Evidence that one subset of anaplastic thyroid carcinomas are derived from papillary carcinomas due to BRAF and p53 mutations

Authors


Abstract

BACKGROUND

Anaplastic thyroid carcinoma (ATC) is the most lethal form of thyroid neoplasia and represents the end stage of thyroid tumor progression. In the current study, genetic alterations in a panel of ATC were profiled to determine the origins of ATC.

METHODS

Eight ATC were analyzed for BRAF mutation at codon 599 by using mutant-allele-specific polymerase chain reaction (PCR) and DNA sequencing of the PCR-amplified exon 15. RAS mutation (HRAS, KRAS, and NRAS) at codons 12, 13, and 61 was analyzed by direct sequencing of PCR-amplified exons 1 and 2 of the RAS gene. RET/PTC rearrangements and p53 mutation were monitored by immunohistochemical (IHC) staining by anti-RET antibodies and an anti-p53 mAb, respectively.

RESULTS

BRAF was mutated in 5 of the 8 ATCs tested. Histologic examination revealed that 4 of these 5 BRAF-mutated ATCs contained a PTC component, suggesting that they may be derived from BRAF-mutated PTC. Of the 3 ATCs with wild-type BRAF, 2 had spindle cell features; one had follicular neoplastic characteristics mixed with papillary structures. Analysis of RAS mutation revealed only an HRAS mutation at codon 11, due to the transversion of GCC to TCC in one ATC with wild-type BRAF. This leads to the substitution of valine to serine. IHC analysis of RET/PTC rearrangements revealed no positive staining of RET in any of 8 ATCs, suggesting that these ATCs are not derived from RET/PTC- rearranged PTC. In contrast, IHC analysis of p53 mutation revealed that p53 was detected in the nuclei of 5 of 5 BRAF-mutated ATCs and 2 of 3 ATCs with wild-type BRAF. p53 staining was present only in anaplastic thyroid tumor cells but not in neighboring papillary thyroid tumor cells.

CONCLUSIONS

These results suggest that many ATCs with papillary components are derived from BRAF-mutated PTC, because of the addition of p53 mutation. Cancer 2005. © 2005 American Cancer Society.

Thyroid cancer is the most common endocrine neoplasm in the United States. Approximately 2–5% of individuals will develop a clinically palpable thyroid nodule during their lifetime and be at risk for developing thyroid cancer.1 Anaplastic thyroid carcinomas (ATCs), which account for less than 5% of all thyroid cancers, is the most malignant thyroid neoplasm and is almost invariably fatal.2 A large portion of ATCs are identified in patients who have longstanding goiters or incompletely treated papillary or follicular thyroid cancers.3, 4 Careful histopathologic examination of ATC reveals that many of them contain a papillary structure or follicular components in focal areas. It is believed that ATC is derived from the follicular epithelial cells and represents a terminal dedifferentiation of preexisting differentiated carcinoma.2 Molecular genetic studies suggest that RAS oncogenes are frequently mutated in ATC as a result of progression from RAS-mutated follicular thyroid carcinomas (FTCs).5–8 Because RET/PTC rearrangements have not been detected in ATC,9 it has been postulated that ATC does not progress from RET/PTC-rearranged PTC but may derive from other oncogene-mutated PTC. A tumor suppressor gene, p53, that is frequently and highly mutated in ATC, may play a critical role in promoting dedifferentiation of well differentiated PTC or FTC.10, 11

Recent serial studies have consistently demonstrated that BRAF is mutated at the hotspot codon 599 in approximately 40% of PTC (Table 1).12–21 In contrast, BRAF is not mutated in several other thyroid neoplasms, including medullary carcinomas (MTC), follicular and Hurthle cell neoplasms (Table 1). A few studies also analyzed BRAF mutation in ATC but did not reach a consensus on the rate of BRAF mutation in this tumor type. For example, Fukushima et al.19 did not detect BRAF mutation in 7 ATC specimens. In contrast, Namba et al.18 reported that BRAF mutation occurred in 2 (33%) of 6 undifferentiated thyroid carcinomas. While this article was being prepared, 2 recent studies demonstrated that BRAF is mutated in 3 (10%) of 29 ATCs17 and in 2 (20%) of 10 ATCs,12 respectively. Therefore, it remained to be established whether BRAF-mutated PTC is able to progress to ATC, and whether different frequencies of BRAF mutation in ATC are due to different origins, e.g., papillary versus follicular or Hurthle cell carcinomas.

