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Departments of Endocrine Neoplasia and Hormonal Disorders (L.S., G.J.C., S.I.S.), of Pathology (A.K.E.-N.), and of Head and Neck Surgery and Cancer Biology (J.N.M.), The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030-4009
Address all correspondence and requests for reprints to: Steven I. Sherman, Department of Endocrine Neoplasia and Hormonal Disorders, Unit 435, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030-4009. E-mail: sisherma{at}mdanderson.org.
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Abstract |
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Objective: Our objective was to explore the potential contributions of these two pathways in ATC pathogenesis.
Design, Setting, and Subjects: The mutational status of BRAF, PIK3CA, PTEN, and RAS genes was analyzed in genomic DNA from microdissected tumor specimens of 36 cases of ATC, and in 16 samples of paired-matched lymph node metastases. PIK3CA copy number gain was assessed by real-time quantitative PCR. We performed immunohistochemistry for phospho-ERK and phospho-AKT in 26 cases of ATC.
Results: DTC was present in half of the cases. BRAF V600E mutation was identified in nine of 36 (25%) ATCs; seven cases had identical mutations in both the ATC and DTC components. PIK3CA kinase domain mutations were found in five (14%) ATCs, one of which had mutations in both differentiated and anaplastic areas. RAS and PTEN mutations were each found in two (6%) ATCs. PIK3CA gain copy number was found notably increased in 14 (39%) ATCs.
Conclusions: BRAF mutations appear to play a role in the tumorigenesis of a subset of ATCs, and the majority of lymph node metastases. PIK3CA alterations occur preferentially in the later stages of ATC and were the most relevant events during thyroid cancer progression. The activation of both pathways suggests an important role in ATC dedifferentiation.
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Introduction |
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A role for abnormalities in Ras/Raf-MAPK signaling has been established in DTC tumorigenesis, but a similar role in ATC pathogenesis is less clear. RAS family oncogenes are important regulators of cell growth (8) and have a role in thyroid tumor differentiation (9). Downstream of RAS are multiple signal transduction pathways responsible for the execution of RAS-mediated cellular effects. The most widely validated ones that contribute to human tumorigenesis include the Raf-MAPK and phosphatidylinositol 3-kinase (PI3K)/Akt pathways (10).
The BRAF gene belongs to the RAF family of serine/threonine kinase, playing a central role in the regulation of cell growth, division, and proliferation (11). The gene has been variably mutated in PTC and ATC (12). Recent reports of the restriction of BRAF mutations to a subset of ATC derived from PTC did not agree on the real rate of BRAF mutation in this type of tumor, and previous studies have shown a variable mutation frequency in BRAF of 10, 62.5, and 20% (3, 4, 7).
Alterations in components of the PI3K/Akt pathway have been demonstrated in a variety of human cancers (13). The PIK3CA gene encoding the catalytic subunit p110
of class IA PI3K has acted as an oncogene because of its elevated kinase activity and frequent genomic amplification in cancer (13, 14). Common PIK3CA gene amplifications and mutations have been demonstrated in thyroid cancer (15, 16, 17, 18), providing evidence that aberrant activation of the PI3K/Akt pathway plays an important role in thyroid tumorigenesis, but the role of these genetic alterations in the progression from DTC to ATC is still unclear.
PTEN is a tumor suppressor gene that antagonizes signaling through the PI3K pathway. The function of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein is to dephosphorylate the phosphatidylinositol-3,4,5-triphosphate, which reduces the downstream activity of protein kinase B/Akt kinase, thereby inducing cell-cycle arrest, apoptosis, or both (19). PTEN has been mutated or deleted in different types of thyroid tumors (20), but the real incidence of these alterations and their role in ATC have not been well established.
The pathogenesis of ATC and its possible progression from preexisting DTC are subjects of debate. No published studies have simultaneously addressed the mutational activation of RAS, BRAF, PIK3CA, and PTEN, in the PI3K and RAS pathways in the malignant transformation of ATC. The purpose of this study was to evaluate how these events are involved in ATC and in dedifferentiation from DTC to ATC. Understanding the function and the interaction of these signaling pathways is crucial to comprehending the regulation of subsequent signaling events: cell metabolism, cell cycle progression, cell survival, and cell growth in thyroid cancer progression.
