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Malignant melanoma arising from a perianal fistula and harbouring a BRAFgene mutation: a case report
© Martinez-Cadenas et al; licensee BioMed Central Ltd. 2011
Received: 28 December 2010
Accepted: 9 August 2011
Published: 9 August 2011
Melanoma of the anal region is a very uncommon disease, accounting for only 0.2-0.3% of all melanoma cases. Mutations of the BRAF gene are usually absent in melanomas occurring in this region as well as in other sun-protected regions. The development of a tumour in a longstanding perianal fistula is also extremely rare. More frequent is the case of a tumour presenting as a fistula, that is, the fistula being a consequence of the cancerous process, although we have found only two cases of fistula-generating melanomas reported in the literature.
Here we report the case of a 38-year-old male who presented with a perianal fistula of four years of evolution. Histopathological examination of the fistulous tract confirmed the presence of malignant melanoma. Due to the small size and the central location of the melanoma inside the fistulous tract, we believe the melanoma reported here developed in the epithelium of the fistula once the latter was already formed. Resected sentinel lymph nodes were negative and the patient, after going through a wide local excision, remains disease-free nine years after diagnosis. DNA obtained from melanoma tissue was analysed by automated direct sequencing and the V600E (T1799A) mutation was detected in exon 15 of the BRAF gene.
Since fistulae experience persistent inflammation, the fact that this melanoma harbours a BRAF mutation strengthens the view that oxidative stress caused by inflammatory processes plays an important role in the genesis of BRAF gene mutations.
Malignant melanomas of the anal region are a rare occurrence, accounting for only around 0.2-0.3% of all melanoma cases and 4% of anal cancers . Diagnosis may be challenging, since perianal melanomas may be disguised as haemorrhoids  and more rarely as fissures  or perianal abscesses . Prognosis is generally very poor , with five-year survival rates ranging between 6.7% and 16% [6, 7].
The BRAF gene encodes a serine/threonine kinase involved in the mitogen-activated protein kinase pathway (MAPK) . Activating BRAF mutations are present in approximately 70% of cutaneous malignant melanomas  and 82% of melanocytic naevi . More than 90% of BRAF mutations involve a single point mutation, T1799A, in codon 600 of exon 15, leading to a V600E amino acid substitution [9, 10]. However, several studies have shown that BRAF mutations are very uncommon in melanomas arising in sun-protected areas [11–13]. These findings have suggested an association between the presence of BRAF mutations in malignant melanomas and ultraviolet (UV) light exposure.
In this report we describe a case of malignant melanoma presenting in a perianal fistula. To the authors' knowledge, this clinical presentation has not been previously described in the scientific literature. To add to our knowledge of this rare entity, we have determined the mutational status of the BRAF gene in tumour tissue of the patient.
In June 2002, once the diagnosis of melanoma in perianal fistula (Clark level III) had been confirmed by the pathologist, the patient went through a surgical extension of excision margins (1 cm) and a sentinel lymph node biopsy (one right and two left inguinal nodes). Both the extended margins and the sentinel lymph nodes produced negative results for tumour cells.
In order to discard the possibility of metastatic melanoma, the patient was also subjected to an abdominal ultrasound examination and a whole body CT scan, both without relevant findings. Serum levels of S-100 protein, a widely used melanoma marker, were also negative. The patient went through his latest follow-up in December 2010, and at the time of this report he remains free of disease.
Five μm sections from tumour tissue were stained with a standard Haematoxylin-Eosin protocol. Histological slides were examined with a Leica DM2000 microscope and micrographs were taken using a Leica DMD108 microimaging device with integrated camera (Leica Microsystems, Wetzlar, Germany). Slides were then cleaned with 99% ethanol for 15 minutes and left to air-dry overnight. Melanoma tissue was then manually dissected from surrounding normal tissue with a sterile blade. DNA extraction from dissected melanoma tissue was performed using the ChargeSwitch gDNA Tissue Kit (Invitrogen, Carlsbad, USA) according to the manufacturer's instructions.
Detection of the V600Emutation
Melanoma DNA, a control sample (from peripheral blood of the same patient) and DNA extracted from melanoma cell line A375 were analysed for the V600E mutation at nucleotide 1799 of the BRAF gene by direct automated DNA sequencing. PCR primer sequences used to amplify a 102-bp fragment containing nucleotide 1799 of exon 15 were: 5'-GAAGACCTCACAGTAAAAATAGG TGA-3' (sense) and 5'-CCACAAAATGGATCCAGACA-3' (antisense). PCR reactions were carried out using about 100 ng of melanoma DNA as template. Thermocycling conditions included a denaturation step at 95°C for 8 minutes, followed by 40 cycles of 95°C for 30 seconds, 56°C for 45 seconds and 72°C for 1 minute, plus a final extension cycle of 72°C for 10 minutes. Amplified products were purified using ExoSAP-IT (USB Corporation, Cleveland, USA) according to the manufacturer's instructions. Purified PCR products were then run on an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, USA) and analysed using software supplied by the manufacturer.