Table 1. BRAF Mutation In Various Thyroid Neoplasms
ReferenceFTAFTCMTCPTC (%)ATC (%)
  1. FTA: follicular thyroid adenoma; FTC: follicular thyroid carcinoma; MTC: medullary thyroid carcinoma; PTC: papillary thyroid carcinoma; ATC: Anaplastic thyroid carcinoma.

Cohen et al.210/200/130/324/35 (69) 
Kimura et al.200/140/10 28/78 (36) 
Fukushima et al.19 0/80/940/76 (53)0/7 (0)
Soares et al.150/510/18 23/50 (46) 
Namba et al.180/200/11 49/170 (29)2/6 (33)
Trovisco et al.14   28/53 (53) 
Xing et al.120/430/140/1417/38 (45)2/10 (20)
Nikiforova et al.170/460/320/1345/119 (38)3/29 (10)
Xu et al.160/18  21/56 (38) 
Total0/2120/1060/37275/675 (40.7)5/52 (10)

MATERIALS AND METHODS

Tumor Specimens and Patient Information

Specimens from 8 ATC patients with adequate clinical and pathologic information treated at Rush Presbyterian St. Luke's Medical Center from 1994 to 2003 were studied. Paraffin-embedded tumor blocks from thyroidectomy specimens of ATC patients were retrieved from the Department of Pathology after approval by the Institutional Review Board of Rush University Medical Center. ATC specimens embedded in paraffin blocks were sectioned, restained, and reviewed by a pathologist (P. Gatusso) who has extensive experience in thyroid disease.

Mutant-Allele-Specific Amplification (MASA) Analysis of BRAF Mutation

Genomic DNA was extracted from microdissected tumor sections as previously reported.16 MASA was used to identify BRAF mutation. A forward primer flanking the sequence of exon 15 (5′–TAGGTGATTTTGGTCTAGCTACA-GT–3′) was used as a positive control to amplify wild-type as well as mutant BRAF. A second primer with substitution of two bases at the 3′ end (5′-GGTGATTTTGGTCTAGCTACAAA-3′) was designed to amplify the mutant BRAF gene only. The sequence of the reverse primer is 5′-GGCCAAAATTTAATCAGTGGA-3′. The PCR conditions were 94 °C for 2 minutes, (94 °C 30 seconds; 52 °C 45 seconds; 72 °C 45 seconds) × 40 cycles at 72 °C for 8 minutes. The PCR product was a 129 base pairs (bp) fragment. PCR products were analyzed in a 3% agarose gel and stained with ethidium bromide. Genomic DNA extracted from DRO81 cells, a BRAF-mutated anaplastic thyroid tumor cell line, was used as a positive control (PC).

Sequence Analysis of BRAF and HRAS Mutations

The sequence of primers used to amplify exon 15 of the BRAF gene and the three RAS genes, and the PCR conditions are listed in Table 2. PCR fragments were extracted by using a Qiagen PCR Product Purification kit (Qiagen Inc., Santa Clarita, CA). BRAF and RAS mutations were detected by direct sequencing of the PCR products at the CRC–DNA sequencing facility of the University of Chicago.

Table 2. Primers and PCR Conditions Used to Amplify the BRAF and RAS Genes
GeneExonPCRPrimerSequenceT (°C)CycleSize (bp)
  1. PCR: polymerase chain reaction; T: temperature; bp: base pairs.

BRAF15FF5′-TCATAATGCTTGCTCTGA-TAGGA-3′5538225
   R5′-GGCCAAAATTTAATCAGTGGA-3′   
NRAS1 F5′-GACTGAGTA-CAAACTGGTGG-3′5538118
   R5′-GGGCCTCACCTCTATGGTG-3′   
 2 F5′-GGTGAAACCTGTTTGTTGGA-3′5538102
   R5′-ATACACAGAGGAAGCCTTCG-3′   
HRAS1primaryF5′-GGAGACCCTGTAGGAGGACC-3′5538282
   R5′-GAGGAAGCAGGAGACAGGG-3′   
  secondaryF5′-ATGACGGAATATAAGCTGGT-3′5840237
   R5′-GAGGAAGCAGGAGACAGGG-3′   
 2primaryF5′-GAGAGGTACCAGGGAGAGGC-3′5538358
   R5′-ACATGCGCAGAGAGGACAG-3′   
  secondaryF5′-AGGTGGTCATTGATGGGGAG-3′5840259
   R5′-ACATGCGCAGAGAGGACAG-3′   
KRAS1primaryF5′-AGGCCTGCTGAAAATGACTG-3′5538171
   R5′-AAAGAATGGTCCTGCACCAG-3′   
  secondaryF5′-GGCCTGCTGAAAATGACTGAA-3′5840163
   R5′-GGTCCTGCACCAGTAATATGC-3′   
 2primaryF5′-GTCTTTTCAAGTCCTTTGCCC-3′5538250
   R5′-CACAAAGAAAGCCCTCCCCA-3′   
  secondaryF5′-CAGGATTCCTACAGGAAGCAAGTAG-3′5840132
   R5′-CACAAAGAAAGCCCTCCCCA-3′   

IHC Analysis of RET/PTC Rearrangements and p53Mutation

RET/PTC rearrangements were analyzed by using IHC staining with anti-RET mAb for RET-PTC expression. Briefly, tissue sections were dewaxed with xylene and rehydrated. Slides were then heat-inactivated in 10 mM sodium citrate (pH 6.0) in a microwave for 3 minutes. Cooled slides were rinsed with phosphate buffered saline (PBS) and then incubated with 1% H2O2 in methanol for 30 minutes at room temperature. RET/PTC expression was probed with a monoclonal antibody against the extreme C-terminus of RET (1:40) (Novocastra Laboratories Ltd., Burlingame, CA) using a Catalyzed Signal Amplification system (DAKO Corp., Carpinteria, CA) following the manufacture's protocol. Slides were counterstained with Mayer hemotoxylin for 2 minutes, dehydrated and mounted. RET/PTC expression was graded by two investigators in a blinded fashion. IHC analysis of p53 mutation was conducted by using an ABC kit (Novocastra Laboratories Ltd., Burlingame, CA). Briefly, the rehydrated sections of paraffin-embedded blocks were heated at 121 °C for 10 minutes in an autoclave oven. The sections were inactivated with 1% H2O2 for 30 minutes. Expression of p53 was detected by IHC staining with a monoclonal antibody (clone DO-7, Novocastra Laboratories Ltd., Burlingame, CA) (1:60) following the manufacturer's instruction. Normal mouse immunoglobulin G (IgG) was included as a negative control.

RESULTS: DETECTION OF BRAF MUTATION IN ATC

We analyzed BRAF mutation in 8 ATCs by using mutant-allele-specific amplification (MASA). As shown in Figure 1A, the mutant BRAF gene was amplified in 5 of 8 ATCs, indicating that BRAF is mutated at T1796. To confirm BRAF mutation in these samples, PCR-amplified exon 15 was directly sequenced using two primers flanking it. Shown in Figure 1B are two representative DNA sequences of BRAF-mutated samples (Specimen nos. 1 and 7); one had almost equal peak areas of A and T nucleotides, indicating a high homogeneity of tumor cells in these microdissected sections. A second sample had a smaller A peak than T peak, reflecting heterogeneity of tumor cells and nontumor cells in the microdissected section.

Figure 1.

BRAF mutation in ATC. (A) Mutant-allele-specific amplification (MASA) analysis of BRAF mutation. Genomic DNA extracted from microdissected tumor sections were analyzed for BRAF mutation by MASA-PCR as described in Materials and Methods. PCR products were analyzed in a 3% agarose gel and stained with ethidium bromide. Genomic DNA extracted from DRO81 cells, a BRAF-mutated anaplastic thyroid tumor cell line, was used as a positive control (PC). (B) BRAF and HRAS mutations detected by DNA sequencing. Exon 15 of the BRAF gene was amplified with two primers in the introns flanking it. The amplified PCR product was sequenced with a forward primer used to amplify exon 15 of the BRAF gene. Two representative DNA sequences of mutated BRAF gene are shown. PCR-amplified RAS exons 1 and 2 were analyzed for RAS mutations by direct sequencing. Left and middle panels: sequence of mutant BRAF gene at codon 599 in Specimen nos. 1 and 7 by using a forward primer in the sequencing reaction; Right panel, sequence of mutant HRAS gene at codon 11 in Specimen no. 5 by using a reverse primer in the sequencing reaction.

Because our group and other researchers have demonstrated that BRAF is only mutated in PTC but not in FTC,15, 16, 20, 21 it is likely that these BRAF-mutated ATCs are derived from BRAF-mutated PTC. Histologic examination revealed that 4 (Specimen nos.1, 5, 7, 8) of 5 BRAF-mutated ATCs indeed contained papillary components (Figs. 2A, D, E; blue arrows); the fifth specimen was a giant cell carcinoma (Table 3). In contrast, among 3 ATCs with wild-type BRAF, 2 were squamous or spindle cell carcinoma (Specimen nos.2 and 4), and the third ATC specimen (Specimen no. 6) originated from a follicular variant of papillary carcinoma.

Table 3. Summary of the Genetic Alterations in ATC
PatientAgeGenderHistologyRASBRAFRET/PTCp53
164FSpindle cell with papillary component++
276FSquamous and spindle cell+
386FGiant cell anaplastic++
479MKeratin-positive spindle cellHRAS VIIS++
563MPapillary background++++
654MWith follicular neoplasia and papillary structure
767FPapillary background++
884MPapillary background++++
Figure 2.

IHC analysis of RET/PTC and p53 expression in ATC. RET/PTC rearrangements were analyzed for RET/PTC expression by using IHC staining with anti-RET mAb (A–C) as detailed in Materials and Methods. (A, B) Lack of RET/PTC expression in two ATC specimens was detected by IHC with an anti-RET mAb. (C) Strong RET/PTC expression in the cytoplasm of a PTC specimen was detected by IHC with an anti-RET mAb. However, no signal was present in the cytoplasm and membrane of normal follicular cells. (D) Hemotoxylin and eosin staining showing focal papillary-like structure in Specimen no. 5. Green arrow shows strong RET-PTC signal; Red arrows show no signal present in normal thyroid follicles. Blue arrows show papillary structure. (E–F) IHC analysis of p53 expression. (E) Strong p53 staining is seen in the nuclei of the anaplastic tumor cells but lack of p53 signal is seen in the neighboring papillary thyroid tumor cells (Specimen no. 8). (F) Negative p53 staining is seen in ATC Specimen no. 6. Normal mouse IgG was included as a negative control; no nonspecific staining was present (picture not shown).

Analysis of RAS Mutations

In vitro studies have demonstrated that the transformation potential of BRAF mutant is about 50-fold lower than that of RAS.22 Clinical studies suggest that BRAF mutation alone is not sufficient to transform melanocytes because BRAF is mutated in approximately 70% of nevi.23 It is possible that BRAF mutation may cooperate with other genetic alterations to induce thyroid carcinogenesis. All three RAS genes (NRAS, KRAS, HRAS) can be mutated in PTC,8 we, therefore, tested whether progression of ATC from PTC is due to double mutations of the BRAF and RAS oncogenes. RAS mutation at codons 12, 13, and 61 of RAS genes were analyzed by direct sequencing of PCR-amplified exons 1 and 2 of RAS genes. No mutation was detected at codons 12, 13, 61 of all 3 RAS genes in any of 8 ATCs. Interestingly, we found that HRAS was mutated at nucleotide position 31 by a transversion of G to T in ATC Specimen no. 4 (Fig. 1B), leading to the substitution of valine at codon 11 to serine. It is not clear whether HRAS mutation at this site results in the activation of Ras. Nevertheless, these observations are consistent with several other studies that show BRAF and RAS mutations to be mutually exclusive in melanomas, colon adenocarcinomas, and papillary thyroid carcinomas.15, 21

