Abstract
Context:
Medullary thyroid carcinoma (MTC) is characterized by proto-oncogene RET mutations in almost all hereditary cases as well as in more than 40% of sporadic cases. Recently, a high prevalence of RAS mutations was reported in sporadic MTC, suggesting an alternative genetic event in sporadic MTC tumorigenesis.
Objective:
This study aimed to extend this observation by screening somatic mutational status of RET, BRAF, and the three RAS proto-oncogenes in a large series of patients with MTC.
Materials and Methods:
Direct sequencing of RET (exons 8, 10, 11, 13, 14, 15, 16), BRAF (exons 11 and 15), and KRAS, HRAS, and NRAS genes (exons 2, 3, and 4) was performed on DNA prepared from 50 MTC samples, including 30 sporadic cases.
Results:
Activating RET mutations were detected in the 20 hereditary cases (germline mutations) and in 14 sporadic cases (somatic mutations). Among the 16 sporadic MTC without any RET mutation, eight H-RAS mutations and five K-RAS mutations were found. Interestingly, nine RAS mutations correspond to mutation hot spots in exons 2 and 3, but the other four mutations were detected in exon 4. The RET and RAS mutations were mutually exclusive. No RAS gene mutation was found in hereditary MTC, and no BRAF or NRAS mutation was observed in any of the 50 samples.
Conclusions:
Our study confirms that RAS mutations are frequent events in sporadic MTC. Moreover, we showed that RAS mutation analysis should not be limited to the classical mutational hot spots of RAS genes and should include analysis of exon 4.
Medullary thyroid carcinoma (MTC) is a rare tumor, accounting for less than 5% of all thyroid cancers, that arises from neuroendocrine cells secreting calcitonin called “C cells” or parafollicular cells. MTC appears in a sporadic context in 70% of cases or in an inherited context in the other 30% of cases. Activating mutations of the RET (REarranged during Transfection) (1) proto-oncogene are identified in almost all familial cases and in about 40% of sporadic forms. Therefore, nearly 45% of cases are not associated with an oncogenic RET mutation (2). RET tyrosine kinase receptor is involved in the regulation of differentiation, proliferation, survival, and cell motility processes through several intracellular signaling pathways, including MAPK and PI3K/AKT/mTOR pathways.
Oncogenic mutations in the RAS proto-oncogenes are frequently detected in follicular thyroid tumors (3, 4). For years, mutations in RAS or BRAF genes were considered absent in MTC (5–8). However, mutations in HRAS and KRAS genes were recently found in a significant proportion of non-RET-mutated MTC (9, 10), suggesting that RAS mutations could represent alternative genetic events in sporadic MTC tumorigenesis. This observation could be of value for defining therapeutic strategies in non-RET-mutated tumors. Indeed, a lack of response to tyrosine kinase inhibitors (TKI) of the epidermal growth factor receptor has been reported in colorectal or lung tumors presenting a RAS mutation (11).
Thus, the aim of this study was to analyze the frequency of RET, RAS, and BRAF mutations in a large series of familial and sporadic MTC tumors. Mutational status of HRAS, KRAS, and NRAS genes was examined not only on classical hot spot codons 12, 13, and 61 (located in exons 2 and 3), but also on codons 117, 146, and 147 in exon 4 of the three RAS genes. Interestingly, we found that mutations are also present in this particular region that has never been investigated.
Patients and Methods
Patients
A total of 50 tumoral tissues were collected at the Institut Gustave Roussy (Villejuif, France) and stored in the tumor biobank according to local ethics recommendations (Table 1). These tissues correspond to 42 frozen samples and eight formalin-fixed, paraffin-embedded samples, including 37 primary tumors and 13 metastasis. There were 26 females and 24 males, with a mean age at diagnosis of 43 ± 5 yr; 20 were inherited MTC (seven MEN2A, three MEN2B, and 10 FMTC), and 30 were sporadic cases without any familial history and without any detected germline RET mutation. A germline RET mutation was found for all MTC cases occurring in a family context. Among the other cases, 26 showed no germline RET mutation in the usually tested loci (exons 8, 10, 11, 13, 14, 15, and 16). Only four individuals (patients 22, 23, 35, and 37) were not screened for all exons due to a lack of blood sample, but these cases revealed no clinical clues in favor of a familial origin of the tumor. Before extraction, all samples were hematoxylin-eosin stained, and immunochemistry was performed using anti-calcitonin antibodies. Histological control was achieved by a pathologist, and all selected samples contained more than 50% of tumor cells.
