Rearrangement in Follicular Thyroid Tumors

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Rearrangement in Follicular Thyroid Tumors

  1. 1. Involvement of the PAX8/Peroxisome Proliferator-Activated Receptor {gamma} Rearrangement in Follicular Thyroid Tumors Trisha Dwight, Srinivasan R. Thoppe, Theodoros Foukakis, Weng O. Lui, Göran Wallin, Anders Höög, Tony Frisk, Catharina Larsson and Jan Zedenius J. Clin. Endocrinol. Metab. 2003 88: 4440-4445, doi: 10.1210/jc.2002-021690 To subscribe to Journal of Clinical Endocrinology & Metabolism or any of the other journals published by The Endocrine Society please go to: http://jcem.endojournals.org//subscriptions/ Copyright © The Endocrine Society. All rights reserved. Print ISSN: 0021-972X. Online
  2. 2. 0021-972X/03/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 88(9):4440 – 4445 Printed in U.S.A. Copyright © 2003 by The Endocrine Society doi: 10.1210/jc.2002-021690 Involvement of the PAX8/Peroxisome Proliferator- Activated Receptor Rearrangement in Follicular Thyroid Tumors TRISHA DWIGHT, SRINIVASAN R. THOPPE, THEODOROS FOUKAKIS, WENG O. LUI, ¨ ¨ ¨ GORAN WALLIN, ANDERS HOOG, TONY FRISK, CATHARINA LARSSON, AND JAN ZEDENIUS Endocrine Tumor Unit (T.D., S.R.T., T.Fo., W.O.L., T.Fr., C.L.), Department of Molecular Medicine, Karolinska Hospital CMM L8:01, Stockholm, SE-171 76 Sweden; Centre for Metabolism and Endocrinology (T.D., T.Fo., T.Fr., J.Z.), Department of Surgery, Karolinska Institutet at Huddinge University Hospital, Stockholm, SE-141 86 Sweden; Department of Surgery (T.Fo., G.W., T.Fr.), Karolinska Hospital P9:03, Stockholm, SE-171 76 Sweden; and Department of Oncology and Pathology (A.H.), Karolinska Hospital P1:02, Stockholm, SE-171 76 Sweden Recently, a translocation t(2; 3)(q13;p25), involving the fusion carcinomas studied. In addition, seven of the 87 thyroid tu- of PAX8 and peroxisome proliferator-activated receptor mors exhibited involvement of PPAR alone. Our findings (PPAR ) was suggested to arise only in follicular thyroid car- suggest that PAX8/PPAR occurs frequently in follicular thy- cinomas. In this study, a group of 87 thyroid tumors were roid carcinomas, and the presence of this rearrangement is analyzed to determine the involvement of the PAX8/PPAR likely to prove highly suggestive of a malignant tumor. Lack fusion gene in these tumors, and also to determine whether of the PAX8/PPAR rearrangement in the anaplastic thyroid this rearrangement can be used as a diagnostic marker for the carcinoma group suggests that the tumorigenic pathway in differentiation between follicular thyroid carcinoma and ad- these tumors is likely to be independent of this fusion. Fur- enoma. The PAX8/PPAR rearrangement was detected by RT- thermore, the results suggest that other rearrangements, in- PCR, fluorescence in situ hybridization, and/or Western anal- volving PPAR and other unidentified genes, may be involved ysis in 10 of 34 (29%) follicular thyroid carcinomas and in one in follicular thyroid tumorigenesis. (J Clin Endocrinol Metab of 20 (5%) atypical follicular thyroid adenomas, but not in any 88: 4440 – 4445, 2003) of the 20 follicular thyroid adenomas or 13 anaplastic thyroid F OLLICULAR THYROID CARCINOMA (FTC) accounts for 10 –20% of all thyroid malignancies and normally arises sporadically in the fifth to sixth decades of life (1). gene (PAX8) and the peroxisome proliferator-activated re- ceptor (PPAR ). This fusion gene was present in the ma- jority of FTC but was not seen in any of the papillary thyroid Although the use of fine needle aspiration biopsies has been carcinoma, FTA, or multinodular goiters (5), suggesting that of great benefit in the preoperative diagnosis of other thyroid it may be a useful marker for the preoperative differentiation malignancies, such as papillary and medullary thyroid car- between FTC and FTA. The occurrence of PAX8/PPAR re- cinoma, it has proved to be of limited use for the differen- arrangements in a significant proportion of FTC cases was tiation of FTC and follicular thyroid adenoma (FTA) (2). recently