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Institutes of Molecular Radiobiology (K.U., H.Z., H.B., P.K.) and Pathology (P.H., G.T.), GSF-National Research Center for Environment and Health, D-85764 Neuherberg, Germany; Department of Cellular and Molecular Pathology/Instituto di Endocrinologia ed Oncologia Sperimentale Consiglio Nazionale delle Ricerche Universita di Napoli Federico II, 80131 Naples, Italy (G.S., M.S.); Institute of Endocrinology and Metabolism (T.B., L.Z., N.T.), Academy of Medical Sciences of the Ukraine, 254114 Kiev, Ukraine; and South West Wales Cancer Institute, Singleton Hospital, SA2 8QA Swansea, United Kingdom (P.H., G.T.)
Address all correspondence and requests for reprints to: Dr. Horst Zitzelsberger, GSF-Forschungszentrum für Umwelt und Gesundheit GmbH, Institute of Molecular Radiobiology, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany. E-mail: Zitzelsberger{at}gsf.de.
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Abstract |
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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To investigate the distribution of RET/PTC-positive cells within a set of post-Chernobyl PTCs, we used fluorescence in situ hybridization (FISH) analysis on paraffin-embedded tissue sections. The same samples were also analyzed by expression analysis of the TK domain of RET and RT-PCR analysis for RET/PTC1 and 3. We found that in a subset of cases, the distribution of RET/PTC-positive cells within the tumors was not homogeneous. Rather, in this subset of cases, positive cells appeared as clusters mixed with tumor cells scored negative for the rearrangement.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Thirty-two patients (18 female, 14 male, mean age 12.9 yr, range 9.1–18.7 yr) with histologically verified thyroid tumors operated between 1995 and 1998 in the Institute of Endocrinology and Metabolism (Kiev, Ukraine) were studied for the presence of RET rearrangements in the tumors. Appropriate informed consent was obtained from the patients concerned. The tumors were classified according to the World Health Organization classification of thyroid tumors (11). Of these, 29 were diagnosed as papillary carcinoma, two as follicular adenoma, and one as follicular cancer. Patient data are summarized in Table 1
. RNA was extracted from frozen specimens of paired samples of normal and tumor tissue from operative specimens. The presence of tumor was verified on a frozen section of the tissue before extraction. Aliquots of RNA extracted from frozen tissue were analyzed in separate laboratories for RET expression by RT-PCR and for RET/PTC3 and RET/PTC1 rearrangements by rearrangement-specific RT-PCR. Serial paraffin sections were cut from routine histological blocks from the same cases as used for RET analysis using RT-PCR. The presence of tumor was verified by staining with hematoxylin and eosin, and sections were microdissected to provide sections entirely composed of tumor or normal thyroid. The researchers in each center were blinded to the results of the other centers until completion of the study.
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FISH analysis
Yeast artificial chromosome (YAC) DNA probes used in this study were selected as previously described (12, 13). YAC clones 313F4 and 214H10 map proximal to and include the RET locus, whereas clone 344H4 contains DNA sequences distal to RET. These three YACs were always used in combination to investigate the RET locus. Total yeast DNA was extracted according to standard procedures and was labeled either with digoxigenin-11-dUTP (344H4) or with biotin-16-dUTP (214H10, 313F4) using nick translation. Serial 10-µm sections of the tissue blocks were used for FISH analysis that had been hybridized with the above-mentioned DNA probes. Tissue sections were deparaffinized in xylene and rehydrated. They were then pretreated in citrate buffer at 100 C for 15 min in a microwave and subsequently digested with pronase E (0.5 µg/ml) in PBS solution at 37 C for variable time (microscopic control). Sections were incubated in glycine solution (2 mg/ml in PBS) and in 4% formalin solution for 5 min each. Afterward, they were denatured in 70% formamide-2x saline sodium citrate for variable time and dehydrated in an ethanol series (70, 90, and 100%). Hybridization was performed for 48–72 h with labeled RET-specific YAC probes that had been denatured before use. Slides were washed in 50% formamide-2x SSC (three times, 15 min, 42 C), 2x SSC (15 min, 42 C), phosphate buffer with 0.1% nonidet P40 (PN) buffer (15 min, 42 C), and PN buffer (15 min, room temperature). Bound labeled DNA probes have been detected with streptavidin-fluorescein isothiocyanate (FITC) (Dianova, Hamburg, Germany) and antidigoxigenin-Cy3 (Dianova). Signals were amplified by sequential incubation in biotinylated antistreptavidin (Dianova) and rat-antimouse-Cy3/mouse-antirat-Cy3 (Dianova) antibodies. Each antibody was incubated for 20 min at 37 C. Slides were incubated for 10 min at 37 C with PN buffer with 5% nonfat dry milk and 0.02% sodium azide before each antibody incubation and were washed in PN buffer (twice for 2 min at room temperature) after each incubation step. Tissue sections were counterstained with TOPRO-3 (Molecular Probes, Leiden, The Netherlands; 0.1 µM in PBS diluted 1:5 with purified water) and mounted with Vectashield (Vector Laboratories, Burlingame, CA).