In this study we analyzed a series of 36 ATC cases, 50% of which showed coexisting DTC, for alterations in the PI3K and RAS pathways. We found evidence of different and increasing molecular alterations during thyroid cancer progression.
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Materials and Methods |
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ATC specimens paraffin embedded from 36 patients were obtained from the Department of Pathology at The University of Texas M. D. Anderson Cancer Center. The study protocol was approved by the institutional review board. The tumor tissue specimens were evaluated for histological features. Among the 36 ATCs, 18 coexisting DTCs (15 PTCs, two FTCs, and one Hürthle cell carcinoma) and four concomitant PDTCs were present. Moreover, 10 cases of PTC and 10 cases of FTC without an undifferentiated component were examined as controls.
Areas within the primary tumor that had different microscopic appearances with the same histological grade (primarily spindle cell/sarcomatoid, giant cell epithelioid, or mixed) or that were high grade and poorly differentiated were separately analyzed. Sixteen samples of matched lymph node metastases (LN) (11 from primary tumors containing both ATC and PTC and five from primary tumor containing ATC) and 10 noncontiguous foci of PTC within the ATC samples with coexisting PTC were also analyzed in this study.
The paraffin-embedded tumor sections were microdissected by laser capture in a way to ensure that the cancer cells represented at least 80% of the cells in each section. DNA was isolated from both undifferentiated and differentiated components of the tissue sections as well as from paired lymph nodes. A total of 84 tumor specimens for the ATC cases was analyzed: 36 undifferentiated components, 18 differentiated components, 16 lymph nodes, 10 additional foci of PTC, and four foci of PDTC. Samples of normal thyroid tissue from 10 of the cases were available and analyzed as well. All slides were reviewed according to established histological criteria (5).
Genomic DNA was isolated from 5-µm sections containing both thyroid tumor and normal thyroid tissue when available. The sections were dewaxed with two xylene washes (30 min at 55 C each), one 100% ethanol wash, two 70% ethanol washes, and two distilled water washes (6 min at 55 C for each step), and then incubated with proteinase K (20 mg/ml) and digestion buffer [100 mM NaCl/10 mM Tris-HCl (pH 8.0) 25 mM EDTA (pH 8.0), and 0.5% sodium dodecyl sulfate]. We analyzed the DNA sequences of the genes BRAF (exon 15), PIK3CA (exons 9 and 20), PTEN (exon 5–8), and H-, N-, and K-RAS (exons 1 and 2) in all 84 tumor specimens and the 10 paired normal thyroid tissue samples.
Mutational analysis for BRAF, PIK3CA, RAS, and PTEN genes
Genes were amplified by PCR, and direct sequenced with forward and reverse amplification primers. The BRAF gene was amplified with the primers 5'-TCATGAAGACCTCACAGTAAAAAT-3' (forward) and 5'-TGGATCCAAGACAACTGTTCAA-3' (reverse). PCR was performed in a 25-µl mixture containing 20 pmol each primer, 200 µM each deoxynucleotide triphosphate, 0.5 U Taq DNA polymerase, and 30- to 50-ng genomic DNA. PCR conditions consisted of the initial denaturation (95 C for 5 min), followed by 36 cycles of denaturation at 94 C for 30 sec, annealing at 54 C for 60 sec, and extension at 72 C for 30 sec. For PIK3CA, we restricted mutation analyses to the helical and kinase domain "hot spots," in which 80–90% of mutations are reported to cluster. The PIK3CA gene was amplified for exon 9 as previously reported (16) and for exon 20 with the primers 5'-TGACATTTGAGCAAAGACCTG-3' (forward) and 5'-ATCAAACCCTGTTTGCGTTT-3' (reverse). PCR was performed in a total volume of 25 µl containing 20 pmol each primer pair, 50- to 60-ng genomic DNA, 200 µM deoxynucleotide triphosphates, 50 mM PCR buffer containing 2.0 mM MgCl2, and 0.5 U Taq DNA polymerase. The PCR consisted of the initial denaturation step of 8 min at 95 C, followed by 36 cycles of 1 min at 94 C, 30 sec at 55 C, and 30 sec at 72 C. The PTEN gene was amplified for exons 5–8, where most identified mutations are reported to cluster (17, 18). For the three RAS genes, we used direct sequencing after PCR amplification of exons 1 and 2, as previously described (21). All PCR products were run onto 2% agarose gel to establish the quality of the PCR reaction and purified using a DNA clean-up kit (Promega, Madison, WI).