confirmed by three studies, which also reported the Diagnosis of follicular thyroid malignancy is based on the PAX8/PPAR fusion in a few cases of FTA (6 – 8). presence of capsular or vascular invasion (3), which can only In the present study, we have determined the involvement be assessed by histopathological examination of the surgi- of the PAX8/PPAR rearrangement in a series of 34 regular cally removed specimen. Hence, although FTA arises much FTC and 20 FTA, as well as in 20 FTA diagnosed as atypical more frequently than FTC (2), most individuals diagnosed (AFTA). In addition, 13 anaplastic thyroid carcinomas (ATC) with follicular thyroid neoplasia undergo surgery. The pre- were similarly analyzed, because this most aggressive form operative differentiation between FTA and FTC is therefore of thyroid cancer has been proposed to originate from dif- of great importance in the clinical setting. ferentiated papillary or follicular forms. These analyses were Translocations or inversions can give rise to the activation performed using RT-PCR followed by sequencing, fluores- of an oncogene through its positioning near a strong pro- cence in situ hybridization (FISH), and Western analysis. moter or its fusion with another gene, endowing the fused transcript with tumorigenic properties (4). Recently, a trans- Patients and Methods location between chromosomes 2 and 3, t(2;3)(q13;p25), was Patients and tumor samples reported to be specific to FTC (5). Cloning of the translocation breakpoint revealed a fusion between the DNA binding do- Seventy-four follicular tumors, 13 anaplastic carcinomas, and two normal thyroid samples were obtained from 89 patients who underwent main of the thyroid-specific transcription factor paired box thyroidectomy at the Karolinska Hospital (Stockholm, Sweden). The tumors were classified by routine histopathological examination, ac- Abbreviations: AFTA, Atypical FTA; ATC, anaplastic thyroid carci- cording to the criteria of the World Health Organization committee (9), nomas; FISH, fluorescence in situ hybridization; FTA, follicular thyroid as follows: 1) FTA, a benign encapsulated tumor showing evidence of adenoma; FTC, follicular thyroid carcinoma; PPAR , peroxisome pro- follicular cell differentiation; 2) AFTA, an encapsulated tumor with liferator-activated receptor . follicular cell differentiation. The tumor has an irregular architecture and 4440
  3. 3. Dwight et al. • PAX8/PPAR in Follicular Thyroid Tumors J Clin Endocrinol Metab, September 2003, 88(9):4440 – 4445 4441 cellular pattern, with signs of considerable proliferation and pleomor- were confirmed by hybridization onto normal male human metaphase phism. Frequent mitoses are often seen. Invasion into the capsule, or chromosomes (Vysis, Inc., Downers Grove, IL). vascular invasion must be carefully excluded; 3) FTC, minimally inva- sive (MI) type, a malignant tumor with follicular cell differentiation but Interphase FISH analysis. Interphase nuclei were prepared as previously lacking the diagnostic features of papillary carcinoma. Unequivocal described (12). Briefly, a small piece of each tumor or normal tissue was vascular invasion in few vessels occur and/or invasion through the full cut and gently touched onto a glass slide, fixed in methanol-acetic acid thickness of the capsule; 4) FTC, widely invasive (WI) type, as per MI (3:1), air dried, and stored at 20 C. Dual-color FISH was performed type but with widespread infiltration of several blood vessels and/or using standard methods. Interphase nuclei were denatured in 70% for- broad invasion of surrounding tissues; 5) ATC, a highly malignant mamide/2 saline sodium citrate (SSC) (pH 7.0) at 75 C for 3 min, tumor composed in part or wholly of undifferentiated cells. The tumor followed by dehydration in 70, 85, and 100% ethanol for 2 min each. The is typically composed of varying proportions of spindle, polygonal, and hybridization mixture, containing 10 ng/ l of each of the labeled probes giant cells. (either 57D6 and 368K23, or 402P11 and 434I13), was denatured for 7 min