Table 1.
Mutational status of 50 MTC samples analyzed in this study
| No. | MTC tissue type | Transmission | Sex | Age at diagnosis (yr) | RET mutational status | KRAS, HRAS, NRAS, and BRAF mutational status | |||
|---|---|---|---|---|---|---|---|---|---|
| 1 | Lymph node | Frozen | Familial | F | 38 | p.Cys634Arg | Germline | WT | |
| 2 | Lymph node | Frozen | Familial | M | 48 | p.Cys634Tyr | Germline | WT | |
| 3 | Primary tumor | Frozen | Familial | F | 4 | p.Cys634Ser | Germline | WT | |
| 4 | Primary tumor | Frozen | Familial | F | 49 | p.Lys666Asn | Germline | WT | |
| 5 | Primary tumor | Frozen | Familial | F | 53 | p.Cys531Arg | Germline | WT | |
| 6 | Primary tumor | Frozen | Familial | F | 73 | p.Leu790Phe | Germline | WT | |
| 7 | Primary tumor | Frozen | Familial | M | 8 | p.Cys634Ser | Germline | WT | |
| 8 | Primary tumor | Frozen | Familial | M | 10 | p.Cys634Arg | Germline | WT | |
| 9 | Primary tumor | Frozen | Familial | M | 39 | p.Cys620Tyr | Germline | WT | |
| 10 | Primary tumor | Frozen | Familial | M | p.Ser891Ala | Germline | WT | ||
| 11 | Primary tumor | Frozen | Familial | F | 18 | p.Cys634Ser | Germline | WT | |
| 12 | Primary tumor | Frozen | Familial | F | 24 | p.Cys634Tyr | Germline | WT | |
| 13 | Primary tumor | Frozen | Familial | F | 32 | p.Cys634Arg | Germline | WT | |
| 14 | Primary tumor | Frozen | Familial | F | 59 | p.Cys618Ser | Germline | WT | |
| 15 | Primary tumor | Frozen | Familial | F | 11 | p.Met918Thr | Germline | WT | |
| 16 | Primary tumor | Frozen | Familial | F | 25 | p.Met918Thr | Germline | WT | |
| 17 | Primary tumor | Frozen | Familial | M | 8 | p.Met918Thr | Germline | WT | |
| 18 | Lymph node | FFPE | Familial | F | 35 | p.Cys634Arg | Germline | WT | |
| 19 | Lymph node | FFPE | Familial | F | 66 | p.Gln780Arg | Germline | WT | |
| 20 | Lymph node | FFPE | Familial | M | 29 | p.Cys634Arg | Germline | WT | |
| 21 | Lymph node | Frozen | Sporadic | F | 30 | WT | HRAS c.182A>G | p.Gln61Arg | |
| 22 | Lymph node | Frozen | Sporadic | F | 45 | WT | HRAS c.182A>G | p.Gln61Arg | |
| 23 | Lymph node | Frozen | Sporadic | M | 46 | p.Met918Thr | Somatic | WT | |
| 24 | Lymph node | Frozen | Sporadic | M | 61 | WT | KRAS c.35G>T | p.Gly12Val | |
| 25 | Primary tumor | Frozen | Sporadic | F | 37 | WT | HRAS c.181C>M | p.Gln61Lys | |
| 26 | Primary tumor | Frozen | Sporadic | F | 47 | WT | HRAS c.37G>C | p.Gly13Arg | |
| 27 | Primary tumor | Frozen | Sporadic | F | 49 | p.Cys634Tyr | Somatic | WT | |
| 28 | Primary tumor | Frozen | Sporadic | F | 49 | p.Met918Thr | Somatic | WT | |
| 29 | Primary tumor | Frozen | Sporadic | F | 51 | WT | HRAS c.181C>M | p.Gln61Lys | |
| 30 | Primary tumor | Frozen | Sporadic | F | 55 | WT | KRAS c.437C>T | p.Ala146Val | |
| 31 | Primary tumor | Frozen | Sporadic | F | 56 | WT | KRAS c.437C>T | p.Ala146Val | |
| 32 | Primary tumor | Frozen | Sporadic | F | 63 | WT | HRAS c.182A>G | p.Gln61Arg | |