Signals from 100–150 cell nuclei per specimen (for tumor and normal tissue each) were scored using a confocal laser scanning microscope (Zeiss LSM 510; Zeiss, Jena, Germany). Nuclei from samples of normal thyroid epithelium from the same patient were scored as control. Cell nuclei were scored for the presence of a split FISH signal (separated red and green signal) in addition to an overlapping signal. Only cells with either two overlapping signals or one split and one overlapping signal were counted to ensure that only complete cell nuclei had been scored. Up to 20 viewing areas per case were scanned in 0.5-µm steps for three different channels (FITC, Cy3, and TOPRO-3) throughout the thickness of the respective section. Images were then superimposed by the laser scanning microscopy (LSM) software (Fig. 1A). Aberrant cell nuclei were identified by scoring of captured images. The analysis software allowed a step-wise scoring every 0.5 µm to ensure that signals in different layers of the sections were evaluated accurately.
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Statistical analysis
For an analysis of the distribution of aberrant FISH signals within different viewing areas of a particular case, a binomial homogeneity test was applied. Using this test, a dispersion factor was calculated, and P values were derived that were used to determine whether the distribution of aberrant FISH signals was significantly different from a homogeneous distribution, indicating a clustering of aberrant cells.
To determine the cutoff level that represents a significantly elevated frequency of RET-rearranged cells compared with the baseline frequency in nontumorous tissues, mean value (
) and SD of RET-rearranged cells in nontumorous tissues were calculated. The resulting mean value was 2.4 ± 2.1. For calculation of the cutoff level, a value of 3 equivalent to 7.1% of aberrant cells was accepted.
Analysis of RET expression by RT-PCR
Expression of the RET TK domain and extracellular (EC) domain. We studied the expression of the RET TK domain as well as the RET EC domain. Samples were considered positive for rearrangement whenever they presented expression of RET TK and no expression of RET EC. The RT reaction was performed on 1 µg RNA. The RT reaction was performed in a final volume of 25 µl containing 0.1 OD units of random hexamers (Amersham Biosciences Inc., Piscataway, NJ), 2.5 µl of 10x RT buffer [50 mM 1x RT Tris-HCl (pH 8.3), 50 mM KCl, 4 mM dithiothreitol, 10 mM MgCl2, 2.5 µl of 10 mM dNTPs, 1 U/µl RNasin, and 200 U/ml superRT], and incubated at 41 C for 60 min. The reaction was terminated at 95 C for 5 min. Two microliters of the reaction mixture was used for PCR amplification with primers 5'-CACCGGATGGAGAGGCCAGACAA CTGCAGC-3' and 5'-ACCGGCCTTTTGTCCGGCTC-3' (TK) and 5'-GTGCAGTTCTTGTGCCCCAACATCAG-3' and 5'-CCCAGCGCGTG CTCACCT-3' (EC) in a final volume of 50 µl. Primers for the 3' end of the RET TK corresponded to exons 16 (5' upstream) and 7 (3' downstream) spanning a 1150-bp intron. Primers for the EC domain were designed to span intron 4, giving a product of 106 bp. PCR consisted of 30 cycles of amplification at 95 C for 30 s, 50 C for 30 s, and 72 C for 1 min. Precautions were taken to prevent PCR contamination. Fifteen microliters of each PCR was run on a 2% agarose gel together with a 100-bp ladder. The specificity of the PCR product had been previously confirmed by Southern blotting using a probe specific for the 3' region of the RET gene and by direct sequencing. RNA extracted from the neuroblastoma cell line SHSY was used as a positive control, and RNA extracted from the 293 kidney cell line, known not to express RET, was used as a negative control. S6 ribosomal mRNA was used as a reporter target for amplification to monitor the presence of RNA; a negative amplification result indicated RNA degradation. The primers 5'-ATTCCGCAACGTTTCAATCTCT-3' and 5'TGAATCTTGGGTGCTTTGGTCCTA-3' were chosen in different exons to generate a 106-bp product from mRNA, readily distinguishable from an S6 genomic amplification that would result in a PCR product of 1826 bp. The same reaction conditions were used as for the RET TK and EC primer sets.