Using the above-mentioned criteria, the 87 thyroid tumors were clas- at 80 C followed by preannealing at 37 C for 60 min. Ten microliters of sified as follows: 28 FTC-MI, six FTC-WI, 20 FTA, 20 AFTA, and 13 ATC. the hybridization mixture were then applied to the interphase nuclei. All AFTA samples were reevaluated by further sectioning, at different After hybridization at 37 C for 48 h, the slides were washed in 0.4 depths through the tumor sample, to exclude capsular or vascular in- SSC/0.3% Tween 20 at 74 C for 2 min, 2 SSC/0.1% Tween 20 at room vasion. In no case was the original diagnosis changed. All tissue from temperature for 2 min, followed by water at room temperature for 2 min, the thyroid tumors was frozen in liquid nitrogen immediately after before being air dried. After counterstaining with 4,6-diamino-2- surgical removal and stored at 70 C until required. For each frozen phenylindole and mounting in antifade, hybridization signals were sample used, a representative section was cut, embedded in paraffin, analyzed by use of a Zeiss Axioplan 2 (Carl Zeiss Jena GmbH, Jena, and subjected to histopathological evaluation to confirm the high purity Germany) epifluorescence microscope and documented using the of tumor cells ( 70%) in the tumor samples and the lack of neoplastic Metasystems Isis imaging system (Metasystems, Altlussheim, Ger- cells in the normal controls. None of the patients had previously been many). Nuclei in which the two probes were fused, touched, or were exposed to irradiation, with the exception of the 13 ATC patients who close to each other (distance 1 probe signal) were scored as positive underwent intensive irradiation therapy 3– 6 wk before surgery. All for gene fusions. From each case, 100 nonoverlapping nuclei were in- patients gave informed consent according to a protocol approved by the cluded in the scoring without other selection criteria. All discrete hy- ethical committee at the Karolinska Hospital. bridization signals were counted, and manual adjustment was applied to detect signals in slightly different focal planes. The specificity of the fusion assay was confirmed by hybridization to the interphase nuclei of RT-PCR and cDNA sequencing two normal thyroids, which showed falsely positive signals in less than Total RNA was isolated from the 87 tumor samples and two normal 10% of the nuclei. thyroid samples using either the BIOTECX Ultraspec-II (Biotecx Labo- ratories, Houston, TX) or TRIzol (Invitrogen AB, Lidingo, Sweden) RNA ¨ Western analysis isolation system and was used for RT-PCR-based detection of the PAX8/ PPAR fusion transcript. Briefly, cDNA was synthesized from 3 g of Protein was extracted from the tumor and normal thyroid tissues total RNA using oligo (dT) primers and Superscript II reverse tran- according to standard procedures using RIPA buffer. A total of 100 g scriptase (Invitrogen AB, Lidingo, Sweden), according to the manufac- ¨ of protein in 1 sodium dodecyl sulfate buffer containing 50 mm di- turer’s instructions. The PCR amplifications were performed using the thiothreitol was heated to 85 C for 5 min and then electrophoresed, with Advantage 2 PCR Enzyme System (Clontech, Palo Alto, CA) according 5 l of kaleidoscope protein size standard (Bio-Rad Laboratories, Her- to the manufacturer’s instructions and with the following primers: 5 - cules, CA), on 7.5% SDS-PAGE gels in a mini-Protean 2 electrophoresis GCATTGACTCACAGAGCAGCA-3 (PAX8, c.786 – 806, accession no. system (Bio-Rad). Proteins were blotted onto 0.45- m nitrocellulose X69699) or 5 -GCCACCAAGTCCCTGAGTCC-3 (PAX8, c.605– 624, ac- membranes (Protran; Schleicher & Schuell, Dassel, Germany), stained cession no. X69699) and 5 -CATTACGGAGAGATCCACGG-3 (PPAR , with Ponceau S, and scanned into the computer to use as a loading c.158 –177, accession no. X90563). In short, 2 l of each cDNA was control. After blocking in 4% nonfat milk, the membranes were incu- amplified in 20- l reactions containing 0.4 l of 50 dNTP Mix, 0.4 l bated with anti-PPAR (sc-7273; 1:500 dilution) (Santa Cruz Biotech- of 50 