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Low Frequency of BRAF Mutations in Endometrial and in Cervical Carcinomas

Laboratory of Breast and Gynaecological Cancer, Molecular Pathology Programme, Centro Nacional de Investigaciones Oncológicas, Madrid, Spain

David Hardisson

Department of Pathology, Hospital Universitario La Paz, Madrid, Spain

To the Editor: BRAF, which encodes a RAF family member that functions downstream of RAS, has been reported to be somatically mutated in a number of human cancers, most frequently at codon 599 (1). Activating mutations of BRAF have been frequently observed in colorectal carcinomas with microsatellite instability (MSI; ref. 2). In addition, mutations of BRAF and KRAS have been reported as being mutually exclusive in colorectal carcinomas (2). Mutations in BRAF have been studied in gynecological cancer. They are common in low-grade ovarian serous carcinoma, in which they provide an alternative route for activation of the RAS signaling pathway (3). In contrast, BRAF mutations have not been found in either other histological types of ovarian carcinomas or in cervical carcinomas (4).

In the September 2005 issue of Clinical Cancer Research, Feng et al. reported a 21% incidence of BRAF mutations among 97 endometrial, 78 endometrioid (EEC), and 19 nonendometrioid carcinomas (5). In addition, one out of nine (11%) atypical endometrial hyperplasias also featured a BRAF mutation, most of which were located at previously unreported sites. In this series, there were no apparent differences in the prevalence of BRAF mutations among different stages, histological subtypes, and grades. The authors found 2 mutations in nonendometrioid carcinomas (11%) and 18 mutations in EECs (23%). Interestingly, BRAF mutations were more frequently found among tumors that negatively stained for hMLH1 (12 out of 32; 41%), suggesting an association between these BRAF mutations and MSI.

These findings are in clear contradiction with those of three recent series exhibiting a low frequency of BRAF mutations in EEC. Thus, Mutch et al. (6) reported a single mutation among 146 EECs, which were also evaluated for KRAS mutations and MSI. This mutation occurred in 1 of the 81 MSI-positive cases. Salvesen et al. (7) studied BRAF mutations in 48 endometrial carcinomas and found one mutation in an MSI-negative EEC. In addition, Pappa et al. (4) found no mutations among the 67 endometrial carcinomas they analyzed.

We have determined the incidence of BRAF mutations in exons 11 and 15 by PCR/single-strand conformational polymorphism and sequencing in a series of endometrial lesions previously characterized for KRAS, PTEN, and ß-catenin mutations and MSI status (8). BRAF mutations were found in 1 of 19 atypical endometrial hyperplasias (5.6%) at codon 598 (T598I) as well as in 2 out of 94 EECs (2.1%) at codons 470 (S470F) and 614 (G614E), respectively. We also analyzed a series of 61 cervical carcinomas (19 squamous cell carcinomas and 42 adenocarcinomas) but did not find any BRAF mutations in the exons studied.

With respect to cervical cancer, our results are in accordance with those reported by Pappa et al., who did not find any BRAF mutations among 47 cases, including squamous cell carcinomas and adenocarcinomas. Concerning endometrial carcinomas, our data are in close agreement with those of Pappa et al. (4), Mutch et al. (6) and Salvesen et al. (7), which indicate a low prevalence of BRAF mutation in these tumors. Differences between these series and that reported by Feng et al. (5) are difficult to explain because all of them featured an analysis of exons 11 and 15. One possible explanation may be the different ethnicity of the populations studied, whereby BRAF seems to have a more important role in Chinese patients. This is improbable, however, because the frequency of other molecular alterations, such as RAS and MSI, was very similar in all studies.

In conclusion, most of the series analyzed thus far have shown a low prevalence of BRAF mutations in endometrial carcinomas, and an absence of association with other frequent molecular alterations, such as KRAS, PTEN, and MSI.

References

1. Garnett MJ, Marais R. Guilty as charged: B-RAF is a human oncogene. Cancer Cell 2004;6:313–9.[CrossRef][Medline]
2. Rajagopalan H, Bardelli A, Lengauer C, Kinzler KW, Vogelstein B, Velculescu VE. Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status. Nature 2002;418:934.[CrossRef][Medline]
3. Singer G, Oldt R III, Cohen Y, et al. Mutations in BRAF and KRAS characterize the development of low-grade ovarian serous carcinoma. J Natl Cancer Inst 2003;19:484–6.
4. Pappa KI, Choleza M, Markaki S, et al. Consistent absence of BRAF mutations in cervical and endometrial cancer despite KRAS mutation status. Gynecol Oncol 2005;100:596–600.[Medline]
5. Feng YZ, Shiozawa T, Miyamoto T, et al. BRAF mutation in endometrial carcinoma and hyperplasia: correlation with KRAS and p53 mutations and mismatch repair protein expression. Clin Cancer Res 2005;11:6133–8.[Abstract/Free Full Text]
6. Mutch DG, Powell MA, Mallon MA, Goodfellow PJ. RAS/RAF mutation and defective DNA mismatch repair in endometrial cancer. Am J Obstet Gynecol 2004;190:935–42.[CrossRef][Medline]
7. Salvesen HB, Kumar R, Stefansson I, et al. Low frequency of BRAF and CDKN2A mutations in endometrial cancer. Int J Cancer 2005;115:930–4.[Medline]
8. Moreno-Bueno G, Hardisson D, Sanchez C, et al. Abnormalities of the APC/ß-catenin pathway in endometrial cancer. Oncogene 2002;21:7981–90.[CrossRef][Medline]