| 33 | Primary tumor | Frozen | Sporadic | F | 73 | WT | WT | ||
| 34 | Primary tumor | Frozen | Sporadic | M | 21 | p.Met918Thr | Somatic | WT | |
| 35 | Primary tumor | Frozen | Sporadic | M | 27 | p.Met918Thr | Somatic | WT | |
| 36 | Primary tumor | Frozen | Sporadic | M | 34 | WT | KRAS c.34G>C | p.Gly12Arg | |
| 37 | Primary tumor | Frozen | Sporadic | M | 45 | p.Met918Thr | Somatic | WT | |
| 38 | Primary tumor | Frozen | Sporadic | M | 48 | p.Cys634dela | Somatic | WT | |
| 39 | Primary tumor | Frozen | Sporadic | M | 48 | p.Met918Thr | Somatic | WT | |
| 40 | Primary tumor | Frozen | Sporadic | M | 50 | p.Met918Thr | Somatic | WT | |
| 41 | Primary tumor | Frozen | Sporadic | M | 53 | WT | WT | ||
| 42 | Primary tumor | Frozen | Sporadic | M | 53 | p.Met918Thr | Somatic | WT | |
| 43 | Primary tumor | Frozen | Sporadic | M | 54 | p.cys634insb | Somatic | WT | |
| 44 | Primary tumor | Frozen | Sporadic | M | 69 | WT | KRAS c.187G>A | p.Glu63Lys | |
| 45 | Primary tumor | Frozen | Sporadic | M | 74 | p.Ala883Phe | Somatic | WT | |
| 46 | Lymph node | FFPE | Sporadic | F | 42 | WT | HRAS c.351G>T | p.Lys117Asn | |
| 47 | Lymph node | FFPE | Sporadic | M | 34 | p.Met918Thr | Somatic | WT | |
| 48 | Lymph node | FFPE | Sporadic | M | 46 | WT | WT | ||
| 49 | Lymph node | FFPE | Sporadic | M | 51 | p.Cys634dela | Somatic | WT | |
| 50 | Primary tumor | FFPE | Sporadic | F | 47 | WT | HRAS c.182A>G | p.Gln61Arg | |
Bold, Samples wild-type for all tested genes. WT, Wild-type; FFPE, formalin-fixed, paraffin-embedded; F, female; M, male.
p.Glu632_Ile638del.
p.Cys634_Arg635insProLys.
DNA isolation
DNA was extracted from 20-μm-section tumoral specimens after an overnight digestion by proteinase K, using the DNeasy Tissue Kit and the QIAcube automated extractor (QIAGEN, Hilden, Germany), according to the manufacturer's protocol. Yield and quality of DNA were assessed by Qubit fluorometer (Invitrogen, Carlsbad, CA).
Mutational analysis
Exons 8, 10, 11, 13, 14, 15, and 16 of RET; exons 11 and 15 of BRAF; and exons 2, 3, and 4 of KRAS, HRAS, and NRAS genes were analyzed by direct Sanger's sequencing after a specific amplification by PCR as previously described (12). Briefly, PCR were carried out on 20 ng of DNA in 10 μl final volume and 1 U of Hot Start Taq polymerase (QIAGEN). PCR primer sequences are available on demand. The amplified products were analyzed by direct sequencing after clean-up exonuclease ExoSAP-IT (Affymetrix, Santa Clara, CA) using the Big Dye Terminator Cycle Sequencing Kit and capillary electrophoresis on the automated sequencer ABI3730 (Applied Biosystems, Carlsbad, CA). Sense and antisense sequences were screened for exonic alterations using SeqScape v2.5 software (Applied Biosystems) and compared with the NCBI reference sequences: RET (NM_020975.4), KRAS (NM_033360.2), HRAS (NM_005343.2), NRAS (NM_002524.3), and BRAF (NM_004333.4). All mutations identified were further confirmed by independent PCR amplification.
