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BRAF mutations and RET/PTC rearrangements are alternative events in the etiopathogenesis of PTC

Oncogene volume 22, pages4578–4580(2003)Cite this article

Abstract

Rearrangement of RET proto-oncogene is the major event in the etiopathogenesis of papillary thyroid carcinoma (PTC). We report a high prevalence of BRAFV599E mutation in sporadic PTC and in PTC-derived cell lines. The BRAFV599E mutation was detected in 23 of 50 PTC (46%) and in three of four PTC-derived cell lines. The prevalence of the BRAFV599E mutation in PTC is the highest reported to date in human carcinomas, being only exceeded by melanoma. PTC with RET/PTC rearrangement as well as the TPC-1 cell line (the only one harboring RET/PTC rearrangement) did not show the BRAFV599E mutation. BRAFV599E mutation was not detected in any of 23 nodular goiters, 51 follicular adenomas and 18 follicular carcinomas. A distinct mutation in BRAF (codon K600E) was detected in a follicular adenoma. Activating mutations in RAS genes were detected in 15% of FA, 33% of FTC and 7% of PTC. BRAFV599E mutation did not coexist with alterations in any of the RAS genes in any of the tumors. These results suggest that BRAFV599E mutation is frequent in the etiopathogenesis of PTC. The BRAFV599E mutation appears to be an alternative event to RET/PTC rearrangement rather than to RAS mutations, which are rare in PTC. BRAFV599E may represent an alternative pathway to oncogenic MAPK activation in PTCs without RET/PTC activation.

Main

Thyroid gland presents a wide spectrum of tumors derived from follicular cells. Their behavior varies from the indolent growing, well-differentiated papillary and follicular carcinomas (PTC and FTC, respectively) to the extremely aggressive undifferentiated carcinoma (UC) (Rosai et al., 1992). It is assumed that distinct genetic events underlie PTC and FTC and that UC may develop from well-differentiated carcinomas through a multistep process of mutations and clonal expansion (Wynford-Thomas, 1997).

PTC is the most frequent type of carcinoma of the thyroid gland (Kovacs and Asa, 1991; Rosai et al., 1992) and the most frequent type of endocrine cancer. It is sporadic in about 95% of the cases and familial in the remaining 5%. Somatic rearrangements of the RET proto-oncogene have been detected in 3–60% of PTC sporadic tumors but are rarely found in familial cases (Nikiforov et al., 2002). PTC are usually diploid and microsatellite stable (Soares et al., 1997). Besides RET/PTC rearrangements, no other genetic event has been consistently related to PTC (Grieco et al., 1990).

FTC is far less frequent than PTC (Kovacs and Asa, 1991; Rosai et al., 1992). Rearrangements involving PAX-8 and PPARγ were reported by Kroll et al. (2000) as specific for FTC (63% of the cases). More recently, PAX8/PPARγ was detected by RT–PCR in 25–56% of FTC and in 0–13% of follicular adenomas (Marques et al., 2002; Nikiforova et al., 2002). Mutations of one of the three RAS genes have been reported in 18–52% of FTC and in 24–53% of follicular adenomas (Nikiforova et al., 2002).

BRAF is the first cancer-causing gene identified from the Cancer Genome Project, and in an extensive screen of 530 cancer cell lines, 43 harbored BRAF mutations (Davies et al., 2002). Most of the mutations were found to be in or adjacent to the activation segment of the kinase domain. The mutations increased the kinase activity of BRAF, resulting in activation of ERK. BRAF mutants were transfected into NIH-3T3 cells, and were shown to increase transformation efficiency by 70–138-fold when compared with wild-type BRAF (Davies et al., 2002; Pollock and Meltzer, 2002a,2002b). BRAF mutations have been reported in primary tumors, namely in a high proportion of melanomas and nevi, and, less frequently, in carcinomas (colon, lung, ovarian) (Naoki et al., 2002; Yuen et al., 2002; Pollock et al., 2003). The change from a valine to a glutamate at codon 599 is a hot spot, accounting for more than 80% of the mutations described in BRAF. In all, 5–10% of the colon cancers were shown to have BRAF mutation, but in the setting of the mismatch repair deficient tumors, the frequency rose to 30% of the tumors (Rajagopalan et al., 2002). Moreover, it was advanced that mutations in BRAF and RAS exert equivalent effects in tumorigenesis since they appear to occur as alternative events in colon cancer (Rajagopalan et al., 2002).

To determine the frequency of BRAF mutations in thyroid tumors and to verify if BRAF and RAS may also act as alternative events in thyroid tumorigenesis, we performed mutation analysis in exons 11 and 15 of BRAF and in exons 1 and 2 of N, H, and Ki-RAS in 142 thyroid tumors and tumor-like lesions (23 nodular goiters, 51 follicular adenomas-FA, 50 PTC and 18 FTC) and in four PTC-derived cell lines.

The 1796T-A mutation in exon15 of BRAF, leading to substitution of a valine for glutamate at position 599, was detected in 23 out of the 50 PTC (46%) and in none of the other thyroid lesions (P<0.0001) (Table 1, Figure 1). Only one case of follicular adenoma showed a mutation in exon 15 of BRAF but in a different codon (nucleotide 1798 A-G changing a lysine for a glutamate in codon 600). No alterations were found in exon 11 of BRAF. The BRAFV599E was detected in all types of PTC and no significant association was found between occurrence of the mutation and histopathologic features (data not shown).

