Clinical Cancer Research Bridging the Lab and the Clinic in Cancer Medicine Infection and Cancer: Biology, Therapeutics, and Prevention
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kumar, R.
Right arrow Articles by Hemminki, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kumar, R.
Right arrow Articles by Hemminki, K.
Clinical Cancer Research Vol. 9, 3362-3368, August 2003
© 2003 American Association for Cancer Research

Molecular Oncolgy, Markers, Clinical Correlates

BRAF Mutations in Metastatic Melanoma

A Possible Association with Clinical Outcome

Rajiv Kumar1, Sabrina Angelini, Kamila Czene, Ilari Sauroja, Marjo Hahka-Kemppinen, Seppo Pyrhönen and Kari Hemminki

Department of Biosciences, Karolinska Institute, Novum, 141 57 Huddinge, Sweden [R. K., S. A., K. C., K. H.]; Division of Molecular Genetic Epidemiology, German Cancer Research Center, 69120 Heidelberg, Germany [R. K., K. H.]; Department of Oncology and Radiotherapy, University of Turku, 20520 Turku, Finland [I. S., S. P.]; and Department of Oncology, 00290 Helsinki University Central Hospital, Helsinki, Finland [M. H-K.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: The RAS-RAF-mitogen-activated protein kinase pathways mediate the cellular response to growth signals. In melanocytes, BRAF is involved in cAMP-dependent growth signals. Recently, activating mutations in the BRAF gene, were reported in a large proportion of melanomas. We have studied mutations in the BRAF gene and their association with clinical parameters.

Experimental Design: We analyzed exons 1, 11, and 15 of the BRAF gene and exons 1 and 2 of the N-ras gene for mutations in 38 metastatic melanomas by PCR-single-strand conformation polymorphism and direct sequencing. Kaplan-Meier survival and multivariate analyses were used to correlate mutations with various clinical parameters.

Results: Mutations in exon 15 of the BRAF gene were detected in 26 (68%) melanomas. In 25 cases, mutation involved the "hot spot" codon 6002of the BRAF gene. Three melanomas without a BRAF mutation carried amino acid substituting base changes at codon 61 of the N-ras gene. In a multivariate proportional hazard (Cox) model, BRAF mutation, along with the stage of metastatic melanomas, showed a statistically significant hazard ratio of 2.16 (95% confidence interval 1.02–4.59; {chi}2 for the model 6.94, degrees of freedom 2, P = 0.03) for diminished duration of response to the treatment. In a Kaplan-Meier survival model, cases with BRAF mutation showed longer disease-free survival (median of 12 months) than cases without mutation (median of 5 months), although this association was not statistically significant (Log-rank test P = 0.13).

Conclusions: Our results, besides confirming the high frequency of BRAF mutations in metastatic melanomas, also underline the potential importance of these mutations in disease outcome.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cutaneous malignant melanoma is a potentially fatal neoplasm with complex and heterogeneous etiology (1) . The sporadic form, which constitutes >90% of all cases, is linked to sunlight exposure (2 , 3) . The inherited form of melanoma is associated with a melanoma susceptible gene on chromosome 9p21; on this locus, germ-line mutations in the CDKN2A gene, which encodes two cell cycle inhibitors p16INK4a and p14ARF, are found in a proportion of melanoma prone families (4 , 5) . However, somatic alterations in sporadic melanoma are heterogeneous. Allelic loss at chromosome 9p21 locus is the most prevalent genetic occurrence in sporadic melanoma; however, mutations and other alterations in the CDKN2A gene are rather rare (6) .

The predominant oncogenic changes associated with malignant melanoma have been the activating mutations in the N-ras gene, most commonly involving codon 61 (7) . Recently, a genome-wide mutation detection strategy revealed alterations in a high proportion of melanoma cell lines and primary tumors in the BRAF gene (8) . An overwhelming proportion of these mutations affected a single residue (V600E; previously V599E2) in the kinase activation domain of BRAF. RAF serine/threonine kinases are the key signaling components in the RAS pathways. The key relevance of BRAF in melanoma is caused by its melanocyte-specific activation in cAMP-dependent signaling cascade as a consequence of {alpha}-melanocyte-stimulating hormone and related peptide binding to melanocortin receptor 1 (9) .