Results
Mutational screening of exons 2, 3, and 4 of RAS genes and exons 11 and 15 of BRAF was performed on the 50 MTC samples. All familial cases (n = 20) showed an activating germline mutation of RET: 10 in exon 11 (MEN2A), three in exon 16 (MEN2B), and seven in other exons (FMTC). Among the sporadic cases (n = 30), 14 carried a somatic RET mutation—four at codon 634, one at codon 883, and nine at codon 918. No tumor presenting a RET mutation was found with any RAS or BRAF mutation. Details of all detected mutations are reported in Table 1.
A RAS mutation was found in 13 of 16 RET-negative MTC samples (81%) and in 13 of 50 tumors, considering the entire collection (26%). Only three tumors appear to be wild-type for RET, RAS, and BRAF genes (6% of the collection). KRAS mutations were identified in five RET-negative sporadic tumors (10% of the samples and 31% of RET wild-type tumors) and were located at codon 12 (n = 2, exon 2), at codon 63 (n = 1, exon 3), and at codon 146 (n = 2, exon 4). Mutations in the coding sequence of HRAS were found in eight tumors (16% of the samples and 50% of RET wild-type tumors): one at codon 13 (exon 2), six at codon 61 (exon 3), and one at codon 117 (exon 4). No mutation was found in NRAS and in BRAF genes in any tumor sample.
Discussion
Somatic mutations in genes involved in the Ras/Raf kinase pathway are common in several types of tumors, including differentiated thyroid cancers. Hitherto, with the exception of two controversial studies (5–13), it was admitted that RAS and RAF mutations were not involved in MTC oncogenesis. However, recent studies reported HRAS and KRAS mutations in two independent MTC series (9, 10) (Table 2), and among 47 MTC, an HRAS mutation was found in two and a KRAS mutation in one other patient (14).
Table 2.
Previous studies of RAS and BRAF genes in various collections of MTC
| First author, year (Ref.) | Gene(s) | Locus studied | Method | n | Mutated samples |
|---|---|---|---|---|---|
| Okazaki, 1989 (5) | HRAS | Codon 61 | NA | 18 | 1/18 |
| Yang, 1990 (20) | NRAS | NA | Southern blot | 7 | 0/7 |
| Horie, 1995 (3) | HRAS, KRAS, NRAS | Exon 2 | Direct sequencing | 9 | 0/9, 0/9, 0/9 |
| Fenton, 1999 (21) | HRAS, KRAS, NRAS | Codons 12, 13, 61 | Specific hybridization | 2 | 0/2, 0/2, 0/2 |
| Bockhorn, 2000 (6) | HRAS, KRAS | Codons 12, 13, 61 | Direct sequencing | 15 | 0/15, 0/15 |
| Nikiforova, 2003 (7) | BRAF | Exon 15 | Direct sequencing | 13 | 0/13 |
| Xing, 2004 (4) | BRAF | Exon 15 | Direct sequencing | 14 | 0/14 |
| Perren, 2004 (8) | BRAF | Exons 11, 15 | Direct sequencing | 25 | 0/25 |
| Goutas, 2008 (13) | KRAS | Codon 12 | PCR-RFLP | 44 | 18/44 |
| Goutas, 2008 (13) | BRAF | Codon 600 | PCR-RFLP | 44 | 30/44 |
| Ameur, 2009 (12) | BRAF | Exon 15 | Direct sequencing | 25 | 0/25 |
| Schulten, 2001 (10) | HRAS, KRAS | Exons 2, 3 | Direct sequencing | 13 | 1/13, 0/13 |
| Schulten, 2001 (10) | NRAS, BRAF | Exon 15 | Direct sequencing | 13 | 0/13, 0/13 |
| Moura, 2011 (9) | HRAS, KRAS | Exons 2, 3 | Direct sequencing | 65 | 15/65, 3/65 |
| Moura, 2011 (9) | NRAS, BRAF | Exon 11, 15 | Direct sequencing | 65 | 0/65, 0/65 |
| Current study | HRAS, KRAS | Exons 2, 3, 4 | Direct sequencing | 50 | 8/50, 5/50 |
| Current study | NRAS, BRAF | Exons 11, 15 | Direct sequencing | 50 | 0/50, 0/50 |
