endocrine Oncology
Right arrow Thyroid

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals