BRAF mutations distinguish anorectal from cutaneous melanoma at the molecular level
2004; Elsevier BV; Volume: 127; Issue: 6 Linguagem: Inglês
10.1053/j.gastro.2004.08.051
ISSN1528-0012
AutoresBurkhard Helmke, Jan Mollenhauer, Christel Herold‐Mende, Axel Benner, Marianne Thome, Nikolaus Gaßler, Wolfgang Wahl, Stefan Lyer, Annemarie Poustka, Herwart F. Otto, Martin Deichmann,
Tópico(s)Immunotherapy and Immune Responses
ResumoBackground & Aims: Anorectal melanoma (AM) is a rare but highly malignant tumor, displaying histologic and immunohistochemical features very similar to cutaneous melanoma (CM). Because BRAF mutations were recently identified in the majority of CM and nevi, we investigated AM for BRAF mutations and mutations of NRAS, an additional component of the MAPK-signalling pathway. Methods: DNA from formalin-fixed and paraffin-embedded AM was PCR amplified and sequenced. Results: We detected BRAF mutations in 2 of 19 cases and NRAS mutations in none of the cases. Mutations in exon 15 of BRAF were present in only 1 tumor (1 of 19 cases). The A1800T base exchange represented a novel mutation and resulted in a K600N transition in an AM from a 96-year-old white man who presented with rectal bleeding and painful sitting of a few weeks’ duration. The second positive AM case, a 69-year-old white man who presented with painless rectal bleeding and clinical symptoms of an intestinal constipation showed a novel missense mutation (C1327T leading to R443W conversion) in BRAF exon 11. None of the AM cases displayed the oncogenic V599E mutation preponderating in CM. Conclusions: With regard to the frequency of V599E BRAF mutations, AM significantly differs from CM (P ≤ .0001), which suggests that BRAF mutations distinguish anorectal from cutaneous melanoma at the molecular level. Background & Aims: Anorectal melanoma (AM) is a rare but highly malignant tumor, displaying histologic and immunohistochemical features very similar to cutaneous melanoma (CM). Because BRAF mutations were recently identified in the majority of CM and nevi, we investigated AM for BRAF mutations and mutations of NRAS, an additional component of the MAPK-signalling pathway. Methods: DNA from formalin-fixed and paraffin-embedded AM was PCR amplified and sequenced. Results: We detected BRAF mutations in 2 of 19 cases and NRAS mutations in none of the cases. Mutations in exon 15 of BRAF were present in only 1 tumor (1 of 19 cases). The A1800T base exchange represented a novel mutation and resulted in a K600N transition in an AM from a 96-year-old white man who presented with rectal bleeding and painful sitting of a few weeks’ duration. The second positive AM case, a 69-year-old white man who presented with painless rectal bleeding and clinical symptoms of an intestinal constipation showed a novel missense mutation (C1327T leading to R443W conversion) in BRAF exon 11. None of the AM cases displayed the oncogenic V599E mutation preponderating in CM. Conclusions: With regard to the frequency of V599E BRAF mutations, AM significantly differs from CM (P ≤ .0001), which suggests that BRAF mutations distinguish anorectal from cutaneous melanoma at the molecular level. Anorectal melanoma (AM) is a rare mucosal melanocytic malignancy in the anal canal and is associated with an extremely poor prognosis. 1Helmke B.M. Otto H.F. Das anorektale Melanom Eine seltene und hochmaligne Tumorentität des Analkanals.Pathologe. 2004; 25: 171-177Crossref PubMed Scopus (16) Google Scholar The question of its relationship to cutaneous melanoma (CM) is unsolved, but of fundamental importance for the treatment of AM patients, because limited patient numbers hardly allow for obtaining data on optimal AM therapeutic strategies. Presently, the patients are treated according to strategies used in CM therapy because AM displays histologic and immunohistochemical features similar to CM. However, the 5-year survival rates of AM patients are worse compared with stage IV CM, and, a priori, a distinct etiology must be assumed. 