IHC Analysis of RET/PTC Rearrangements and p53 Mutation

IHC staining with an anti-RET antibody has been used by some investigators as a surrogate method to monitor RET/PTC rearrangements in thyroid neoplasms.9, 24–26 The RET gene is found to be rearranged in approximately 40% of PTCs. Our recent study using this experimental approach demonstrated that 33% of BRAF-mutated PTCs are RET/PTC positive.16 Here we tested whether BRAF mutation overlaps with RET/PTC rearrangements. As shown in Figures 2A, B, no RET/PTC expression was detected using anti-RET mAb in two ATC specimens (Specimen nos. 1 and 5). All 7 ATC specimens were negative for RET expression, which was also confirmed with an anti-RET rabbit antiserum (data not shown). A RET-rearranged PTC specimen was included as a positive control (Fig. 2C). Strong signal was present in the cytoplasm of papillary carcinoma cells but not in the neighboring normal follicular epithelial cells. Though these results suggest that RET/PTC is not rearranged in these ATCs, and that BRAF-mutated ATC is not due to the accumulation of BRAF mutation and RET/PTC arrangements, the data should be interpreted with caution because the rearranged RET/PTC could be silenced during the progression of PTC to ATC,27 and because the RET/PTC rearrangements detected by IHC staining and other methods such as RT-PCR and Southern blot have not been reproduced by all investigators.

Early studies have demonstrated that p53 is frequently mutated in ATC but rarely mutated in PTC and FTC. Wild-type p53 protein has a very short half-life and remains undetectable when analyzed by IHC staining, whereas mutated p53 protein can be readily detected in various p53-mutated malignancies because of its relatively long half-life. Therefore, IHC analysis with several well characterized specific antibodies has been routinely used to detect p53 mutations. Our group conducted IHC analysis using a commercially available, well defined mAb (clone DO-7) against p53. As shown in Figure 2E, an intense p53 signal was detected in nuclei of a BRAF-mutated, PTC-derived ATC.28 However, a p53 signal was not present in either neighboring papillary thyroid tumor cells (blue arrows) or normal follicular thyroid epithelial cells (not shown). Shown in Figure 2F is an ATC specimen with wild-type BRAF; no p53 signal was detected, which suggests that p53 is not mutated in this sample. Overall, we found that p53 was detected in 5 of 5 BRAF-mutated ATCs and in 2 of 3 ATCs with wild-type BRAF. In the p53-mutated ATC specimens, the p53 mutation was detected only in ATC cells but not in neighboring PTC cells. These observations confirm that p53 is frequently mutated in ATC but not in its PTC precursor. This indicates that development of ATC is due to further p53 mutation in the setting of an oncogenic mutation such as BRAF.

DISCUSSION

In the current study, we profiled several genetic alterations, including 3 oncogenes (RAS, BRAF, RET/PTC) and 1 tumor suppressor gene (p53) in 8 ATC specimens. We found that 5 of the 8 ATC specimens harbored BRAF mutation. Among them, 4 had structural components of papillary thyroid carcinomas, suggesting that this subset of ATC is derived from BRAF-mutated PTC. This observation is consistent with a recent study17 showing that BRAF is mutated in 3 of 5 anaplastic thyroid carcinomas with papillary components but not mutated in 24 anaplastic thyroid carcinomas without papillary components (Table 4). Because the numbers of the ATC specimens with a papillary component in our and Nikiforova's17 studies are relatively small, combination of the data from these two studies reveals that BRAF is mutated in 78% of 9 ATCs with papillary background (Table 4); however, BRAF is only mutated in 1 (4%) of 28 ATCs without PC background (Table 4). Thus, the frequency of BRAF mutation in ATC with papillary features (78%) is significantly higher than in PTC (approximately 40%). This observation suggests that the majority of ATCs with papillary components are derived from BRAF-mutated PTC, and this indirectly supports the notion that PTC with RET/PTC rearrangements do not progress to ATC.

