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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.
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Abstract |
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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.
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Introduction |
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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.
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Materials and Methods |
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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 [
-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).
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Results |
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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. 1
). All other samples had sufficient quality and quantity of RNA, as confirmed by amplification of the PGK gene, but were negative for RET/PTC.
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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. 2A). 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. 2B). 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.
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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. 3). 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.
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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. 4). All tumors tested positive by SB were also positive by SS RT-PCR, HS RT-PCR, and LC RT-PCR.
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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. 5). 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.
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The efficiency of RET/PTC detection in individual tumor cases by the five methods is summarized in Table 1. 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.
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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 1
). 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Acknowledgments |
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Footnotes |
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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.
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References |
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-radiation: a model of carcinogenic chromosomal rearrangement induced by ionizing radiation. J Clin Endocrinol Metab 90:2364–2369This article has been cited by other articles:
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