1Helmke B.M. Otto H.F. Das anorektale Melanom Eine seltene und hochmaligne Tumorentität des Analkanals.Pathologe. 2004; 25: 171-177Crossref PubMed Scopus (16) Google Scholar Although ultraviolet (UV) radiation has been postulated to induce CM, this environmental carcinogen is unlikely to contribute to AM. Moreover, CM are thought to often arise from melanocytic nevi, 1Helmke B.M. Otto H.F. Das anorektale Melanom Eine seltene und hochmaligne Tumorentität des Analkanals.Pathologe. 2004; 25: 171-177Crossref PubMed Scopus (16) Google Scholar but no evidence for corresponding precursor lesions exists for AM. The search for genes with major causal contributions to sporadic CM did not retrieve promising candidates for several years. However, recent studies revealed activating BRAF mutations in 66% of melanoma cell lines, 80% of primary melanomas, and 68% of melanoma metastases. 2Davies 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. 2002; 417: 949-954Crossref PubMed Scopus (8397) Google Scholar, 3Gorden A. Osman I. Gai W. He D. Huang W. Davidson A. Houghton A.N. Busam K. Polsky D. Analysis of BRAF and NRAS mutations in metastatic melanoma tissues.Cancer Res. 2003; 63: 3955-3957PubMed Google Scholar Remarkably, also 82% of cutaneous nevi showed mutations within BRAF, 4Pollock 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. 2003; 33: 19-20Crossref PubMed Scopus (1387) Google Scholar whereas no mutations were identified in pedigrees with familial melanoma. 5Lang J. Boxer M. MacKie R. Absence of exon 15 BRAF germline mutations in familial melanoma.Hum Mutat. 2003; 21: 327-330Crossref PubMed Scopus (58) Google Scholar In sporadic CM and nevi, activating mutations within BRAF typically target exon 15, which encodes the kinase domain. Besides melanocytic tumors, benign colorectal lesions and colorectal carcinomas 6Yuen 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. 2002; 62: 6451-6455PubMed Google Scholar; serous ovarian cancer (including borderline tumors) 7Singer G. Oldt III, R. Cohen Y. Wang B.G. Sidransky D. Kurman R.J. Shih IeM. Mutations in BRAF and KRAS characterize the development of low-grade ovarian serous carcinoma.J Natl Cancer Inst. 2003; 95: 484-486Crossref PubMed Scopus (716) Google Scholar; papillary thyroid carcinoma 8Cohen 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. 2003; 95: 625-627Crossref PubMed Scopus (787) Google Scholar, 9Kimura 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. 2003; 63: 1454-1457PubMed Google Scholar; cholangiocarcinoma 10Tannapfel A. Sommerer F. Benicke M. Katalinic A. Uhlmann D. Witzigmann H. Hauss J. Wittekind C. Mutations of the BRAF gene in cholangiocarcinoma but not in hepatocellular carcinoma.Gut. 2003; 52: 706-712Crossref PubMed Scopus (311) Google Scholar; and lung, head, and neck cancer 2Davies 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. 2002; 417: 949-954Crossref PubMed Scopus (8397) Google Scholar, 8Cohen 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. 2003; 95: 625-627Crossref PubMed Scopus (787) Google Scholar, 11Brose 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. 2002; 72: 6997-7000Google Scholar, 12Naoki K. Chen T.H. Richards W.G. Sugarbaker D.J. Meyerson M. Missense mutations of the BRAF gene in human lung adenocarcinoma.Cancer Res. 2002; 62: 7001-7003PubMed Google Scholar also show exon 15 BRAF mutations. BRAF is located on chromosome 7q34 and encodes a serine/threonine kinase, regulating proliferation and differentiation via the MAPK pathway. 4Pollock 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. 2003; 33: 19-20Crossref PubMed Scopus (1387) Google Scholar, 13Satyamoorthy 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. 2003; 63: 756-759PubMed Google Scholar The V599E mutation leads to hyperactivation of BRAF kinase activity. 2Davies 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. 