In the present study, we analyzed exons 1, 11, and 15 of the BRAF for mutations in 38 metastatic melanomas, and additionally, we also screened for mutations in exons 1 and 2 of the N-ras gene. In the BRAF gene, we detected mutations in 26 melanomas in exon 15, and most of the mutations involved "hot spot" codon 6002. Three melanomas without BRAF mutations carried mutations in the codon 61 of the N-ras gene. Mutations in the BRAF gene showed association with poor treatment response.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The material for this study consisted of patients treated for progressive metastatic melanoma at the Helsinki University Central Hospital between 1988 and 1996 and which have been described earlier (10) . In 38 cases, tumor material was available for the present study. All of the cases included in the study had entered into a chemoimmunotherapy trial, consisting of dacarbazine, vincristine, bleomycin, and lomustine in combination with human leukocyte IFN. Twenty-three of these cases were males, and 15 were females; the median age of patients was 54 (range 33–75 years). Clinical stage described for the patients was assessed at the diagnosis of metastatic disease and at time of initiation of chemoimmunotherapy according to the TNM3 classification by International Union against Cancer (11, 12, 13) . The localization of metastasized melanomas in the cases included in this study was cutaneous, lymphnode, lung, liver, brain, bone, and spleen. The mean disease-free survival (progression time from the diagnosis of primary melanoma to the appearance of first metastasis) of the patients was 35.5 months (range 0–200 months). Response to chemoimmunotherapy has been assessed previously according to WHO (12, 13, 14) . Among the cases presented here, 6 (16%) achieved CR, 15 (39%) patients experienced PR, 6 (16%) presented SD, and 11 (29%) had PD. The duration of response varied from 1 to 114+ months (median 4.9 months), which included 11 cases that did not respond to the treatment at all (response time 0 month; Table 1Citation ). The duration of CR was determined from the date CR was first recorded to the date PD was noted. In those who achieved PR, the duration of overall response was calculated from the 1st day of the treatment to the date of first observation of PD (14) .


View this table:
[in this window]
[in a new window]
  		Table 1 BRAF and N-ras mutations in metastatic melanomas

 
DNA Extraction and SSCP Analysis.
DNA was extracted from frozen tissues by incubating the sections removed from the slides with sterile scalpels in a digestion buffer containing 200 µg/ml Proteinase K. Control DNA samples were extracted from total WBCs obtained from two healthy individual donors. Exons 1, 11, and 15 of the BRAF gene and exons 1 and 2 of the N-ras gene were amplified for SSCP analysis using primers described in Table 2Citation . PCR was carried in 10-µl volume reactions containing 50 mM KCl, 0.11 mM each deoxynucleotide triphosphate, 1 µCi [{alpha}-32P]dCTP, 0.3 units of TaqDNA polymerase, 1–2 mM MgCl2, and 0.15–0.3 µM each primer. For the amplification of exon 1 of the BRAF gene, 10% DMSO was included as cosolvent. The temperature for PCR was set as denaturation at 95°C for 1 min, annealing (at temperatures specific for each exon as given in Table 2Citation ) for 1 min, and polymerization at 72°C for 1 min for three cycles, followed by 27–33 cycles at same temperatures with the segment time of 30 s each. Amplified products were electrophoresed on a 0.5 x mutation detection enhancement gel in at least three different conditions.


View this table:
[in this window]
[in a new window]
  		Table 2 Primers used for the amplification of the BRAF and N-ras genes

 
Sequence Analysis.
Mutations detected by SSCP in different exons of the BRAF and N-ras genes were identified and confirmed by direct sequencing using Rhodamine dye terminator cycle sequencing kit (Big Dye; Applied Biosystems). DNA samples from melanomas containing mutations in different exons were amplified by PCR. Different fragments (exons 1, 11, and 15 of the BRAF gene and exons 1 and 2 of the N-ras gene) were also amplified using DNA from healthy controls as templates and were sequenced as controls. The amplified products were purified using Sephadex micro-spin columns (Amersham-Pharmacia) and subjected to 26 cycles of sequencing reaction using forward or reverse primers separately (Table 2)Citation . The precipitated sequencing reaction products were electrophoresed on a denaturing polyacrylamide gel in an automated sequencer (ABI 377; Applied Biosystems) and analyzed using Prism and Edit View 1.0.1 software. The sequencing data were analyzed using Align software in DNA star package.