Copy number analysis of PIK3CA by real-time quantitative PCR
Genomic DNA was assessed for copy number gain of PIK3CA in all 36 ATCs, in the concurrent DTC when present, and in three ATC cell lines (ARO, KAT4, and C643).
Real-time PCR was run on the StepOne system (Applied Biosystems, Foster City, CA), and the reactions were all performed in triplicate using: forward primer, 5'-AGTGAATTGAATTCTGTCCCTTCCAA-3'; primer reverse, 5'-CGATTTCTGGATAGTGGGCAATAAGG-3'; and probe, 5'-FAM-GAATTTACCAGTGGAT-NFQ-3'. Ribonuclease P (Applied Biosystems) was used as a reference control. Data were analyzed by the 
Ct method.
Immunohistochemical staining for phospho-ERK p44/42 and phospho-AKT
To examine the activation status of MAPK/Erk1/2 and Akt, 4-µm paraffin-embedded sections from 26 cases of ATC, including DTC when present, were immunostained with antibodies to phospho-p44/42 MAPK (Thr202/Tyr204) (dilution 1:200) and phospho-Akt (Ser473) (dilution 1:100) (Cell Signaling Technology, Beverly, MA). After deparaffinization and rehydration, sections were incubated at 4 C overnight with the antibodies. Staining was performed using the Vectastain Universal Quick kit (Vector Laboratories, Burlingame, CA). The peroxidase reaction was developed with 3,3'-diaminobenzidine, and the slides were counterstained with hematoxylin.
Immunoreactivity was expressed as the percentage of positive-stained cells in different categories: –, staining; +, low/weak; 2+, moderate/distinct; and 2++, high/intense. The immunohistostaining score for the intensity of the staining was assigned in a blind fashion without knowing the mutational status of the genes.
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Results |
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and 2).
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). The remaining two BRAF-mutated ATC cases did not contain any papillary architecture, although a focus of PTC was noted in a lymph node in one case (case 18). The BRAF mutations were not uniformly identified within different PTC foci with similar morphological features. Among 10 noncontiguous foci of PTC present within the ATC tumors, four had the BRAF mutation, whereas the remaining six had wild-type BRAF, suggesting different clonal cell origins within the same tumor.
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). The analysis of phenotypically different areas with the same histological grade within the same tumor showed that the PIK3CA mutations were heterogeneously distributed within the specimen, illustrating the intratumor heterogeneity of the ATC.
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Only two (6%) cases of K-RAS mutations were found. One mutation was the transversion mutation GGT>GAT at codon 12 (Gly>Asp) within an ATC, including the coexistent PDTC. The mutation GGC>GAC at codon 13 (Gly>Asp) was found in another tumor only within the undifferentiated component.
Finally, two cases (cases 7 and 15) harbored both BRAF and PIK3CA mutations (Fig. 3), and one case harbored both PIK3CA and RAS mutations (case 14) (Table 2
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). Mutations of both BRAF and PIK3CA were found in one lymph node (case 15). Among the 20 cases of DTC with no coexisting undifferentiated components, the BRAF V600E mutation was found in four cases of PTC (40% of PTC cases); no mutations were found in FTC. No mutations in the PIK3CA or PTEN genes were found in both PTC and FTC.