2002; 417: 949-954Crossref PubMed Scopus (8397) Google Scholar BRAF activation probably contributes to the development of sporadic CM, but the V599E mutation may furthermore represent a specific molecular marker for melanocytic tumors. Thus, BRAF represents an excellent candidate gene to investigate the relationship between AM and CM. A 96-year-old white man presented with rectal bleeding and painful sitting of a few weeks’ duration. His past medical record indicated a potential prostate neoplasm, which was treated with high-energy thermotherapy twice in the previous 3 years. Four and 5 months before presentation, he was hospitalized because of hematuria and hemorrhagic cystitis. At that time, his serum total prostate specific antigen (PSA) was 138.3 ng/dL, compared with 68.8 ng/dL at the time of anorectal symptoms. A sessile, ulcerated, and bleeding polyp (4.0 × 3.0 × 1.5 cm) was observed in the anal canal at 5–6 cm during rectoscopy. Because of the advanced age of the patient, a minimal invasive local excision was performed. The histologic diagnosis indicated a melanotic anorectal melanoma. The tumor cells showed positive immunohistochemical reactions, including cytoplasmatic staining with anti-HMB-45 and anti-Melan-A, nuclear, and cytoplasmatic staining against the S-100 protein and nuclear reaction with antibodies directed against MiTF. External examination did not provide evidence for a primary cutaneous melanoma or atypical melanocytic skin lesions. The results of the metastatic workup were negative. Because of his advanced age, no postoperative chemotherapy was applied. Twelve months after resection, diffuse liver metastasis was diagnosed, and a local recurrence was suspected. The patient died 15 months after local excision from progressive metastatic disease at age 97 years. A 69-year-old white man presented with painless rectal bleeding and severe constipation. Upon clinical examination, a polypoid lesion was found in the anal canal. Biopsy specimens were taken, and a melanoma was diagnosed histologically. Computed tomography imaging performed prior to surgery showed diffuse metastasis of the liver. A perineal rectum amputation with total mesorectal extirpation was performed. Gross examination revealed a polypoid lesion (5.5 × 4.0 × 2.0 cm) of the anal canal undermining the squamous epithelium. Histologic examination of the tumor confirmed the diagnosis of a melanotic anorectal melanoma with venous and lymphatic invasion and metastases to the regional lymph nodes. Immunohistochemistry revealed the same immunoprofile as seen in case 1. A primary cutaneous melanoma was ruled out clinically. The postoperative chemotherapy included 3 cycles of DVC (dacarbacin, vindesin, and cisplatin) and 6 cycles of fotemustine. The patient died 19 months after surgery from hemorrhagic shock because of extended metastases in the liver, lung, and stomach. To investigate AM for the presence of BRAF mutations that have recently been reported to occur frequently in CM, we examined 19 AM samples from 13 female and 6 male patients with a mean age of 69.5 years (range, 38–96 years) at diagnosis and matched normal DNA from the same patients. This cohort of AM patients represents one of the largest collectives of this rare tumor type worldwide. Immunohistochemical diagnosis of AM was performed using standard protocols, 14Sheffield M.V. Yee H. Dorvault C.C. Weilbaecher K.N. Eltoum I.A. Siegal G.P. Fisher D.E. Chhieng D.C. Comparison of five antibodies as markers in the diagnosis of melanoma in cytologic preparations.Am J Clin Pathol. 