Statistical Analysis.
The cumulative survival curves for two outcomes, disease-free survival and duration of response (in months), in cases with and without BRAF mutations were drawn with Kaplan-Meier method. The differences between the curves were analyzed with Log-rank test. The association between BRAF mutations, duration of response to the treatment in months, and clinical stage of the disease was carried out using proportional hazard regression (Cox) model. For the latter analysis, the clinical stage at the initiation of chemoimmunotherapy was used, because the analysis refers to post-treatment variables. All of the statistical tests were carried out using Statistica software.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mutations in the BRAF Gene.
Exons 1, 11, and 15 were analyzed for mutations in the BRAF gene using the PCR-SSCP technique. Exon 1 was included in the analysis because in the original report on mutations in the BRAF gene, it was reported that this exon could not be amplified (8) . However, we amplified a 276-bp fragment containing exon 1 in DNA from metastatic melanomas and blood samples drawn from healthy controls that were included in this study. No mutational bandshift in SSCP was detected in any of the samples in this fragment. DNA samples from three melanomas (cases 1, 2, and 10) and two healthy controls were sequenced to check that we have amplified the correct fragment. Alignment of the obtained sequence with the reported sequence in the NCBI gene databank (accession number NT_007914; version 03 Jan. 2003) showed that the sequence of the amplified fragment was correct. However, alignment of NT_007914 BRAF sequence with mRNA sequence in the databank with accession no. NM_004333 showed a discrepancy of three nucleotides in exon 1 sequence (Fig. 1)Citation . The codon and nucleotide numbering in the BRAF gene reported thus far in all publications has been based on the NM_004333 mRNA sequence, which originates from an earlier study and has not been subjected to review by NCBI (15 , 16) . Our sequencing results show that the correct version of the sequence is given in the NCBI databank sequence with accession no. NT_007914. Therefore, the exon 1 of the BRAF gene comprises of 46 codons instead of 45, and the total number of amino acid residues in the BRAF gene is 766 instead of 765. As a consequence, we propose a change in nucleotide numbering after nucleotide 94 (starting from ATG codon) by +3 and consequently the codon numbers correspondingly change by +1. This numbering will reflect the correct sequence of the BRAF gene. The amino acid sequence from codon 30–35, according to the corrected version, is Ala-Gly-Ala-Ala-Ala-Ser, whereas in the incorrect version, the sequence from codon 30–34 is Arg-Pro-Ala-Ala-Ser. Therefore, in the correct version, the Ser residue is at codon 35 and not 34, and numbering of all residues, thereafter, changes by +1. Accordingly, in this study, we have used the corrected nucleotide and codon numbers throughout, e.g., the mutational hot spot codon and nucleotide reported in earlier reports as 599 and 1796, respectively, are codon 600 and nucleotide 1799 in the correct version (8 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) .



View larger version (34K):
[in this window]
[in a new window]
  		Fig. 1. A, alignment of partial coding part of exon 1 of BRAF sequences from the NCBI gene bank NM_004333 and NT_007914 shows three extra nucleotides in the latter sequence. B, part of exon 1 sequence of the BRAF gene obtained from a control DNA isolated from whole blood drawn from a healthy donor. This analysis shows that the BRAF sequence reported in the NCBI gene bank database with accession no. NT_007914 is the correct version. Consequently, the nucleotide and codon numbering used in various studies thus far requires a revision by +3 and +1, respectively. In this study, we have, accordingly, used the corrected codon and nucleotide numbers.

 
In exon 11 of the BRAF gene, one base change in codon 444 was detected in one melanoma (case 27), which did not result in any amino acid change (CGG>CGA). In exon 15, we detected mutations in 26 of 38 cases (Table 1Citation ; Fig. 2ACitation ). In 25 melanomas, mutation involved hot spot codon 600 (previously 599). Twenty-two melanomas carried T1799A (V600E; previously called T1796A; V599E) mutation (Fig. 2B)Citation , whereas in three cases, the same codon carried a tandem mutation GT1798–99AA (previously GT1795–96AA), causing valine to lysine change in the amino acid (Fig. 2C)Citation . This tandem mutation has been reported in melanocytic nevi and is different from the TG1799–1800AT (previously TG1796–97AT) mutation reported earlier (8 , 22) . Additionally, in one melanoma, we detected A1801G (K601E; previously A1798G; K600E) mutation (data not shown), similar to one reported in a case of colon cancer (17) .