Table 1 Frequency of BRAF mutation in thyroid tumors and tumor-like lesions
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Figure 1
figure1

(Left) SSCP pattern of wild-type DNA sample (N) and mutated DNA (T) samples (V599E); (right) sequencing pattern of the sample with aberrant band in the SSCP gel, showing a thymine-to-adenine transversion in nucleotide 1796 (arrow), resulting in a substitution of a valine for a glutamate. DNA was purified from frozen tumor tissue (n=76), microdissected tumor from paraffin blocks (n=66) and cell lines (n=4) using standard procedures. The complete coding sequence of exons 11 and 15 of BRAF, exon 2 of H- and N-RAS and exons 1 and 2 of Ki-RAS were amplified by polymerase chain reaction as described (Davies et al., 2002). PCR products were subjected to SSCP analysis: electrophoresis of samples of BRAF exons 11 and 15 and H-RAS exon 2 was performed in 0.8% MDE gels, while N-RAS exon 2 samples were separated in 12% polyacrylamide gels. Run conditions were at 180 V, for 16 h, at 8°C and the gels were silver stained. Samples presenting aberrant bands in the SSCP gels as well as Ki-RAS exons 1 and 2 were analysed for mutations by direct sequencing of the PCR products: each sample was subjected to an enzymatic purifying treatment and subjected to automatic sequencing. Sequencing was performed on both strands using the described primers (Davies et al., 2002). Microsatellite instability and RET/PTC rearrangements had been evaluated in previous works (Soares et al., 1997,1998)

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Seven of 39 PTC (18%) had RET/PTC rearrangement; none of these 7 PTC cases had the BRAFV599E mutation (P=0.03) (Table 1). Three of the four cell lines had the BRAFV599E mutation; the remaining cell line (TPC-1), negative for BRAFV599E, is the only one displaying an RET/PTC rearrangement (Table 1). The 16 cases presenting BRAFV599E mutation belonged to the group of PTC cases negative for RET/PTC rearrangement.

Activating mutations in RAS genes were detected in 15% of FA, 33% of FTC and 7% of PTC (Table 1). BRAFV599E mutation did not coexist with alterations in any of the RAS genes in any of the tumors.

The rare occurrence of microsatellite instability in PTC prevents us to predict the existence of any significant association between BRAFV599E mutation and MSI (Table 1).

The present study shows that the BRAFV599E mutation is a frequent event in papillary thyroid carcinogenesis. Excluding melanoma, PTC displays the highest frequency of BRAF mutation in human cancer. The mutation was detected in PTC, and not in any of the other benign and malignant follicular lesions, thus showing its histotype specificity. The only mutation different from BRAFV599E (K600E) occurred in a follicular lesion and had already been described in colorectal cancer (Brose et al., 2002; Rajagopalan et al., 2002). BRAFK600E mutation is also located in the BRAF activation segment and its functional effect is still unknown. In colon and lung cancer, it was observed that BRAF mutations other than BRAFV599E coexisted with RAS mutation (Davies et al., 2002). These observations suggest that different BRAF mutations can have distinct transforming potential in tumorigenesis.

We did not find any case with coexistence of RAS mutation and the BRAFV599E mutation. This finding fits with data obtained in other tumor models (Davies et al., 2002; Rajagopalan et al., 2002), but its significance is limited by the rarity of RAS mutations in PTC. Instead, our findings suggest that the BRAFV599E mutation appears to be, in PTC, an alternative to RET/PTC. Further studies are necessary to clarify if BRAFV599E and RET/PTC genetic events are equivalent in their tumorigenic effects as was hypothesized for BRAF and RAS mutations in colon cancer (Rajagopalan et al., 2002).

The signaling pathways of wild-type RET and its activated forms remain largely unknown. It was demonstrated that RET/PTC can signal through RAS/ERK pathway, but it is still unclear if it follows the classic MAPK signaling cascade (Ludwig et al., 2001; Castellone et al., 2003). Our data suggest that a signaling pathway involving BRAF can be activated in PTC opening a new perspective towards the genetic mechanisms and pathways underlying the etiopathogenesis of PTC.

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Acknowledgements

We thank the contribution that Carla Oliveira gave to this work. We are grateful to the ‘Fundação para a Ciência e Tecnologia’ for grant support to Vítor Trovisco, Ana Rocha, Jorge Lima, Patrícia Castro, Ana Preto and Tiago Botelho. This work was supported by Fundação para a Ciência e Tecnologia (FCT), project POCTI/CBO/38567/2001.

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  1. Paula Soares and Vítor Trovisco: These authors contributed equally to this work

Affiliations

  1. Institute of Molecular Pathology and Immunology of the University of Porto, (IPATIMUP), Rua Roberto Frias s/n, Porto, 4200-465, Portugal
    • Paula Soares
    • , Vítor Trovisco
    • , Ana Sofia Rocha
    • , Jorge Lima
    • , Patrícia Castro
    • , Ana Preto
    • , Valdemar Máximo
    • , Tiago Botelho
    • , Raquel Seruca
    •  & Manuel Sobrinho-Simões
  2. Department of Pathology, Medical Faculty of Porto, Porto, Portugal
    • Paula Soares
    •  & Manuel Sobrinho-Simões
  3. Department of Pathology, Hospital S. João, Porto, Portugal
    • Manuel Sobrinho-Simões
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Correspondence to Manuel Sobrinho-Simões.

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Soares, P., Trovisco, V., Rocha, A. et al. BRAF mutations and RET/PTC rearrangements are alternative events in the etiopathogenesis of PTC. Oncogene 22, 4578–4580 (2003). https://doi.org/10.1038/sj.onc.1206706

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Keywords

  • BRAF
  • RET/PTC
  • thyroid
  • papillary carcinoma

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