Advantage 2 Polymerase Mix, 2.0 l of 10 Advantage 2 PCR nology, Santa Cruz, CA) or anti-Pax-8 (kindly provided by Prof. Roberto Buffer, and 0.4 m of each primer. The amplifications were performed Di Lauro, Stazione Zoologica A. Dohrn, Napoli, Italy; 1:2000 dilution) at using the following thermocycling conditions: an initial denaturation at 4 C overnight. The membranes were then incubated with secondary 95 C for 1 min, was followed by 35 step cycles of denaturing at 95 C for horseradish peroxidase-conjugated antibodies (1:2000 dilution; Santa 10 sec, annealing at 60 C for 10 sec, and extension at 72 C for 15 sec, with Cruz Biotechnology). Detection was carried out by enhanced chemilu- a final extension at 72 C for 1 min. RNA integrity and efficiency of cDNA minescence (Amersham Pharmacia Biotech, Uppsala, Sweden) for 1 h, synthesis were assessed by parallel amplification with the housekeeping and the proteins were visualized by a charge coupled device camera gene -actin (ACTB, accession no. XM037239), as previously described using the luminescent image analyzer LAS-1000plus (Fujifilm, Tokyo, (10). PCR products were directly sequenced and analyzed on a 377XL Japan) and analyzed using the Image Gauge V3.3 program (Fujifilm). To automated DNA sequencer (Applied Biosystems, Perkin-Elmer Corp., confirm that approximately equal amounts of protein were blotted in Foster City, CA), as previously described (11). each sample, the filters were reversibly stained with a 3% Ponceau S solution (Sigma-Aldrich, St. Louis, MO) in 3% trichloroacetic acid, before incubating with the primary antibody, as described by Wautot et al. (13). FISH Specific blocking peptides for PPAR (Sc7752p) and PAX8 (Sc7273p) (Santa Cruz Biotechnology) were used to confirm the identity of bands Probes. The genomic clones used for FISH analyses originate from the for fusion transcripts. Peptide neutralization was done according to the human male BAC library RPCI-11 (http://www.chori.org/bacpac). A protocol recommended by the manufacturer. The blocking peptides fusion assay was applied to detect the presence of the PAX8/PPAR were incubated with the primary antibody at room temperature for 2 h fusion gene, whereby one clone from the PAX8 locus in 2q13 was and then used for filter hybridization. In all cases, the Western signal cohybridized with a second clone from the PPAR locus in 3p25. Spe- (enhanced chemiluminescence) was blocked, thus confirming the iden- cifically, RP11– 434I13 centromeric of PAX8 was combined with RP11– tity and specificity of the PPAR and PAX8 signals. A positive control 402P11 telomeric to PPAR , or RP11–368K23 telomeric of PAX8 was with a known PAX8-PPAR fusion was also used. combined with RP11–57D6 centromeric of PPAR (see Fig. 2). DNA was isolated from the BAC clones using the Qiagen Plasmid Midi Kit (VWR International AB, Stockholm, Sweden), according to the manufacturer’s Results instructions, and labeled by nick translation with fluorescein-12-dUTP or Texas Red-5-dUTP (NEN, Life Science Products, Boston, MA). In The involvement of the PAX8/PPAR fusion oncogene was control experiments, the accuracy and specificity of all four BAC clones evaluated on the genomic, RNA, and protein levels in a panel
  4. 4. 4442 J Clin Endocrinol Metab, September 2003, 88(9):4440 – 4445 Dwight et al. • PAX8/PPAR in Follicular Thyroid Tumors of 87 thyroid tumors. The results for the different histopatho- followed by the entire coding part of PPAR , with the ad- logical subtypes are summarized in Table 1. dition of a new amino acid (glutamine) in between the two partner genes. The AFTA (T11), in which multiple transcripts RT-PCR and cDNA sequencing of the PAX8/PPAR rearrangement were detected by RT- Five of the 87 thyroid tumors analyzed by RT-PCR were PCR, was unable to be successfully sequenced. found to express PAX8/PPAR (Tables 1 and 2). Four of the five tumors exhibiting PAX8/PPAR were minimally inva- FISH sive FTC (T2, T3, T8, and T10), whereas one was an AFTA Seven of the 73 informative thyroid tumors analyzed by (T11). Multiple transcripts