View larger version (41K):
[in this window]
[in a new window]
  		Fig. 2. Results of SSCP and sequencing analysis of exon 15 of the BRAF gene are shown. A, a representative SSCP analysis of exon 15 of the BRAF gene. Samples in Lanes 1 (case 38), 3 (case 36), and 4 (case 35) show the aberrant bands caused by T1799A (V600E2) mutation; aberrant band in Lane 2 (case 37) is caused by GT1798–99AA (V600K) tandem mutation. Lanes 5 and 6 contained DNA from melanoma cases 34 and 33. B, part of exon 15 of the BRAF gene sequence showing T1799A (V600E) mutation in case 38 of metastatic melanoma (shown by an arrow). C, part of exon 15 of the BRAF gene sequence obtained from DNA extracted from case 37 of melanoma showing GT1798–99AA (V600K) sequence change (shown by a thick line). D, corresponding wild-type sequence of exon 15 of the BRAF gene obtained from a control DNA sample (from a healthy donor). Thick line, the position of mutated bases shown in two preceding cases. E, Kaplan-Meier survival analysis showing difference in disease-free survival between melanoma cases with and without BRAF mutations. Median survival in cases with BRAF mutations was 12 months against 5 months without mutations, although this difference was not statistically significant. F, Kaplan-Meier curves showing difference in response time to the treatment in cases with and without BRAF mutations. Median response period in months was 3.4 against 9.8 in cases with and without BRAF mutations, respectively.

 
Association of BRAF Mutations with Clinical Parameters.
In Kaplan-Meier survival model, cases with BRAF mutations showed median disease-free survival of 12 months, whereas cases without mutation had median survival of 5 months, although this difference between the two groups was not statistically significant (Log-rank test P = 0.13; Fig. 2ECitation ). The inclusion of data on stage at diagnosis of metastatic disease in a multivariate model did not affect the outcome on association between BRAF mutations and disease-free survival (data not shown). The cases with BRAF mutations showed a shorter duration of the response to the treatment than cases without any BRAF mutations (median 3.4 versus 9.8 months; Log-rank test P = 0.03; Fig. 2FCitation ). In a multivariate proportional hazard (Cox) model, where the effect of BRAF mutations along with clinical stage of the melanomas on the duration of response to the treatment was determined, cases with BRAF mutations showed a statistically significant hazard ratio 2.16 (95% confidence interval 1.02–4.59; {chi}2 for the model 6.94, degrees of freedom 2, P = 0.03) for diminished duration of response to the treatment. In another model, where values for age, p53, MDM2, MIB expression, and metastatic stage were included, cases with BRAF mutations retained significance for shorter response period to the treatment than cases without BRAF mutations (data not shown). We could not assess association between duration of response to the treatment and location of metastasized tumors because of the small number of cases in the subgroups (Table 1)Citation . However, when melanomas were grouped into two, those with only s.c. metastasis (10 cases) and all others (28 cases), the location had no effect on the association between mutations in BRAF gene and duration of response to the treatment (data not shown).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study on metastatic melanomas, we searched for mutations in exon 1, 11, and 15 of the BRAF gene. The earlier study had revealed distribution of mutations in regions of BRAF kinase domain that are encoded by exons 11 and 15. Exon 1 was included for the reason that it had not been screened in earlier studies, and sequencing analysis showed that the correct version of the sequence has an additional three nucleotides and, therefore, one additional codon in this exon. Consequently, on the basis of our sequencing results, we have proposed and used a revised numbering of nucleotides (changed by +3 after nucleotide 94 in exon 1) and codons (changed by +1) of the BRAF gene, which is based on the correct version of the sequence given in the NCBI gene bank database (accession no. NT_007914, version 03 Jan. 2003).

Furthermore, our results confirm the presence of mutations in the BRAF gene in sporadic melanomas. The level of BRAF mutations detected in the metastatic melanomas in this study corresponds to that reported earlier, and in all cases, except one, the mutations involved hot spot codon 600 (reported as 599 in all earlier studies) in the kinase domain. The V600E BRAF mutant has been reported to possess 10-fold greater basal activity, and it induces focus formation in NIH3T3 cells with much higher efficiency than the wild-type BRAF (8) . Our detection of a V600K (GT1798–1799AA) amino acid substitution, found in melanocytic nevi in an earlier study (reported as V599K; GT1795–96AA), confirms the possibility of BRAF activation through substitution with both positively and negatively charged residues (22) . The residue 600 (previously 599), which accounts for 25 out of 26 mutations in the BRAF gene in this study and >90% of all mutations in all reported studies, is identical at corresponding positions in RAF1 and ARAF1, and it is conserved through the evolution with a single exception of Drosophila Raf homologue (8 , 21) . The hypothesis that in melanocytes, the activation of RAS or BRAF leads to the same phenotype was further strengthened by the mutual exclusivity of mutations. N-ras mutations were detected only in melanomas that had wild-type BRAF, which is in conformation with earlier data on colorectal cancer and melanomas (17 , 21 , 22) .