Table 4. Comparison of BRAF Mutation in ATC in Earlier and Current Studies
ReferenceNo. casesBRAF mutation (%)
ATC with unknown background  
 Namba et al.1862 (33)
 Fukushima et al.1970
 Xing et al.12102
 Total234 (17)
ATC with papillary structure  
 Current study44
 Nikiforova et al.1753
 Total97 (78)
ATC without papillary structure  
 Current study41
 Nikiforova et al.17240
 Total281 (4)

The tumor suppressor gene, p53, is rarely mutated in well differentiated PTC and FTC but is mutated at a moderate rate in poorly differentiated PTC and at a very high rate in ATC. For example, Donghi et al.10 reported that p53 is mutated in 5 of 7 undifferentiated thyroid carcinomas; Fagin et al.11 reported that p53 is mutated in 5 of 6 ATCs. It has been proposed that with the addition of p53 mutation, well differentiated thyroid carcinomas progress to poorly differentiated or anaplastic thyroid cancer. Consistently, our present study using IHC analysis demonstrated that 7 of 8 ATCs were immunoreactive with an anti-p53 mAb, suggesting that p53 is mutated in these ATCs. These observations confirmed previous studies showing that p53 mutation is a frequent event in ATC but rarely occurs in PTC. Because p53 mutation was detected in 5 of 5 BRAF-mutated ATCs, it suggests that accumulation of p53 mutation in BRAF-mutated PTC contributes to the progression of PTC to ATC.

Whereas our current study demonstrated that simultaneous BRAF and p53 mutations constitute a subset of ATC derived from preexisting, well differentiated PTC, it did not provide direct evidence that p53 mutation leads to thyroid tumor cell dedifferentiation and development of ATC. Previous studies with attempt to define the role of p53 on the dedifferentiation of PTC have not reached a consensus conclusion. Wynford-Thomas29 reported that inactivation of p53 on well differentiated thyroid tumors resulted in the loss of some tissue markers but did not result in an anaplastic phenotype. Blagosklonny et al.30 reported that introduction of p53 into p53-mutant anaplastic thyroid tumor cell lines did not result in the redifferentiation of anaplastic thyroid tumor cell line. In contrast, Moretti et al.31 reported that introduction of p53 into ARO81 cells enabled these cells to reacquire the ability to respond to thyroid stimulating hormone. In a separate study, Fagin et al.32 reported that one of the p53-transfected papillary thyroid cell clones expressed thyroid peroxidase. It appears that p53 does not directly control the expression of thyroid differentiation markers; however, p53 mutation may cause thyroid tumor cell dedifferentiation by a secondary mechanism such as genomic instability.

To our knowledge, this is the first comprehensive study to profile multiple genetic alterations, including RAS, BRAF, RET/PTC, and p53 in ATC and to analyzed their relation to their pathologic behavior in tumors. We found that 4 of 5 BRAF-mutated ATCs in 8 specimens contained structural components of PTC, suggesting that BRAF-mutated PTC can advance to BRAF-mutated ATC. BRAF mutation was overlapping with p53 mutation but not with two oncogenes, RAS and RET/PTC, supporting the prevailing hypothesis that ATC progresses from differentiated thyroid carcinoma because of the accumulation of p53 mutation. The high rate of BRAF mutation in ATCs with papillary components is in sharp contrast to previous studies showing that RET/PTC-rearranged PTC does not progress to ATC.2, 9 This raises an intriguing question why PTC with RET/PTC rearrangements, which activate multiple downstream signaling pathways, including the RAS-RAF-MAPKK-MAPK pathway, do not progress to ATC; whereas PTC with BRAF mutation, which activates fewer signaling pathways than RET/PTC, can progress to ATC. The transformation potential of BRAF mutation and RET/PTC rearrangements are both lower than that of RAS mutation,22, 33 so it is not clear whether the quantitative and/or qualitative difference in BRAF- and RET/PTC-mediated signaling pathways leads to different clinical outcomes. Nevertheless, our study demonstrated that BRAF and p53 mutations are overlapping in a large number of ATCs, suggesting that BRAF can serve as a new molecular target for target-specific chemotherapy and, possibly, in combination with p53 gene therapy.

Ancillary