Identification of RET/PTC rearrangements by rearrangement-specific RT-PCR. Positive controls were tumor samples harboring RET/PTC rearrangements. Forward primers, designed on the coiled-coil domains of the RET fusion partners, were as follows: RET/PTC1, 5'-ATTGTCATCTCGCCGTTC-3'; and RET/PTC3, 5'-TGGAGAAGAGAGGCTGTATC-3'. Reverse primers were: RET/PTC1, 5'-TGCTTCAGGACGTTGAAC-3'; and RET/PTC3: 5'-CGTTGCCTTGACTTTTC-3'.
Five hundred nanograms of RNA was reverse-transcribed and subjected to 40 cycles of PCR (Perkin-Elmer, Norwalk, CT) (94 C for 30 s, 55 C for 2 min, and 72 C for 2 min). The product was analyzed on a 2% agarose gel, blotted onto a nylon membrane, and hybridized with a radioactive-labeled RET probe covering the TK domain. The amplified products were sequenced to confirm the rearrangement (Sequenase, USB, Cleveland, OH).
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Results |
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We initially investigated post-Chernobyl PTCs by FISH interphase analysis. FISH has the possibility of detecting RET rearrangements regardless the specific fusion partner involved and, in addition, allows examining the presence of the rearrangement at a single-cell level. FISH was carried out on paraffin-embedded tissue sections using a combination of three YAC probes that were labeled in two different colors. Cell nuclei exhibiting a rearranged RET gene show a split FISH signal in red and green in addition to a overlapping signal, whereas normal cells show two overlapping signals (Fig. 1
). The paraffin sections have been microdissected prior use to ensure that only tumor tissue was present on the slide for subsequent LSM scoring.
In total, we analyzed tumor tissues and corresponding normal tissues of 32 specimens using FISH. The frequency of aberrant cells in tumor and normal tissues is indicated in Table 1. Twenty-three of 32 post-Chernobyl cases (72%) showed RET rearrangements diagnosed by FISH interphase analysis. The highest frequency of rearranged cells after FISH interphase analysis was 46% (case 21). For comparison, two tumor tissues and three nontumorous tissue without radiation history as well as normal colon mucosa (negative control) and paraffin-embedded TPC-1 cells have been investigated by FISH. In the negative control, no rearranged cells were detected of 118 cells. The positive control showed 100% positivity. For the nonradiated samples, the mean frequency of rearranged cells for the nontumorous tissues was 0.7% (S24N, 1.5%; S34N, 0.0%; and S50N, 0.7%) revealed from 417 cells evaluated. The nonradiated PTC samples were known from RT-PCR analysis to harbor a RET/PTC1 rearrangement (P50T) or to be negative for RET rearrangements (S34T). S50T exhibited 12.7% rearranged cells (137 cells investigated), whereas S34T showed 4.6% rearranged cells (104 cells investigated).
Intriguingly, none of the post-Chernobyl tumors showed 100% rearranged cells. It became obvious that some cases showed a clustering of rearranged cells on LSM evaluation within individual areas. This situation is demonstrated in Fig. 1 showing examples of two areas within a tumor with rearranged cells and one area composed of nuclei with no rearrangements. A statistical test for homogeneity (distribution homogeneity test) was performed, and individual cases that show either a homo- or heterogeneous distribution of nuclei positive for rearrangement were identified. The latter were detected by P values < 0.05 and are indicated in Table 1. Statistically, a nonhomogeneous distribution can be randomly expected within one to two cases of 32 cases. In our analysis, nine cases showed a P value < 0.05 indicating a nonhomogeneous distribution and, therefore, subclones of tumor cells either with or without RET rearrangement.