The recent discovery in melanocytic nevi of BRAF mutations supports their potential role as the initiating somatic changes, which require additional genetic changes for melanoma development (22) . A majority but not all melanomas carry mutations in the BRAF gene; however, not all melanomas arise from melanocytic nevi. Nevertheless, the mutations in the BRAF gene (and also N-ras gene) persist from primary through metastatic melanomas, which suggests that probably these mutations are early events but may also have a role in tumor maintenance (29) .4 Association of a diminished response time to the treatment, detected here in patients with BRAF mutations, therefore, indicates the involvement of activated BRAF beyond the initiation. However, an independent effect of BRAF mutations on response to the treatment would require further evaluation because we found that this association was dependent on the clinical stage.

We also found a nonsignificant association between BRAF mutations and longer disease-free survival. These associations between cases with BRAF mutations and clinical parameters may be seemingly contradictory, but the plausible explanation could be that the effect of BRAF mutations can potentially override the effect of widespread genomic destabilization and thus prolong the disease progression period. Thus, it is possible that the subset of melanomas with BRAF mutations differ in their biology from tumors without BRAF mutations. Activating BRAF mutations could overcome one or several extra and intracellular growth inhibitory signals produced by cytotoxic drugs and IFNs. Indeed, it has been shown in cell lines and severe combined immune deficiency mice that constitutive activation of the MAP/ERK kinase/ERK/MAP kinase pathway induces resistance to cisplatin and radiotherapy. In addition, it has been shown that ERK1/2 inhibitor U0126 decreases proliferation and invasion of melanoma cell lines with BRAF mutations (18 , 27 , 28) . These findings, taken together with ours, suggest that the role of BRAF and other MAP kinase pathway oncogenes should be further studied in larger patient populations, in reference to treatment outcome. This, together with in vitro studies, would produce vital information on whether specific inhibitors of these oncogenes could be used for targeted therapy of some very chemoresistant malignancies, such as melanoma and non-small cell lung carcinoma.

The discovery of mutations in the BRAF gene in melanomas in high proportion has already led to the suggestions for the potential role of kinase inhibitors in treatment, which can counter the kinase activity of the activated BRAF (8) . The possible association of these mutations with clinical parameters as indicated in this study further augments the potential role for such treatment. However, any such strategy would require consideration of other complexities in advanced tumors. In conclusion, our study on metastatic melanomas confirms the presence of somatic mutations in the BRAF gene in melanoma, which functions in the same pathways as RAS, indicated by mutual exclusivity of BRAF and N-ras mutations. Furthermore, our results show a possible role of these mutations in the disease outcome.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Center for Nutrition and Toxicology, Karolinska Institute, Novum, 141 57 Huddinge, Sweden. E-mail: rajiv.kumar{at}cnt.ki.se Back

2 In all communications on mutations in the BRAF gene, the nucleotide and codon numbers have been based on the NCBI gene bank nucleotide sequence NM_004333. However, according to a sequence given in NCBI gene bank sequence accession no. NT_007914, version 03 Jan. 2003, there is a discrepancy of one codon (three nucleotides) in exon 1 sequence in the sequence NM_004333. The sequence analysis of exon 1 of the BRAF gene in this study has shown that the sequence derived from NT_007914 is correct. Because of correctness of the latter, sequence numbering of codons and nucleotides after exon 1 are changed by +1 and +3, respectively. The details are given in the text. Back

3 The abbreviations used are: TNM, Tumor-Node-Metastasis; CR, complete response; SD, stable disease; PD, progressive disease; PR, partial response; SSCP, single-strand conformation polymorphism; MAP, mitogen-activated protein; NCBI, National Center for Biotechnology Information; ERK, extracellular signal-regulated kinase. Back