2002; 118: 930-936Crossref PubMed Scopus (87) Google Scholar including investigation with antibodies against HMB-45 antigen (clone gp100, Enzo Life Sciences, Farmingdale), Melan-A (clone A103; DAKO, Glostrup, Denmark), MiTF (clone, C5+D5, Neomarkers, Fremont), and S-100 protein (clone Z0311, DAKO, Glostrup, Denmark). Extraction of genomic DNA was performed from formalin-fixed, paraffin-embedded, malignant and flanking normal tissues (17 primary AM, with 1 local recurrence and 2 lymph node metastases), using a QIAamp DNA Mini Kit (Qiagen, Valencia, CA), according to manufacturer’s instructions. Analyzing these genomic DNA samples by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) gel electrophoresis, followed by DNA cloning and sequencing, we screened for the presence of BRAF exon 11 and 15 mutations. BRAF exon 11 was amplified using primers BRAF-11-forward-98239 and BRAF-11-reverse-98552 before and BRAF-11-forward-98307 and BRAF-11-reverse-98526 following SSCP gel electrophoresis. BRAF exon 15 was amplified using primers BRAF-15-forward-69998 and BRAF-15-reverse-70221 before and BRAF-15-forward-70023 and BRAF-15-reverse-70221 following SSCP. We further investigated the samples for mutations in exons 2 and 3 of NRAS, a second component of the MAPK-signalling pathway. NRAS exon 2 was amplified using primers NRAS-2-forward-2659 and NRAS-2-reverse-2817 before and NRAS-2-forward-2678 and NRAS-2-reverse-2804 following SSCP. NRAS exon 3 was amplified using primers NRAS-3-forward-4902 and NRAS-3-reverse-5025 before and NRAS-3-forward-4922 and NRAS-3-reverse-5005 following SSCP. According to previous reports, 2Davies 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. 2002; 417: 949-954Crossref PubMed Scopus (8397) Google Scholar, 3Gorden A. Osman I. Gai W. He D. Huang W. Davidson A. Houghton A.N. Busam K. Polsky D. Analysis of BRAF and NRAS mutations in metastatic melanoma tissues.Cancer Res. 2003; 63: 3955-3957PubMed Google Scholar and to the database of the National Center for Biotechnology Information (NCBI, Bethesda, MD; http://ncbi.nlm.nih.gov/BLAST), the primer sequences were BRAF-11-forward-98239, 5′-GCG AAC AGT GAA TAT TTC CTT TG-3′ (nucleotide numbering according to NCBI accession number AC006344.2); BRAF-11-forward-98307, 5′-GAC TTG TCA CAA TGT CAC CAC A-3′; BRAF-11-reverse-98552, 5′-TCC CTC TCA GGC ATA AGG TAA-3′; BRAF-11-reverse-98526, 5′-TAC TTA GGG TGA AAC ATA AGG TTT-3′; BRAF-15-forward-69998, 5′-GGC CAA AAA TTT AAT CAG TGG A-3′ (primer identical with exon-15-reverse according to Gorden et al 3Gorden A. Osman I. Gai W. He D. Huang W. Davidson A. Houghton A.N. Busam K. Polsky D. Analysis of BRAF and NRAS mutations in metastatic melanoma tissues.Cancer Res. 2003; 63: 3955-3957PubMed Google Scholar); BRAF-15-forward-70023, 5′-ATA GCC TCA ATT CTT ACC ATC C-3′; BRAF-15-reverse-70221, 5′-TCA TAA TGC TTG CTC TGA TAG GA-3′ (identical with primer exon-15-forward according to Pollack et al 4Pollock 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. 2003; 33: 19-20Crossref PubMed Scopus (1387) Google Scholar); NRAS-2-forward-2659, 5′-CTG GTT TCC AAC AGG TTC TTG C-3′ (nucleotide numbering according to NCBI accession number AY428630.1); NRAS-2-forward-2678, 5′-TGC TGG TGT GAA ATG ACT GAG TA-3′; NRAS-2-reverse-2817, CTA CCA CTG GGC CTC ACC T-3′; NRAS-2-reverse-2804, 5′-TCA CCT CTA TGG TGG GAT CA-3′; NRAS-3-forward-4902, 5′-GGT GAA ACC TGT TTG TTG GAC-3′; NRAS-3-forward-4922, 5′-CAT ACT GGA TAC AGC TGG ACA A-3′; NRAS-3-reverse-5025, 5′-ACT TGC TAT TAT TGA TGG CAA A-3′; NRAS-2-reverse-5005, 5′-AAT ACA CAG AGG AAG CCT TCG-3′. The amplified DNA was mixed with an equal volume of formamide loading dye (94% formamide, 0.05% xylene cyanol, and 0.05% bromphenol blue), denatured at 95°C for 5 minutes, chilled on ice for 1 minute, and loaded onto Gene Excel 12.5/24 polyacrylamide gels (Pharmacia Biotech, Freiburg, Germany). Following nondenaturing electrophoresis at 600 V, 25 mA, 15 W, 6°C for 80 minutes, DNA fragments were visualized by silver staining using a DNA silver-staining kit according to the manufacturer’s instructions (Pharmacia Biotech). DNA fragments that reproducibly showed mobility shifts were isolated from the acrylamide gels and subjected to seminested PCR, performed under the same conditions as described previously. The nested PCR products were then purified using a QIAquick PCR purification kit (QIAGEN, Hilden, Germany) and cloned into pCR2.1-TOPO vector (Invitrogen, NV Leek, The Netherlands) according to the manufacturer’s instructions. In brief, 10 ng secondary PCR product were ligated with 10 ng vector and transformed