PIK3CA gains (with a gene copy number of three or more) were found in 22 (61%) ATC samples. PIK3CA gene copies were notably increased (four copies or more) in 14 (39%) ATCs. Four of five (80%) PIK3CA mutated cases also had an increase in gene copy number (Table 2). Among the DTC within the ATC, only two of 18 (11%) showed moderate gene copy gain. Two ATC cell lines, ARO and KAT4, showed a high level of PIK3CA amplification, whether C643 did not show any significant gene copy gain.
Activation of AKT (phosphorylation) was detected in 22 of 26 (85%) ATCs. High levels of phosphorylated AKT (p-AKT) were found in five (23%) ATCs, moderate staining in 12 (54%) ATCs, and low levels in five (23%) ATCs. In all positive cases, the activation of AKT was demonstrated in the cytoplasm. In 17 (77%) ATC (and in one PTC) samples, nuclear p-AKT was noted in conjunction with cytoplasmic staining (Fig. 4). Only cytoplasmatic localization of p-AKT was observed in DTC.
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Discussion |
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In our study, when DTC coexisted with ATC, the only mutations that overlapped were BRAF and PIK3CA. Increasing of any genetic mutations became more common as the dedifferentiation progressed. We found the BRAF V600E mutation mainly in the anaplastic area and in the accompanying differentiated areas (PTC) within the same tumor specimen. The transversion T1799A, leading to a Glu substitution for Val at codon 600 (V600E), accounts for approximately 90% of BRAF mutations in thyroid carcinomas, and it has been found to be mutated in approximately 44% of PTC cases and 24% of ATC cases (11). The BRAF V600E mutation appears to play a pivotal role in thyroid cancer initiation because it has been mutated in microcarcinomas (11), and in vitro and in vivo models have demonstrated that overexpression of activated BRAF induces malignant transformation and aggressive tumor behavior (22, 23, 24). We have confirmed that some cases of ATC can occur from anaplastic transformation of DTC. Our data support findings from prior studies (2, 3, 4).
However, we found that two of the nine (22%) cases of ATC with BRAF mutations had no apparent coexisting differentiated component, which was probably due to the loss of papillary or any follicular-epithelial architecture or to the DTC component being completely obscured by the extensive anaplastic tumor. Of note, one of these tumors was mutated for BRAF in the lymph node (case 18). When they were present, other distinct noncontiguous foci of PTC within the same primary tumor were analyzed, and we found that BRAF mutations were restricted to only some of the foci, supporting the idea that PTC multifocality is due to different cell clone origins instead of spreading from the same tumor. Our finding that 19% of ATC cases had BRAF mutations in both the differentiated and undifferentiated areas of thyroid tumor specimens suggests a role for oncogenic BRAF in disease progression as well. However, the fact that BRAF mutations were found in both components of ATC and in control PTC suggests that these mutations are most likely an early event during dedifferentiation, and that these events are probably insufficient by themselves to initiate anaplastic dedifferentiation but may predispose the tumor cells to gain additional genetic alterations that activate other pathways and lead to dedifferentiation. Although, BRAF V600E mutation was the leading event in LN (four of 16, 25%) (Table 2).
We found that the PIK3CA gene was mutated in 14% of the ATC cases and was restricted mainly to the undifferentiated thyroid component. We confirmed the preferential role of PIK3CA mutation in the later stages of thyroid cancer (17), suggesting an important role of this event in ATC rather than DTC.
The importance of the PIK3CA mutation in thyroid cancer might be its ability to activate other genetic markers, such as p53, which was recently activated in a subset of ATC cases derived from PTC (4), and PIK3 signaling could be an important endogenous inducer of p53 in thyroid cancer transformation.
Copy gains of PIK3CA gene seem to be the most relevant event in ATC. Our results show that a change in the gene copy number of PIK3CA occurs almost exclusively in the undifferentiated component compared with paired DTC within the same tumor. This suggests that the activation of the PI3K-Akt pathway through PIK3CA copy gain contributes to thyroid tumor progression in ATC. Moreover, our data provide evidence that gain of gene copy number and somatic mutations may exist in parallel in ATC, most likely reinforcing the activation of the PI3K-Akt pathway.