RT-PCR analysis
Aliquots of RNA, extracted from frozen tissue in all except three cases where frozen samples were not available, were also subjected to RT-PCR analysis for the presence of RET rearrangements. For direct detection of the expression of RET/PTC1 and RET/PTC3, RT-PCR was performed with primers designed on RET and selective fusion partners. Representative examples of this approach are shown in Fig. 2. The sensitivity of the detection was assessed by serial dilutions of a positive control. When positive cells were more than 20%, a clear positive band was detected; in contrast, when positive cells were only 5%, the rearrangement was barely detected (Fig. 2). Furthermore, we performed the assay for unbalanced expression of the RET TK. Such RT-PCR-based assay relies on the detection of the overexpression of the RET TK with respect to its EC domain due to the disruption of the RET gene consequent to the rearrangement. This approach has the advantage of identifying RET rearrangements with novel genes that cannot be directly tested otherwise.
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. Overall, a significantly larger number of samples scored positive at FISH than at RT-PCR. RET/PTC1 rearrangements were detected in two of 29 cases, and RET/PTC3 rearrangements in were detected in three of 29 cases. Two additional cases showed other RET rearrangements after RT-PCR analysis. Elevated expression of the TK domain of RET (unbalanced expression) was identified in 12 of 29 cases. Comparison between FISH and RT-PCR
A comparison between the different approaches used in this study is given in Fig. 3. All six cases showing an elevated expression of the TK domain of RET, and a RET/PTC rearrangement (cases 24–29) also exhibited rearranged cells in FISH analysis above the cutoff level (subgroup 1). Most of the cases belonging to this subgroup showed a large fraction of FISH-positive cells.
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) was not high enough to detect the few rearranged cells. A third subgroup consisted of six cases (cases 18–23) that showed elevated expression of the TK domain of RET but are missing RET/PTC rearrangements after RT-PCR analysis. Three of these cases exhibited split FISH signals in more than 7.1% of cells analyzed by interphase FISH. Thus, likely they harbor RET rearrangements other than H4/RET (RET/PTC1) and RFG/RET (RET/PTC3).
A last case (case 17) revealed a RET/PTC3 rearrangement after RT-PCR analysis and a significant number of rearranged cells (>7.1%) after FISH analysis, but it failed to exhibit an elevated expression of the TK domain of RET. Unfortunately, there was insufficient material to examine this case further.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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), which also became obvious by the fact that areas of 100% nonrearranged cells were observed within a tumor. This observed heterogeneity is unlikely to be an artifact because only cells with either two overlapping or one overlapping and two split signals were scored. Cells in which there was only one signal or no signal were excluded from the analysis. There is a recent paper showing that in 35% of normal human thyroid cells, at least one pair of RET and H4 signals were juxtaposed on FISH. As a consequence, a significant proportion of tumor cells with RET/PTC rearrangement might be therefore misclassified depending on the interphase arrangement of chromosome 10 in the tumor nuclei and the linear distance between RET and the partner gene (15). In this study, we have used a different FISH approach, whereby the three colored probe signals overlap in normal nuclei and are split only when the RET gene sequence is dislocated by rearrangement. Our approach has the disadvantage of losing information regarding which particular RET rearrangement may be present in the nuclei but avoids confusion brought about by the interphase positions of chromosome 10, outlined by Nikiforova et al. (15). Our control studies on nonradiated tissues also argue against artificial results, caused by the overlying of chromosome 10 at interphase. In normal colon mucosa, no rearranged signal could be detected, whereas in nonradiated PTC cases positive for RET/PTC1, clustering of rearranged cells was also present. In a nonradiated PTC case (S34T) shown to be negative for RET by RT-PCR, a frequency of 4.6% of rearranged cells have been detected. Because in the nonradiated nontumorous tissues far lower frequencies (mean 0.7%) became apparent, S34T is in fact a RET-positive case on FISH. The discrepancy between FISH and RT-PCR can be explained by the same phenomena as observed in radiation-induced cases, i.e. clustering of RET-positive cells within the tumor and insufficient sensitivity of RT-PCR less than 5% of RET-positive cells. This may lead to separate subclones being sampled for RT-PCR and for paraffin processing before FISH studies. We would have predicted that if false-negative cells (showing a superimposed signal of two separated FISH signals) had been evaluated in post-Chernobyl samples, the same should have occurred in normal colon mucosa. Thus, technical artifacts due to signal geometry or insufficient confocal steps should have been excluded. The TPC-1 line was used as a positive control because a 100% positivity of cells for RET/PTC1 