into competent TOP10 bacteria by heat shock. The library was plated onto LB plates containing 50 μg/mL ampicillin. Single bacterial clones that were positive in blue/white screening were randomly picked and grown in 2 mL LB medium containing 50 μg/mL ampicillin at 37°C overnight. Subsequent to alkaline lysis, the bacterial lysates were loaded onto silica-gel membranes (QIAprep spin miniprep kit; Qiagen), and plasmid DNA was eluted in low-salt buffer. Plasmid inserts were sequenced at a concentration of 50 ng/μL with a GeneAmp PCR system 9600 using ABI Prism dGTP BigDye Terminator Ready Reaction Kits and AmpliTaq DNA polymerase FS, according to the manufacturer’s protocol (Perkin Elmer; Seqlab, Göttingen, Germany). M13 forward and M13 reverse primers were used for sequencing of both strands of the DNA fragments. The PCR comprised 25 cycles, including a denaturation step at 96°C for 10 seconds, a primer-annealing step at 50°C for 5 seconds, and a chain elongation step at 60°C for 60 seconds. The products were then ethanol precipitated, separated on 4% polyacrylamide 7 mol/L urea gels, and analyzed with an ABI Prism 377 Genetic Analyzer (Perkin-Elmer; Seqlab). Primer sequences were M13 forward primer, 5′-CAA AAG GGT CAG TGC TG-3′; M13 reverse primer, 5′-GTC CTT TGT CGA TAC TG-3′. The resulting sequences were aligned to the known BRAF (accession number NM 004333) and NRAS sequences (accession number AY428630.1) in the NCBI database. The numbering of the BRAF sequence began with the start codon ATG, corresponding to nucleotide positions 1–3. BRAF protein sequences predicted by the cDNA sequences were compared with BRAF wild-type protein sequence (NCBI accession number NP 004324) using BLASTX software at the NCBI. Sequences were confirmed with an independent round of amplification, purification via polyacrylamide gels, and cloning. Statistical analyses were carried out using the 2-tailed Fisher exact test. We detected an exon 15 mutation of the BRAF gene in only 1 of 19 AM cases (case 1). It represented a novel A1800T mutation, resulting in a K600N transition in the BRAF protein (Figure 1). None of the AM displayed the V599E mutation typical for CM and nevi (Table 1). Statistical analysis revealed a highly significant difference of the V599E mutation frequency in AM compared with both CM 2Davies 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. 2002; 417: 949-954Crossref PubMed Scopus (8397) Google Scholar, 4Pollock 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. 2003; 33: 19-20Crossref PubMed Scopus (1387) Google Scholar, 5Lang J. Boxer M. MacKie R. Absence of exon 15 BRAF germline mutations in familial melanoma.Hum Mutat. 2003; 21: 327-330Crossref PubMed Scopus (58) Google Scholar and benign melanocytic lesions 4Pollock 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. 2003; 33: 19-20Crossref PubMed Scopus (1387) Google Scholar (0 of 19 AM vs. 82 of 140 CM and 63 of 77 nevi, respectively; P ≤ .0001). Exon 11 mutation of the BRAF gene was also detected in a single case only (case 2). This also represented a novel mutation, ie, a C1327T mutation leading to R443W conversion (Table 1 and Figure 2). We detected no mutations in exon 2 or 3 of NRAS in the AM.Table 1NRAS and BRAF Mutations in Anorectal MelanomaNo.StageAgeGenderNRASBRAFExon 2Exon 3Exon 11Exon 1560856/94TU68W————19061/96TUaLocal recurrence from 60856/94.70W————30867/96TU86W————37303/89TU58W————17754/00LN60W————31246/01TU82W————12502/97TU45W————05820/02TU59M——C1327T (R443W)—18037/96TU74W————16313/97TU88W————16615/97TU38M————02196/96TU77W————15954/91TU86W————16150/97TU79W————06967/95TU66M————03040/00TU97M———A1800T (K600N)51657/93TU67M————07821/01LN58M————07984/02TU63W————TU, Primary tumor; LN, Lymph node metastases.a Local recurrence from 60856/94. Open table in a new tab Figure 2Analysis of BRAF exon 11 mutations in AM. (A) Histologic appearance of the AM of case 2 with BRAF exon 11 mutation. (B) PCR-SSCP analysis of BRAF exon 11 showing a mobility shift in the tumor (T), not present