All new sequence data contained in this study has been deposited in the GenBank sequence database [GenBank:HQ224878].
Appropriate ethical approval for this research was obtained from the Ethics and Clinical Research Committee (CEIC) of the Castellon Province Hospital. Written informed consent was obtained from the patient for the study and its reportings.
The development of a tumour inside a longstanding fistula is an extremely infrequent event. More frequent is the case of a tumour presenting as a fistula, that is, the fistula being a consequence of the cancerous process. Occasionally, the inflammation and necrosis caused by the development and progression of a tumour stimulates the formation of a fistula. However, there are instances in which the fistula is not a secondary manifestation of the neoplasia, and its formation predates the development of the tumour. In these cases, the neoplastic transformation arises inside the longstanding fistula, probably due to the latter's chronic inflammatory nature . We have found several cases in the literature in which a variety of different tumour types have developed inside a fistula once the latter was already formed, such as squamous cell carcinomas [15–17], basal cell carcinomas [18, 19] and mucinous adenocarcinomas [17, 20–22].
As far as the authors are aware, the occurrence of a melanoma within a fistula has not been previously described in the literature. We have found two published cases of melanomas presenting with fistulous formations, one concerning a biliary tract fistula  and another one presenting as a urethral fistula , in which the appearances of the fistulas were a consequence of the cancerous process.
Anal melanoma is an uncommon disease that affects patients of advanced age, usually appearing in the sixth or seventh decade of life [25, 26]. However, Cagir and cols. found an increased incidence of anorectal melanomas in males aged 25 to 44 years living in the San Francisco area , and they proposed HIV infection as a risk factor for anorectal melanoma. But the HIV test in our patient (who was 38 years of age at diagnosis) was negative, and he had neither a previous history nor signs of other sexually-transmitted diseases.
The fact that this melanoma originated in a perianal location and inside a fistula also ruled out UV light exposure as a possible aetiological factor. Classic genetic mutations caused by UV exposure comprise C>T and CC>TT transitions, although UV exposure generates oxygen-free radicals that also damage DNA and give rise to other types of base substitutions . Even though BRAF mutations occur more frequently in melanomas linked to sun exposure, the BRAF gene does not contain C>T or CC>TT transitions typical of UV-induced damage. The commonest BRAF mutation by far is T1799A (V600E), a T>A transversion present in more than 90% of all BRAF-mutated melanomas and naevi.
Due to the detection of the V600E BRAF mutation in this fistula melanoma, it was decided not to look for alterations in genes such as CCND1 or KIT, more frequently mutated in acral and mucosal melanomas , since concomitant mutations in BRAF and in genes such as KIT or CCND1 are extremely rare . Regarding this, it has to be understood that, although this melanoma arose in the perianal region, it was not a case of mucosal melanoma, since it did not originate in the anal mucous membrane of the patient.
BRAF mutations, including T1799A, have occasionally been found in anal mucosal melanomas  and in other tumours not associated with UV exposure , such as thyroid cancer [31, 32], colorectal cancer [33–35], pancreatic cancer [36, 37] or ovarian cancer [38, 39]. The development of all these tumour types may, in turn, be related to inflammatory processes , in the same way as cutaneous melanoma is associated with inflammation of the skin caused by UV exposure . Accordingly, oxidative stress caused by inflammation may be in part responsible for mutations in the BRAF gene . Since fistulae are environments that experience permanent inflammatory processes, the fact that the fistula melanoma reported here is carrying the T1799A transversion supports the hypothesis that oxidative damage caused by free radicals plays a role in the genesis of BRAF mutations.
Obviously, we are not presuming that all inflammation must cause BRAF mutations, but that persistent or chronic inflammatory processes in the absence of UV exposure (such as thyroiditis in thyroid cancer , chronic pancreatitis in pancreatic cancer , chronic bowel inflammation in colon cancer  and, in our case, a longstanding fistula in a melanoma), can probably make the BRAF gene more susceptible to genetic mutations via an as yet unidentified oxidative stress lesion.