Increased PI3K expression results in the enhanced activity of AKT, which is a marker downstream of PI3K that, once activated, regulates apoptosis, proliferation, and migration in thyroid cells (25) contributing to thyroid cancer progression (26, 27).
A PTEN mutation was present in the 6% of our ATC series. The role of PTEN in tumor suppression has been linked to the down-regulation of the PI3K/Akt pathway by PTEN's lipid phosphatase activity (19). The low prevalence of PTEN mutations in our study may be due to the fact that our series included only two cases of ATC with coexisting FTC. No overlapping mutations between PTEN and PIK3CA were found, suggesting that only one of these mutations is sufficient to promote tumorigenesis and that each event may play a distinct role in thyroid tumorigenesis. PTEN is often lost or down-regulated in ATC, leading to AKT activation, in the same way gain of function mutations in PIK3CA may induce the activation of PI3K with effects on cell proliferation.
We noted a low frequency of RAS mutations in our cases (6%) (one case of ATC with coexisting PDTC, and in one case of ATC only), both in K-RAS. Constitutive activation of H-, K-, and N-RAS mutations has been reported as markers for aggressive thyroid cancer behavior (28).
In a survey study of RAS mutations involving 39 reports and 269 mutations, N-RAS (exon 1) and K-RAS (exon 2) had a mutation frequency less than 1%, and H-RAS mutation (codons 12 and 13) was 2–3% of all thyroid tumors types (18). From this report also emerge the importance of RAS in FTC and the concept that mutation frequency in RAS isoforms is tumor phenotype related, supporting the idea that most likely, the activity of specific RAS oncogenes in thyroid is histotype dependent, and there is a possibility that K-RAS could be more important in PTC-ATC phenotypes.
Our finding of both RAS and PIK3CA mutations in the same tumor specimen implies that RAS mutations can occur coincident with PIK3CA mutations and reinforces the concept that cross talk between the two signaling pathways exists.
An important finding was that the activation of ERK and AKT was noted at higher frequency in ATC, and that sometimes activation of these pathways occurred also in the absence of PI3K or RAS mutations. Furthermore, the presence of mutation or amplification was not correlated with higher staining intensity. These results imply that activation of these pathways play an important role in ATC progression and also may be a target through other mechanisms, e.g. epidermal growth factor or fibroblast growth factor.
The increasing of any genetic alterations in the Ras/Raf-MAPK and PI3K/Akt pathways was noted almost exclusively in the undifferentiated tumor components. Thus, even though only one pathway is needed to initiate thyroid cancer pathogenesis, both pathways seem to be involved during thyroid cancer dedifferentiation. Although we reported a high prevalence (42%) of genetic mutations in ATC, this is likely an underestimate because not all the coding regions of the genes were examined, and other important genes such as p53 were not analyzed.
On the basis of all these considerations and the results of the experimental assays, we conclude that the Ras and PIK3 signal transduction pathways play important roles in thyroid tumor initiation and progression. Cross talk between the PI3K and RAS signaling pathways may occur at several stages, and both pathways are implicated in the anaplastic transformation of thyroid cancer. Elucidating how these pathways interact and clarifying the genetic background of the thyroid tumor components will lead to an understanding of thyroid carcinogenesis and, perhaps, the development of therapeutic strategies based on targeting both pathways.
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Acknowledgments |
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Footnotes |
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Abbreviations: ATC, Anaplastic thyroid carcinoma; DTC, differentiated thyroid carcinoma; FTC, follicular thyroid carcinoma; LN, lymph node metastases; p-AKT, phosphorylated AKT; PDTC, poorly differentiated thyroid carcinoma; p-ERK, phosphorylated ERK; PI3K, phosphatidylinositol 3-kinase; PTC, papillary thyroid carcinoma; PTEN, phosphatase and tensin homolog deleted on chromosome 10.
Disclosure Statement: The authors have nothing to disclose.
Received May 14, 2007.
Accepted October 26, 2007.
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