were seen in one of the FTC (T10) FISH were found to exhibit fusion signals in at least 60% of and the AFTA (T11), which is likely due to alternative splic- the nuclei analyzed, suggestive of a translocation, t(2;3)(q13; ing of PAX8. Representative RT-PCR results of each of the p25), involving chromosomal regions containing both PAX8 thyroid tumor types analyzed in this study are shown in Fig. and PPAR (Tables 1 and 2 and Fig. 2). These seven tumors 1A. Sequencing of the PAX8/PPAR expressing transcripts in included six FTC-MI (T2, T3, T5, T6, T8, and T10) and one all four FTC tumors revealed similar breakpoints in exon 1 AFTA (T11). All of the tumors expressing PAX8/PPAR by of PPAR , all located at nt 2 or 3 immediately 5 of the RT-PCR analysis (T2, T3, T8, T10, and T11) were also shown, initiating codon. However, different breakpoints in PAX8 by FISH analysis, to contain t(2;3)(q13; p25). In addition to were observed, with breaks occurring at the end of exons 7, this, two FTC (T5 and T6) in which PAX8/PPAR expression 8, or 9. In two FTC (T8 and T10), exon 7 of PAX8 was was not detected by RT-PCR analysis exhibited t(2;3)(q13; juxtaposed to exon 1 of PPAR , whereas in another FTC (T3), p25) by FISH analysis. One possible explanation for the dis- exon 9 of PAX8 was joined to exon 1 of PPAR . In the fourth crepancy between RT-PCR and FISH results may be that the FTC exhibiting PAX8/PPAR (T2), exon 8 of PAX8 was de- breakpoint(s) occurring in PAX8 or PPAR may lay outside leted, and exon 9 of PAX8 was positioned next to exon 1 of the regions encompassed by the primers used in this study. PPAR (Fig. 1B). In all four FTC cases, the PAX8/PPAR Another possible explanation may be that the primers used fusion consisted of the first seven to nine exons of PAX8, are localized to regions that may be deleted due to alternative splicing. TABLE 1. Results from RT-PCR, FISH, and Western analysis in the 87 thyroid tumors in this study Western analysis PAX8/PPAR involvement Eleven of the 86 thyroid tumors analyzed by Western Histopathological Cases demonstrated by diagnosis analyzed blotting exhibited altered protein size for both PAX8 and RT-PCR FISH Western PPAR , when compared with the wild-type proteins (Tables Follicular adenoma 40 1 and 2 and Fig. 3). Eight of the tumors exhibiting altered Ordinary 20 0 0 0 protein size for both PAX8 and PPAR were FTC- MI (T2, T3, Atypical 20 1 1 1 T5, T6, T7, T8, T9, and T10), whereas two were FTC-WI (T1 Follicular carcinoma 34 Minimally invasive 28 4 6 8 and T4). In addition to this, one of the AFTA (T11) was also Widely invasive 6 0 0 2 shown to exhibit altered protein size for both PAX8 and Anaplastic carcinoma 13 0 0 0 PPAR . Furthermore, seven of 86 thyroid tumors exhibited Total 87 5 7 11 altered protein size for PPAR , while expressing only the Normal thyroid 2 0 0 0 wild-type PAX8 protein (Table 3 and Fig. 3), suggesting the TABLE 2. Summary of clinical and genetic data for the 11 follicular thyroid tumors showing PAX8/PPAR involvement Case Histopathology Follow-up PAX8/PPAR Age at Western Sex Tumor operation Size Time mRNA FISH 2;3 (aberrant No. ID (M/F) studied Diagnosis Metastasis Outcome (yr) (cm) (yr) expressiona fusionb product)c PAX8 PPAR T1 414 M 47 Primary 6.0 FTC-WI Lung DoD 2 T2 515 M 31 Primary 0.5 FTC-MI No na na T3 805 M 57 Primary 3.0 FTC-MI Skeletal NED 10 T4 1089 F 81 Primary 1.0 FTC-WI (insular) No DwoD 6 T5 1095 M 44 Primary 6.0 FTC-MI No NED 10 T6 1800 F 44 Primary 4.0 FTC-MI No NED 6 T7 1890 F 50 Primary 2.0 FTC-MI No NED 6 T8 2083 F 41 Primary 0.8 FTC-MI No NED 6 T9 2505 M 50 Primary 6.5 FTC-MI No DwoD 2 na T10 2769 F 45 Primary 3.5 FTC-MI No NED 3 T11 2406 F 36 Primary 5.0 AFTA No na na FTC-MI, Minimally invasive FTC; FTC-WI, widely invasive FTC; DoD, died of disease; DwoD, dead without disease; NED, no evidence of disease; na, not available. a , No PAX8/PPAR fusion detected by RT-PCR; , PAX8/PPAR fusion detected by RT-PCR. b , No fusion of 2q13 and 3p25 probes detected; , fusion of 2q13 and 3p25 probes detected. c , Expression of aberrant protein.