4 R. Kumar et al., unpublished results. Back

Received 1/ 6/03; revised 4/21/03; accepted 4/28/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Chin L., Merlino G., DePinho R. A. Malignant melanoma: modern black plague and genetic black box. Genes Dev., 12: 3467-3481, 1998.[Free Full Text]
  2. Gilchrest B. A., Eller M. S., Geller A. C., Yaar M. The pathogenesis of melanoma induced by ultraviolet radiation. N. Engl. J. Med., 340: 1341-1348, 1999.[Free Full Text]
  3. Hemminki K., Lonnstedt I., Vaittinen P. A population-based study of familial cutaneous melanoma. Melanoma Res., 11: 133-140, 2001.[CrossRef][Medline]
  4. Kumar R., Sauroja I., Punnonen K., Jansen C., Hemminki K. Selective deletion of exon 1ß of the p19ARF gene in metastatic melanoma cell lines. Genes Chromosomes Cancer, 23: 273-277, 1998.[CrossRef][Medline]
  5. Bishop D. T., Demenais F., Goldstein A. M., Bergman W., Bishop J. N., Bressac-de Paillerets B., Chompret A., Ghiorzo P., Gruis N., Hansson J., Harland M., Hayward N., Holland E. A., Mann G. J., Mantelli M., Nancarrow D., Platz A., Tucker M. A. Geographical variation in the penetrance of CDKN2A mutations for melanoma. J. Natl. Cancer Inst. (Bethesda), 94: 894-903, 2002.[Abstract/Free Full Text]
  6. Kumar R., Smeds J., Lundh Rozell B., Hemminki K. Loss of heterozygosity at chromosome 9p21 (INK4–p14ARF locus): homozygous deletions and mutations in the p16 and p14ARF genes in sporadic primary melanomas. Melanoma Res., 9: 138-147, 1999.[Medline]
  7. Saida T. Recent advances in melanoma research. J. Dermatol. Sci., 26: 1-13, 2001.[CrossRef][Medline]
  8. Davies H., Bignell G. R., Cox C., Stephens P., Edkins S., Clegg S., Teague J., Woffendin H., Garnett M. J., Bottomley W., Davis N., Dicks E., Ewing R., Floyd Y., Gray K., Hall S., Hawes R., Hughes J., Kosmidou V., Menzies A., Mould C., Parker A., Stevens C., Watt S., Hooper S., Wilson R., Jayatilake H., Gusterson B. A., Cooper C., Shipley J., Hargrave D., Pritchard-Jones K., Maitland N., Chenevix-Trench G., Riggins G. J., Bigner D. D., Palmieri G., Cossu A., Flanagan A., Nicholson A., Ho J. W., Leung S. Y., Yuen S. T., Weber B. L., Seigler H. F., Darrow T. L., Paterson H., Marais R., Marshall C. J., Wooster R., Stratton M. R., Futreal P. A. Mutations of the BRAF gene in human cancer. Nature (Lond.), 417: 949-954, 2002.[CrossRef][Medline]
  9. Busca R., Abbe P., Mantoux F., Aberdam E., Peyssonnaux C., Eychene A., Ortonne J. P., Ballotti R. Ras mediates the cAMP-dependent activation of extracellular signal-regulated kinases (ERKs) in melanocytes. EMBO J., 19: 2900-2910, 2000.[CrossRef][Medline]
  10. Sauroja I., Smeds J., Vlaykova T., Kumar R., Talve L., Hahka-Kemppinen M., Punnonen K., Jansen C. T., Hemminki K., Pyrhonen S. Analysis of G(1)/S checkpoint regulators in metastatic melanoma. Genes Chromosomes Cancer, 28: 404-414, 2000.[CrossRef][Medline]
  11. Ketcham A. S., Moffat F. L., Balch C. M. Classification and staging Balch C. M. Houghton A. N. Milton G. W. Sober A. J. Soong S-J. eds. . Cutaneous Melanoma, 213-220, J. B. Lippincott Co. Philadelphia 1992.
  12. Pyrhonen S., Hahka-Kemppinen M., Muhonen T. A promising interferon plus four-drug chemotherapy regimen for metastatic melanoma. J. Clin. Oncol., 10: 1919-1926, 1992.[Abstract]
  13. Hahka-Kemppinen M., Muhonen T., Virolainen M., Jekunen A., Asko-Seljavaara S., Pyrhonen S. Response of subcutaneous and cutaneous metastases of malignant melanoma to combined cytostatic plus interferon therapy. Br. J. Dermatol., 132: 973-977, 1995.[Medline]
  14. Miller A. B., Hoogstraten B., Staquet M., Winkler A. Reporting results of cancer treatment. Cancer (Phila.), 47: 207-214, 1981.[CrossRef][Medline]
  15. Sithanandam G., Kolch W., Duh F. M., Rapp U. R. Complete coding sequence of a human B-raf cDNA and detection of B-raf protein kinase with isozyme specific antibodies. Oncogene, 5: 1775-1780, 1990.[Medline]