In the present study, analysis of HRAS, KRAS, NRAS, and BRAF coding sequences was performed in a series of 50 tumors, including familial and sporadic forms. In our collection, mutations in RET and RAS genes seem to be mutually exclusive. In fact, these redundant genetic events (Ras protein signal occurs downstream of the RET receptor) do not appear to confer additional oncogenic benefit to parafollicular cells, in contrast to what has been described in other cancers such as colorectal cancer. Among the 16 RET-negative tumors, we observed 13 RAS mutations. Nine mutations correspond to mutation hot spots known in other types of tumors described in the COSMIC database (15), namely at exon 2 (codons 12 and 13) and exon 3 (codon 61) of HRAS and KRAS genes. However, four RAS mutations were rare but known mutations, including mutations at exon 3 of KRAS (n = 1, codon 63) and at exon 4 of HRAS (n = 1, codon 117) and KRAS (n = 2, codon 146). The detection of these mutations in exon 4 could be related to the slightly higher proportion of RAS positive-RET negative sporadic MTC identified in our study (81%) compared with the Moura et al. (9) study (78%). These mutations have been previously described only in digestive cancers and leukemias, and the authors suggest that RAS mutational status should be assessed beyond the most frequently mutated codons (16–18). To our knowledge, these exon 4 mutations have not yet been reported in thyroid cancers. This finding highlights the importance of sequencing MTC not only for the classical hot spots but also for these additional exons. This high frequency of RAS mutations is in total agreement with the previous reports (9, 10), and the paradigm in which the RAS mutations were present in thyroid cancers of follicular origin but absent from medullary carcinomas (6–8) cannot be retained any more. In accordance with previous studies, point mutations in NRAS and BRAF genes do not appear to be involved in MTC oncogenesis.
To date, no Ras-targeted therapies have been successful. However, the presence of such mutations may be an interesting marker for drug management, including TKI. Indeed, they are associated with a lack of efficacy of epidermal growth factor receptor inhibitors in patients with a colorectal or lung cancer (11). Conversely, in a phase III trial using vandetanib (ZD6474) in metastatic MTC, efficacy was observed in patients carrying a RET p.Met918Thr mutation, but also in patients in whom the search for RET mutation was negative or incomplete. However, no RAS analysis was performed, and no conclusion can be made regarding the impact of RAS mutation in non-RET-mutated sporadic tumors (19). Thus, the screening for RAS mutations in tumors should be performed for the assessment of the effectiveness of RET-specific TKI for the treatment of MTC.
In conclusion, we confirm that an activating mutation of known oncogenes is present in 94% of our series (47 of 50 tumors). In sporadic MTC, almost half of the detected mutations are located in HRAS or KRAS genes. Moreover, we showed that RAS mutations are not limited to classical mutational hot spots of RAS genes and suggest that analysis should include exon 4 sequencing. To date, there are no data establishing a link between the presence of these abnormalities and efficacy of TKI drugs in MTC, but this point should be considered for future drug development.
Acknowledgments
The authors dedicate the manuscript in memory of their colleague, Dr. Bernard Caillou.
This work was supported by grants from the Institut Gustave Roussy. A.B. is a recipient a doctoral grant from the Association pour la Recherche sur le Cancer (allocation doctorale ARC–2011/2012).
Disclosure Summary: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Footnotes
- MTC
- Medullary thyroid carcinoma
- TKI
- tyrosine kinase inhibitor.
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