  5. 5. Dwight et al. • PAX8/PPAR in Follicular Thyroid Tumors J Clin Endocrinol Metab, September 2003, 88(9):4440 – 4445 4443 FIG. 1. A, Representative RT-PCR analysis of follicular thyroid tumors demonstrating the presence of the PAX8/ PPAR fusion transcript in a FTC (T8) and an AFTA (T11) with multiple transcripts, but not in a FTA, ATC, or normal thyroid. The positive control ( ) consists of a sample with a known PAX8/PPAR fusion, and the negative control ( ) includes a PCR without template. B, Schematic illustration of the different breakpoints observed in the PAX8/PPAR fusion transcripts in the four FTC. See text for details. involvement of alternative PPAR fusion partners. Indeed, with PAX8/PPAR rearrangements, 10 were FTC, supporting case T15 was shown previously to exhibit a t(3;7) transloca- previous findings of its involvement in FTC (5– 8). The fre- tion (14). quency of PAX8/PPAR rearrangements detected in our group of FTC (29%) is lower than previously reported, Discussion whereby 53– 62% of FTC cases analyzed were shown to con- Currently, the discrimination between follicular thyroid tain this rearrangement (5– 8). An explanation for this may be carcinoma and adenoma can only be made postoperatively that irradiated patients are being analyzed in other studies, and is based on the presence of capsular or vascular invasion whereas none of the thyroid tumors in this study have been (3, 9). The ability to preoperatively differentiate between the derived from previously irradiated patients, with the excep- malignant and benign forms of follicular thyroid tumors is tion of the ATC patients. In one study (7), all FTC patients therefore of great importance in the clinical setting. Opti- previously exposed to irradiation were shown to have the mally, these diagnostic markers should identify all FTC and PAX8/PPAR rearrangement. When these patients are re- those FTA that have a potential for malignant progression. moved from the sample group, the percentage of FTC cases The PAX8/PPAR fusion was originally proposed to occur in exhibiting the PAX8/PPAR fusion gene falls from 53 to 42%. the majority of FTC but not in FTA. In this study, a large One of the 20 (5%) AFTA cases was also found to contain group of follicular thyroid tumors (both benign and malig- a PAX8/PPAR rearrangement, suggesting that this fusion nant), as well as a group of ATC, were analyzed to determine may not occur specifically in FTC as originally hypothesized the involvement of the PAX8/PPAR fusion gene in these (5). More recent studies have also found the presence of the tumors, and also to determine whether this rearrangement PAX8/PPAR rearrangement not only in FTC, but also FTA can be used as a diagnostic marker for the differentiation (6 – 8). None of the ordinary FTA cases analyzed in this study, between FTC and FTA. The PAX8/PPAR fusion gene was however, exhibited the PAX8/PPAR gene fusion. Although detected in approximately 30% of the FTC cases analyzed. In the PAX8/PPAR fusion gene does not occur solely in FTC addition to this, the rearrangement was also detected in one (6 – 8), it is possible, on a purely speculative basis, that benign of the AFTA cases. Although these findings suggest that the follicular thyroid tumors containing this rearrangement may PAX8/PPAR rearrangement is not restricted to the FTC be more likely to transform into their malignant counterpart entity, it is possible that the vascular or capsular invasion (FTC). However, so far no formal proof has been presented indicative of a carcinoma may not be detected at the his- to support the idea that FTA can progress to FTC. Further- topathological level, or that benign follicular thyroid tumors more, if the PAX8/PPAR rearrangement has occurred as an containing this rearrangement are more likely to transform early event in tumorigenesis, it is possible that molecular into their malignant counterpart. genetic techniques, such as those used in this study, may detect In this study, 11 of the 87 (12%) thyroid tumors analyzed the rearrangement, whereas at the histopathological level signs were found to exhibit PAX8/PPAR involvement by RT-PCR, of capsular or vascular invasion may not yet be evident. FISH, and/or Western analysis. Of these 11 thyroid tumors None of the ATC analyzed in this study or others (6, 7) has