in the corresponding normal DNA (N). (C) C1327T mutation in the AM of case 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) TU, Primary tumor; LN, Lymph node metastases. AM is a rare and highly malignant melanocytic tumor of the anal canal, which shows similar histologic and immunohistochemical features as CM but lacks an etiology via melanocytic precursor lesions. Intending to compare AM with CM on a molecular level, we examined the frequency of BRAF exon 11 and 15 and NRAS exon 2 and 3 mutations in 19 human AM cases. Our results demonstrate that the V599E exon 15 mutation of BRAF is not associated with AM. This defines a clear molecular and mechanistic difference with CM. BRAF exon 15 mutations were not only detected in CM but also in nonmelanocytic tumors, with an overall frequency of approximately 4%–5%. This included benign colorectal lesions and colorectal carcinomas 7Singer G. Oldt III, R. Cohen Y. Wang B.G. Sidransky D. Kurman R.J. Shih IeM. Mutations in BRAF and KRAS characterize the development of low-grade ovarian serous carcinoma.J Natl Cancer Inst. 2003; 95: 484-486Crossref PubMed Scopus (716) Google Scholar; serous ovarian cancer and borderline tumors 7Singer G. Oldt III, R. Cohen Y. Wang B.G. Sidransky D. Kurman R.J. Shih IeM. Mutations in BRAF and KRAS characterize the development of low-grade ovarian serous carcinoma.J Natl Cancer Inst. 2003; 95: 484-486Crossref PubMed Scopus (716) Google Scholar; papillary thyroid carcinoma 8Cohen 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. 2003; 95: 625-627Crossref PubMed Scopus (787) Google Scholar, 9Kimura 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. 2003; 63: 1454-1457PubMed Google Scholar; cholangiocarcinoma 10Tannapfel A. Sommerer F. Benicke M. Katalinic A. Uhlmann D. Witzigmann H. Hauss J. Wittekind C. Mutations of the BRAF gene in cholangiocarcinoma but not in hepatocellular carcinoma.Gut. 2003; 52: 706-712Crossref PubMed Scopus (311) Google Scholar; and lung, head, and neck cancer. 2Davies 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. 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The motif TVKS and the preceding amino acids represent hot spots of BRAF mutation, and the threonine and serine residues are crucial sites for regulatory phosphorylation. The V599E substitution in CM is thought to mimic the phosphorylated state of the preceding threonine because a negatively charged amino acid is introduced, and this is considered to result in constitutive BRAF activity. 22Mercer K.E. Pritchard C.A. Raf proteins and cancer B-Raf is identified as a mutational target.Biochim Biophys Acta. 2003; 1653: 25-40Crossref PubMed Scopus (409) Google Scholar It is conceivable that the K600N substitution has a similar effect. The second case showed a R443W mutation in BRAF exon 11. This amino acid residue is also located close to regulatory phopsphorylation sites, 22Mercer K.E. Pritchard C.A. Raf proteins and cancer B-Raf is identified as a mutational target.Biochim Biophys Acta. 2003; 1653: 25-40Crossref PubMed Scopus (409) Google Scholar so this alteration possibly exerts similar effects as proposed for the V599E mutation. However, it also has to be considered that both mutations in AM do not introduce negatively charged residues. In conclusion, the absence of the V599E mutation, which frequently occurs in melanocytic lesions of the skin, clearly distinguishes AM from CM at the molecular level, which points to AM as an entity distinct from CM. The functional consequences of the R443W and K600N mutation remain to be determined for AM, but therapeutic strategies aimed at specific inhibition of the V599E BRAF variant 23Hingorani S.R. Jacobetz M.A. Robertson G.P. Herlyn M. Tuveson D.A. Suppression of BRAF(V599E) in human melanoma abrogates transformation.Cancer Res. 2003; 63: 5198-5202PubMed Google Scholar cannot be projected from CM to AM. The authors thank Dr. Bergman and Dr. Helmchen for proofreading the manuscript.
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