As to the management of the perianal fistula melanoma, this case illustrates the benefits provided by the use of sentinel lymph node biopsy. Similarly to anorectal melanoma cases , the sentinel lymph nodes were located in the groin, not in the presacral region. As mentioned above, all three nodes dissected were free of tumour cells. After ruling out abdominoperineal resection , wide local excision (WLE) appears to obtain general good results when used as the initial treatment of choice in melanomas of the anal region [5, 26]. Our case can only corroborate this, since nine years after WLE our patient remains healthy and without signs of relapse.
The fact that the melanoma described in this report arose inside a fistula may set it apart from other anal and perianal melanomas, as well as from mucosal melanomas in general. In our opinion, and due to the symptoms occasioned by the presence of the fistula, this melanoma was caught at a very early stage compared to other anorectal or perianal melanomas. The presence of a BRAF mutation, usually absent in other anal melanomas, also suggests different aetiological factors contributing to the development of this particular melanoma, with inflammatory processes and oxidative stress damage being possibly among them.
Acknowledgements and Funding
We gratefully acknowledge Dr. Jose Carlos Garcia-Borron and Dr. Maria Carmen Turpin from the Department of Biochemistry and Molecular Biology, University of Murcia, Spain, for kindly providing us with the A375 melanoma cell line.
This work was supported by a grant from the Fundacion de la CV Hospital Provincial de Castellon to C.M-C. The authors declare that the funding body, the Fundacion de la CV Hospital Provincial de Castellon, had no role whatsoever in the design, data collection, data analyses, interpretation of results, and/or manuscript writing or publication of this study.
- Weinstock MA: Epidemiology and prognosis of anorectal melanoma. Gastroenterology. 1993, 104: 174-8.PubMedGoogle Scholar
- Fripp VT, Esquivel J, Cerruto CA: Perianal melanoma disguised as hemorrhoids: case report and discussion. J Natl Med Assoc. 2005, 97: 726-31.PubMedPubMed CentralGoogle Scholar
- Debets JM, Lijnen RL, Willig AP: [Diagnostic image (84). A woman with persisting perianal fissures]. Ned Tijdschr Geneeskd. 2002, 146: 718-PubMedGoogle Scholar
- Yadoo S, Kessler E: Malignant melanoma presenting as peri-anal abscess. Am J Proctol. 1971, 22: 108-10.PubMedGoogle Scholar
- Thibault C, Sagar P, Nivatvongs S, Ilstrup DM, Wolff BG: Anorectal melanoma - an incurable disease?. Dis Colon Rectum. 1997, 40: 661-8. 10.1007/BF02140894.View ArticlePubMedGoogle Scholar
- Cooper PH, Mills SE, Allen MS: Malignant melanoma of the anus: report of 12 patients and analysis of 255 additional cases. Dis Colon Rectum. 1982, 25: 693-703. 10.1007/BF02629543.View ArticlePubMedGoogle Scholar
- Brady MS, Kavolius JP, Quan SH: Anorectal melanoma. A 64-year experience at Memorial Sloan-Kettering Cancer Center. Dis Colon Rectum. 1995, 38: 146-51. 10.1007/BF02052442.View ArticlePubMedGoogle Scholar
- Peyssonnaux C, Eychène A: The Raf/MEK/ERK pathway: new concepts of activation. Biol Cell. 2001, 93: 53-62. 10.1016/S0248-4900(01)01125-X.View ArticlePubMedGoogle Scholar
- Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ, 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 BA, Cooper C, Shipley J, et al: Mutations of the BRAF gene in human cancer. Nature. 2002, 417: 949-54. 10.1038/nature00766.View ArticlePubMedGoogle Scholar
- Pollock PM, Harper UL, Hansen KS, Yudt LM, Stark M, Robbins CM, Moses TY, Hostetter G, Wagner U, Kakareka J, Salem G, Pohida T, Heenan P, Duray P, Kallioniemi O, Hayward NK, Trent JM, Meltzer PS: High frequency of BRAF mutations in nevi. Nat Genet. 2003, 33: 19-20. 10.1038/ng1054.View ArticlePubMedGoogle Scholar