  16. Sithanandam G., Druck T., Cannizzaro L. A., Leuzzi G., Huebner K., Rapp U. R. B-raf and a B-raf pseudogene are located on 7q in man. Oncogene, 7: 795-799, 1992.[Medline]
  17. Rajagopalan H., Bardelli A., Lengauer C., Kinzler K. W., Vogelstein B., Velculescu V. E. Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status. Nature (Lond.), 418: 934 2002.[CrossRef][Medline]
  18. Pollock P. M., Meltzer P. S. A genome-based strategy uncovers frequent BRAF mutations in melanoma. Cancer Cell, 2: 5-7, 2002.[CrossRef][Medline]
  19. Naoki K., Chen T. H., Richards W. G., Sugarbaker D. J., Meyerson M. Missense mutations of the BRAF gene in human lung adenocarcinoma. Cancer Res., 62: 7001-7003, 2002.[Abstract/Free Full Text]
  20. Brose M. S., Volpe P., Feldman M., Kumar M., Rishi I., Gerrero R., Einhorn E., Herlyn M., Minna J., Nicholson A., Roth J. A., Albelda S. M., Davies H., Cox C., Brignell G., Stephens P., Futreal P. A., Wooster R., Stratton M. R., Weber B. L. BRAF and RAS mutations in human lung cancer and melanoma. Cancer Res., 62: 6997-7000, 2002.[Abstract/Free Full Text]
  21. Yuen S. T., Davies H., Chan T. L., Ho J. W., Bignell G. R., Cox C., Stephens P., Edkins S., Tsui W. W., Chan A. S., Futreal P. A., Stratton M. R., Wooster R., Leung S. Y. Similarity of the phenotypic patterns associated with BRAF and KRAS mutations in colorectal neoplasia. Cancer Res., 62: 6451-6455, 2002.[Abstract/Free Full Text]
  22. Pollock P. M., Harper U. L., Hansen K. S., Yudt L. M., Stark M., Robbins C. M., Moses T. Y., Hostetter G., Wagner U., Kakareka J., Salem G., Pohida T., Heenan P., Duray P., Kallioniemi O., Hayward N. K., Trent J. M., Meltzer P. S. High frequency of BRAF mutations in nevi. Nat. Genet., 33: 19-20, 2003.[CrossRef][Medline]
  23. Singer G., Oldt R., III, Cohen Y., Wang B. G., Sidransky D., Kurman R. J., Shih Ie M. Mutations in BRAF and KRAS characterize the development of low-grade ovarian serous carcinoma. J. Natl. Cancer Inst. (Bethesda), 95: 484-486, 2003.[Abstract/Free Full Text]
  24. Lang J., Boxer M., MacKie R. Absence of exon 15 BRAF germline mutations in familial melanoma. Hum. Mutat., 21: 327-330, 2003.[CrossRef][Medline]
  25. Kimura E. T., Nikiforova M. N., Zhu Z., Knauf J. A., Nikiforov Y. E., Fagin J. A. High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma. Cancer Res., 63: 1454-1457, 2003.[Abstract/Free Full Text]
  26. Cohen Y., Xing M., Mambo E., Guo Z., Wu G., Trink B., Beller U., Westra W. H., Ladenson P. W., Sidransky D. BRAF mutation in papillary thyroid carcinoma. J. Natl. Cancer Inst. (Bethesda), 95: 625-627, 2003.[Abstract/Free Full Text]
  27. Smalley K. S. A pivotal role for ERK in the oncogenic behaviour of malignant melanoma?. Int. J. Cancer, 104: 527-532, 2003.[CrossRef][Medline]
  28. Satyamoorthy K., Li G., Gerrero M. R., Brose M. S., Volpe P., Weber B. L., Van Belle P., Elder D. E., Herlyn M. Constitutive mitogen-activated protein kinase activation in melanoma is mediated by both BRAF mutations and autocrine growth factor stimulation. Cancer Res., 63: 756-759, 2003.[Abstract/Free Full Text]
  29. Omholt K., Karsberg S., Platz A., Kanter L., Ringborg U., Hansson J. Screening of N-ras codon 61 mutations in paired primary and metastatic cutaneous melanomas: mutations occur early and persist throughout tumor progression. Clin. Cancer Res., 8: 3468-3474, 2002.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Clin. Pathol.Home page
L. R Rowe, B. G Bentz, and J. S Bentz
Detection of BRAF V600E activating mutation in papillary thyroid carcinoma using PCR with allele-specific fluorescent probe melting curve analysis
J. Clin. Pathol., November 1, 2007; 60(11): 1211 - 1215.
[Abstract] [Full Text] [PDF]