can be presumed. Although this cell line carries a more complex chromosomal aberration than a simple inversion between RET and H4 (12), it is a suitable control because RET/PTC1 is present and the cell line was identically treated and processed (formalin fixation, paraffin embedding, etc.) according to the post-Chernobyl tissue samples. The fact that this cell line has a complex rearrangement, different from that usually observed in papillary carcinomas in patients, is not an issue using the approach we have used (split signal indicates rearrangement), for the reasons given above. It is interesting to note that the nonradiated normal tissue samples show a much lower frequency of rearranged cells than the normal tissue samples of the post-Chernobyl tumors (up to 9% in one case), thus indicating that radiation may indeed cause this type of rearrangement, but it is not sufficient on its own to induce thyroid cancer. The significant difference between the frequencies of RET alterations at FISH (72% aberrant cases) with respect to RT-PCR (41% aberrant cases) is consistent with the clustering of positive cells within given tumors. We have shown that a minimum number of rearranged cells is required for detection on the RT-PCR analysis in this study. However, as in some of the tumors, even low frequencies of rearranged cells could be accurately detected by RT-PCR, and it is likely that the discrepancy between the FISH and RT-PCR findings is not solely a result of a threshold effect in the number of rearranged cells. Heterogeneity and clustering of rearranged cells is also likely to account for this discrepancy. Because the RT-PCR analyses were carried out on frozen material, whereas the FISH on paraffin-embedded tissues representing different regions of the same tumor it is possible that different subclones of the same tumor were sampled for the different analyses. Only 24% of the cases revealed RET/PTC1 or 3, whereas 41% of the cases revealed unbalanced TK expression. This confirms previously published observations showing that a significant proportion of post-Chernobyl carcinomas harbors RET rearrangements other than H4/RET and RFG/RET.
A distinct intratumoral genetic heterogeneity has previously been reported for thyroid lesions (16) as well as for many other solid tumors (17, 18, 19), suggesting that clonal evolution in such tumors is more complex than predicted by linear models (20). This observation raises questions about the clonal development of PTCs, suggesting that post-Chernobyl papillary cancer may harbor multiple clones of follicular cells. This interpretation is supported by other studies in thyroid nodules with incomplete morphological evidence of papillary carcinoma (21) and in papillary carcinomas exhibiting different RET/PTC forms in the same tumor (22, 23). Also, the facts that post-Chernobyl PTCs are not homogeneous morphologically and that their morphology is changing with time postaccident suggest that they are polyclonal. The development of divergent clones during the process of clonal expansion has already been proposed to explain tumor heterogeneity (24). Additional molecular genetic studies are necessary to confirm that post-Chernobyl thyroid carcinomas tumors are in fact of multiclonal origin. The observed genetic heterogeneity in RET/PTC rearrangement can be interpreted in two different ways: RET/PTC was the first event and new clones emerged later, or RET/PTC is, at least in some carcinomas, a late event in radiation-induced thyroid carcinogenesis. Of the two hypotheses, the second seems more likely to be true because the first would require loss of ret rearrangement during clonal progression. The finding that the majority (72%) of papillary carcinomas harbor clones of cells with a RET rearrangement suggest that RET does play a role in the generation of the tumor, perhaps through paracrine interaction with rearrangement-negative cells. However, it is clear from these results that other oncogenes must cooperate with RET in the development of papillary carcinoma. Recent studies suggest that these are unlikely to include either RAS or B-RAF (25). The observation that two follicular adenomas included in the FISH analysis also showed cells positive for RET rearrangement suggests that RET rearrangement is not confined to papillary carcinoma. The finding that only a small proportion of cells harbored RET rearrangement in follicular tumors suggests that RET is unlikely to play a significant role in their development. In conclusion, this study demonstrates that the development of papillary carcinoma is complex and that studies using highly sensitive RT-PCR in isolation may lead to overinterpretation of the role of specific oncogenes in tumorigenesis.
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Acknowledgments |
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Footnotes |
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Abbreviations: EC, Extracellular; FISH, fluorescence in situ hybridization; FITC, fluorescein isothiocyanate; LSM, laser scanning microscopy; PN, phosphate buffer with 0.1% nonidet P40; PTC, papillary thyroid carcinoma; TK, tyrosine kinase; YAC, yeast artificial chromosome.
Received October 27, 2003.
Accepted May 10, 2004.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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