  6. 6. 4444 J Clin Endocrinol Metab, September 2003, 88(9):4440 – 4445 Dwight et al. • PAX8/PPAR in Follicular Thyroid Tumors FIG. 2. Interphase FISH analysis demonstrating the absence of a t(2;3)(q13;p25) translocation in a FTA and its presence in an AFTA (T11). The locations of the BAC probes used for the FISH fusion assays are shown in relation to PAX8 and PPAR next to the ideograms of normal chromosomes 2 and 3. A 2q14 probe (368K23), telomeric of PAX8, was labeled with fluorescein-12- dUTP (green), and a 3p25 probe (57D6), centromeric of PPAR ,was labeled with Texas Red-5-dUTP (red). Nu- clei from a FTA in which t(2;3)(q13;p25) is absent are shown in panel A, whereas panel B demonstrates nuclei from an AFTA (T11) in which t(2;3)(q13;p25) is present, as demonstrated by the adjacently located green (368K23) and red (57D6) hybridization signals. FIG. 3. Western analysis of PPAR (A) and PAX8 (B) on protein extracts from eight FTC. The PAX8/PPAR rearrangement is expected to yield a protein of approximately 98 kDa, whereas the wild-type (WT) proteins of PPAR and PAX8 are estimated to yield proteins of 48 kDa and 34 – 48 kDa, respectively. The pure antibodies for PPAR and PAX8 were used as positive controls ( ). In three cases (T2, T8, and T10), altered proteins were detected by Western analyses with both PAX8 and PPAR , indicating the presence of the PAX8/PPAR fusion product in these tumors. In case T15, aberrant PPAR was revealed together with the wild-type PAX8, supporting a translocation of PPAR with another partner other than PAX8 (14). The four FTC shown on the right panel also express an aberrant PPAR and PAX8, although a PAX8/PPAR translocation was not detected by the other methods. The band marked by an asterisk corresponds to nonspecific binding, present in all samples. exhibited the PAX8/PPAR gene fusion. These findings, However, it is possible, although unlikely, that the PAX8/ taken together, suggest that the tumorigenic pathway of ATC PPAR rearrangement was not detected in any ATC due to is likely to be independent of the PAX8/PPAR fusion gene. gross DNA damage caused by intensive radiation therapy
  7. 7. Dwight et al. • PAX8/PPAR in Follicular Thyroid Tumors J Clin Endocrinol Metab, September 2003, 88(9):4440 – 4445 4445 TABLE 3. Summary of clinical and genetic data for the thyroid tumors showing only PPAR involvement Case Histopathology Follow-up PAX8/PPAR Age at Western Sex Tumor operation Size Time mRNA FISH 2;3 (aberrant No. ID (M/F) studied Diagnosis Metastasis Outcome (yr) (cm) (yr) expressiona fusionb product)c PAX8 PPAR T12 1129 F 83 Primary 3.0 FTC-MI No NED 8 T13 2311 F 31 Primary 2.0 FTC-MI No NED 5 T14 2384 F 70 Primary 6.0 FTC-MI No NED 4 T15 2723 M 72 Primary 7.0 FTC-MI No NED 3 T16 2507 F 49 Primary 2.0 AFTA No NED 4 T17 2288 M 71 Primary 6.5 ATC No na na na T18 222 F 55 Primary 2.0 FTA No NED 14 na FTC-MI, Minimally invasive FTC; NED, no evidence of disease; na, not available. a , No PAX8/PPAR fusion detected by RT-PCR; , PAX8/PPAR fusion detected by RT-PCR. b , No fusion of 2q13 and 3p25 probes detected; , fusion of 2q13 and 3p25 probes detected. c , Expression of aberrant protein. administered to these patients before surgery. It is also un- cine Endocrine Tumor Unit, CMM L8:01 Karolinska Hospital, SE-171 76, clear as to whether ATC develops from papillary or follicular Stockholm, Sweden. E-mail: Trisha.Dwight@cmm.ki.se or Catharina. cancers, respectively, or if they occur by dedifferentiation Larsson@cmm.ki.se. This work was supported by the Swedish Cancer Foundation, the without an intermediate stage as a differentiated carcinoma. Gustav V Jubilee Foundation, the Milton Foundation, the Cancer Society Furthermore, it is possible that the PAX8/PPAR rearrange- in Stockholm, the Emil and Vera Cornell Foundation, the Wenner-Gren ment arrests tumors in a differentiated state. Other genetic Foundation, and the Torsten and Ragnar Soderberg Foundations. ¨ events may then accumulate, driving progression and a clonal loss of the PAX8/PPAR fusion gene, which subse- References quently may lead to dedifferentiation. There is some genetic evidence for such a process (10), but whether or not the 1. Gimm O 2001 Thyroid cancer. Cancer Lett 163:143–156 2. Lawrence Jr W, Kaplan BJ 2002 Diagnosis and management of patients with PAX8/PPAR fusion gene, or its loss, is involved in ATC thyroid nodules. J Surg Oncol 80:157–170 tumorigenesis requires further study. 