Home page
 J. Mol. Diagn.Home page
M. Yancovitz, J. Yoon, M. Mikhail, W. Gai, R. L. Shapiro, R. S. Berman, A. C. Pavlick, P. B. Chapman, I. Osman, and D. Polsky
Detection of Mutant BRAF Alleles in the Plasma of Patients with Metastatic Melanoma
J. Mol. Diagn., April 1, 2007; 9(2): 178 - 183.
[Abstract] [Full Text] [PDF]

Home page
 Arch DermatolHome page
A. Figl, R. K. Thirumaran, S. Ugurel, A. Gast, K. Hemminki, R. Kumar, and D. Schadendorf
Multiple Melanomas After Treatment for Hodgkin Lymphoma in a Non-Dutch p16-Leiden Mutation Carrier With 2 MC1R High-Risk Variants
Arch Dermatol, April 1, 2007; 143(4): 495 - 499.
[Abstract] [Full Text] [PDF]

Home page
 The OncologistHome page
D. Strumberg, J. W. Clark, A. Awada, M. J. Moore, H. Richly, A. Hendlisz, H. W. Hirte, J. P. Eder, H.-J. Lenz, and B. Schwartz
Safety, Pharmacokinetics, and Preliminary Antitumor Activity of Sorafenib: A Review of Four Phase I Trials in Patients with Advanced Refractory Solid Tumors
Oncologist, April 1, 2007; 12(4): 426 - 437.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
M. R. James, T. Dumeni, M. S. Stark, D. L. Duffy, G. W. Montgomery, N. G. Martin, and N. K. Hayward
Rapid Screening of 4000 Individuals for Germ-line Variations in the BRAF Gene
Clin. Chem., September 1, 2006; 52(9): 1675 - 1678.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
A. Sharma, M. A. Tran, S. Liang, A. K. Sharma, S. Amin, C. D. Smith, C. Dong, and G. P. Robertson
Targeting Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Kinase in the Mutant (V600E) B-Raf Signaling Cascade Effectively Inhibits Melanoma Lung Metastases
Cancer Res., August 15, 2006; 66(16): 8200 - 8209.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
I.-J. Kim, H. C. Kang, S.-G. Jang, K. Kim, S.-A Ahn, H.-J. Yoon, S. N. Yoon, and J.-G. Park
Oligonucleotide microarray analysis of distinct gene expression patterns in colorectal cancer tissues harboring BRAF and K-ras mutations
Carcinogenesis, March 1, 2006; 27(3): 392 - 404.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
V. Vasko, S. Hu, G. Wu, J. C. Xing, A. Larin, V. Savchenko, B. Trink, and M. Xing
High Prevalence and Possible de Novo Formation of BRAF Mutation in Metastasized Papillary Thyroid Cancer in Lymph Nodes
J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5265 - 5269.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
S. Bloethner, B. Chen, K. Hemminki, J. Muller-Berghaus, S. Ugurel, D. Schadendorf, and R. Kumar
Effect of common B-RAF and N-RAS mutations on global gene expression in melanoma cell lines
Carcinogenesis, July 1, 2005; 26(7): 1224 - 1232.
[Abstract] [Full Text] [PDF]

Home page
 Endocr Relat CancerHome page
M Xing
BRAF mutation in thyroid cancer
Endocr. Relat. Cancer, June 1, 2005; 12(2): 245 - 262.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
N. Mitsutake, J. A. Knauf, S. Mitsutake, C. Mesa Jr., L. Zhang, and J. A. Fagin
Conditional BRAFV600E Expression Induces DNA Synthesis, Apoptosis, Dedifferentiation, and Chromosomal Instability in Thyroid PCCL3 Cells
Cancer Res., March 15, 2005; 65(6): 2465 - 2473.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. T. Huntington, J. M. Shields, C. J. Der, C. A. Wyatt, U. Benbow, C. L. Slingluff Jr., and C. E. Brinckerhoff
Overexpression of Collagenase 1 (MMP-1) Is Mediated by the ERK Pathway in Invasive Melanoma Cells: ROLE OF BRAF MUTATION AND FIBROBLAST GROWTH FACTOR SIGNALING
J. Biol. Chem., August 6, 2004; 279(32): 33168 - 33176.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kumar, R.
Right arrow Articles by Hemminki, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kumar, R.
Right arrow Articles by Hemminki, K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online