Department of Obstetrics & Gynecology Shinshu University School of Medicine Matsumoto, Japan

In response: We thank Moreno-Bueno et al. for the letter in response to our recent article in Clinical Cancer Research (1). With regard to the differences in the incidence of BRAF mutations compared with other reports, we consider the following reasons:

1. Single-Strand Conformational Polymorphism Analysis

   Single-strand conformational polymorphism was used in two (2, 3) of three previous articles cited (2–4) and in the report by Dr. Moreno-Bueno G et al. Although single-strand conformational polymorphism has been used widely for the screening of gene mutations, its sensitivity and efficiency are not completely satisfactory. There are many factors that influence the efficiency of single-strand conformational polymorphism, including the nucleotide sequence, PCR product size, percentage of polyacrylamide, etc. (5). Several studies have reported that the sensitivity of single-strand conformational polymorphism for detecting mutation is 50% to 60% (6–8). Therefore, the frequency of BRAF mutations in studies using single-strand conformational polymorphism is likely to be lower than those using direct sequencing for all cases.
   

   
   
2. Method of DNA Extraction

   Microdissection of cells is of particular importance when analyzing genetic differences in tumorous compared with normal tissue. Failure to separate normal and tumor cell types can result in masking or false results. For example, use of the PCR to identify a loss of heterozygosity or mutation in tumor cell DNA may be missed without the use of microdissection, as DNA from the normal cell population may greatly dilute DNA from the tumor cells (9). We have done the microdissection technique using paraffin-embedded tissue. However, in ref. (2) and in some cases in ref. (3), DNA was extracted from frozen tissue of which the histologic diagnosis may have been less accurate. In ref. (4), the extraction method was not described in detail. Therefore, we think that differences in the DNA extraction method may have affected the sensitivity of mutation detection.
   

   
   
3. Exons Examined for BRAF Mutation

   Most BRAF mutations have been reported in exons 11 and 15. In a previous article, mutations in only exon 15 were examined (4). This may have resulted in the low detection rate observed.
   

   
   
4. Distribution of the Patients

   We also examined the K-ras/p53 mutation in our study, and the K-ras and p53 mutation ratio was 19% and 23%, being consistent with previous reports. In addition, we have confirmed the absence of these mutations in normal endometria adjacent to affected carcinomas. Collectively, we think that the design of the present study and the results obtained are reasonable. However, as Dr. Moreno-Bueno G et al. have pointed out, we do not deny the possibility of a chance accumulation of patients with BRAF mutations, possibly due to ethnicity.
   

   
   

References

1. Feng YZ, Shiozawa T, Miyamoto T, et al. BRAF mutation in endometrial carcinoma and hyperplasia: correlation with K-ras and p53 mutations and mismatch repair protein expression. Clin Cancer Res 2005;11:6133–8.[Abstract/Free Full Text]
2. Mutch DG, Powell MA, Mallon MA, Goodfellow PJ. RAS/RAF mutation and defective DNA mismatch repair in endometrial cancers. Am J Obstet Gynecol 2004;190:935–42.[CrossRef][Medline]
3. Salvesen HB, Kumar R, Stefansson I, et al. Low frequency of BRAF and CDKN2A mutations in endometrial cancer. Int J Cancer 2005;115:930–4.[Medline]
4. Pappa KI, Choleza M, Markaki S, et al. Consistent absence of BRAF mutations in cervical and endometrial cancer despite KRAS mutation status. Gynecol Oncol 2006;100:596–600.[Medline]
5. Hayashi K, Yandell DW. How sensitive is PCR-SSCP? Hum Mutat 1993;2:338–46.[CrossRef][Medline]
6. Tolbert DM, Noffsinger AE, Miller MA, et al. p53 immunoreactivity and single-strand conformational polymorphism analysis often fail to predict p53 mutational status. Mod Pathol 1999;12:54–60.[Medline]
7. Dong J, Gailani MR, Pomeroy SL, Reardon D, Bale AE. Identification of PATCHED mutations in medulloblastomas by direct sequencing. Hum Mutat 2000;16:89–90.[Medline]
8. Geisler JP, Hatterman-Zogg MA, Rathe JA, Lallas TA, Kirby P, Buller RE. Ovarian cancer BRCA1 mutation detection: protein truncation test (PTT) outperforms single strand conformation polymorphism analysis (SSCP). Hum Mutat 2001;18:337–44.[CrossRef][Medline]
9. Edwards J, Going JJ, Bartlett JM. Microdissection and extraction of DNA from archival tissue. Methods Mol Med 2004;97:71–5.[Medline]

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