3. Vini L, Harmer C 2002 Management of thyroid cancer. Lancet Oncol 3:407– 414 It is of interest to note that PPAR involvement alone was 4. Lengauer C, Kinzler KW, Vogelstein B 1998 Genetic instabilities in human cancers. Nature 396:643– 649 shown by Western analysis in seven follicular thyroid tumors 5. Kroll TG, Sarraf P, Pecciarini L, Chen CJ, Mueller E, Spiegelman BM, (four minimally invasive FTC, one AFTA, one ATC, and one Fletcher JA 2000 PAX8-PPAR 1 fusion oncogene in human thyroid carcinoma. FTA), suggesting that, in some cases, PPAR may be fusing Science 289:1357–1360 6. Marques AR, Espadinha C, Catarino AL, Moniz S, Pereira T, Sobrinho LG, with a non-PAX8 partner. Further analysis in one of these Leite V 2002 Expression of PAX8-PPAR 1 rearrangements in both follicular tumors (T15), revealed a translocation involving chromo- thyroid carcinomas and adenomas. J Clin Endocrinol Metab 87:3947–3952 somes 3 and 7 [t(3;7)(p25;q34)] (Ref. 14). Identification of the 7. Nikiforova MN, Biddinger PW, Caudill CM, Kroll TG, Nikiforov YE 2002 PAX8-PPAR rearrangement in thyroid tumors: RT-PCR and immunohisto- translocation partners has revealed a new fusion oncogene chemical analyses. Am J Surg Pathol 26:1016 –1023 (Lui, W. O., I. Leibiger, B. Leibiger, S. Thoppe, U. Enberg, J. 8. Cheung L, Messina M, Gill A, Clarkson A, Learoyd D, Delbridge L, Went- Liden, A. Hoog, L. O. Farnebo, J. A. Fletcher, C. Larsson, ´ ¨¨ worth J, Philips J, Clifton-Bligh R, Robinson BG 2003 Detection of the PAX8-PPAR fusion oncogene in both follicular thyroid carcinomas and ad- submitted for publication). enomas. J Clin Endocrinol Metab 88:354 –357 In conclusion, our findings suggest that PAX8/PPAR 9. Hedinger CE, Williams ED, Sobin LH 1988 The WHO international classi- shows a high specificity to FTC, and although it was detected fication of tumors. 2nd ed. Berlin: Springer-Verlag 10. Frisk T, Foukakis T, Dwight T, Lundberg J, Hoog A, Wallin G, Eng C, ¨¨ in one AFTA, the presence of this rearrangement is highly Zedenius J, Larsson C 2002 Silencing of the PTEN tumor-suppressor gene in suggestive of a malignant form of follicular thyroid tumor. anaplastic thyroid cancer. Genes Chromosomes Cancer 35:74 – 80 We therefore postulate that detecting the presence of the 11. Dwight T, Twigg S, Delbridge L, Wong FK, Farnebo F, Richardson AL, Nelson A, Zedenius J, Philips J, Larsson C, Teh BT, Robinson BG 2000 Loss PAX8/PPAR rearrangement is of considerable importance of heterozygosity in sporadic parathyroid tumours: involvement of chromo- in the clinical setting. some 1 and the MEN1 gene locus in 11q13. Clin Endocrinol (Oxf) 53:85–92 12. Yang K, Lui WO, Xie Y, Zhang A, Skytting B, Mandahl N, Larsson C, Larsson O 2002 Co-existence of SYT-SSX1 and SYT-SSX2 fusions in synovial sarcomas. Acknowledgments Oncogene 21:4181– 4190 We thank Lisa Ånfalk for excellent technical assistance and Dr. Todd 13. Wautot V, Khodaei S, Frappart L, Buisson N, Baro E, Lenoir GM, Calender Kroll for valuable comments on the manuscript. A, Zhang CX, Weber G 2000 Expression analysis of endogenous menin, the product of the multiple endocrine neoplasia type 1 gene, in cell lines and human tissues. Int J Cancer 851:877– 881 Received October 29, 2002. Accepted May 28, 2003. 14. Lui WO, Kytola S, Ånfalk L, Larsson C, Farnebo LO 2000 Balanced trans- ¨ ¨ Address all correspondence and requests for reprints to: Trisha location (3;7)(p25;q34): another mechanism of tumorigenesis in follicular thy- Dwight, Ph.D., or Catharina Larsson, Department of Molecular Medi- roid carcinoma? Cancer Genet Cytogenet 119:109 –112

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