- Cohen Y, Rosenbaum E, Begum S, Goldenberg D, Esche C, Lavie O, Sidransky D, Westra WH: Exon 15 BRAF mutations are uncommon in melanomas arising in non sun-exposed sites. Clin Cancer Res. 2004, 10: 3444-7. 10.1158/1078-0432.CCR-03-0562.View ArticlePubMedGoogle Scholar
- Edwards RH, Ward MR, Wu H, Medina CA, Brose MS, Volpe P, Nussen-Lee S, Haupt HM, Martin AM, Herlyn M, Lessin SR, Weber BL: Absence of BRAF mutations in UV-protected mucosal melanomas. J Med Genet. 2004, 41: 270-2. 10.1136/jmg.2003.016667.View ArticlePubMedPubMed CentralGoogle Scholar
- Wong CW, Fan YS, Chan TL, Chan AS, Ho LC, Ma TK, Yuen ST, Leung SY: BRAF and NRAS mutations are uncommon in melanomas arising in diverse internal organs. J Clin Pathol. 2005, 58: 640-4. 10.1136/jcp.2004.022509.View ArticlePubMedPubMed CentralGoogle Scholar
- Welch GH, Finlay IG: Neoplastic transformation in longstanding fistula-in-ano. Postgrad Med J. 1987, 63: 503-4. 10.1136/pgmj.63.740.503.View ArticlePubMedPubMed CentralGoogle Scholar
- Abboud B, Ingea H, Tayar C, Abadjian G: [Epidermoid carcinoma developing in a chronic anal fistula]. Presse Med. 2000, 29: 786-7.PubMedGoogle Scholar
- Seya T, Tanaka N, Shinji S, Yokoi K, Oguro T, Oaki Y, Ishiwata T, Naito Z, Tajiri T: Squamous cell carcinoma arising from recurrent anal fistula. J Nippon Med Sch. 2007, 74: 319-24. 10.1272/jnms.74.319.View ArticlePubMedGoogle Scholar
- Thomas M, Bienkowski R, Vandermeer TJ, Trostle D, Cagir B: Malignant transformation in perianal fistulas of Crohn's disease: a systematic review of literature. J Gastrointest Surg. 2010, 14: 66-73. 10.1007/s11605-009-1061-x.View ArticlePubMedGoogle Scholar
- Mees K, Löhrs U: [Basal cell carcinoma developing in a preauricular fistula (author's transl)]. Laryngol Rhinol Otol (Stuttg). 1978, 57: 359-62.Google Scholar
- Kulaylat MN, Doerr RJ, Karamanoukian H, Barrios G: Basal cell carcinoma arising in a fistula-in-ano. Am Surg. 1996, 62: 1000-2.PubMedGoogle Scholar
- Erhan Y, Sakarya A, Aydede H, Demir A, Seyhan A, Atici E: A case of large mucinous adenocarcinoma arising in a long-standing fistula-in-ano. Dig Surg. 2003, 20: 69-71. 10.1159/000068857.View ArticlePubMedGoogle Scholar
- Venclauskas L, Saladzinskas Z, Tamelis A, Pranys D, Pavalkis D: Mucinous adenocarcinoma arising in an anorectal fistula. Medicina (Kaunas). 2009, 45: 286-90.Google Scholar
- Iesalnieks I, Gaertner WB, Glaβ H, Strauch U, Hipp M, Agha A, Schlitt HJ: Fistula-associated anal adenocarcinoma in Crohn's disease. Inflamm Bowel Dis. 2010, 16: 1643-1648. 10.1002/ibd.21228.View ArticlePubMedGoogle Scholar
- Larmi TK: Malignant melanoma of the gallbladder. Report of a case resulting in an external biliary fistula. Acta Chir Scand. 1960, 119: 502-5.PubMedGoogle Scholar
- Morita T, Suzuki H, Goto K, Hirota N, Tokue A: Primary malignant melanoma of male urethra with fistula formation. Urol Int. 1991, 46: 114-5. 10.1159/000281796.View ArticlePubMedGoogle Scholar
- Droesch JT, Flum DR, Mann GN: Wide local excision or abdominoperineal resection as the initial treatment for anorectal melanoma?. Am J Surg. 2005, 189: 446-9. 10.1016/j.amjsurg.2005.01.022.View ArticlePubMedGoogle Scholar
- Homsi J, Garrett C: Melanoma of the anal canal: a case series. Dis Colon Rectum. 2007, 50: 1004-10. 10.1007/s10350-007-0242-5.View ArticlePubMedGoogle Scholar
- Cagir B, Whiteford MH, Topham A, Rakinic J, Fry RD: Changing epidemiology of anorectal melanoma. Dis Colon Rectum. 1999, 42: 1203-8. 10.1007/BF02238576.View ArticlePubMedGoogle Scholar
- Jhappan C, Noonan FP, Merlino G: Ultraviolet radiation and cutaneous malignant melanoma. Oncogene. 2003, 22: 3099-112. 10.1038/sj.onc.1206450.View ArticlePubMedGoogle Scholar
- Curtin JA, Busam K, Pinkel D, Bastian BC: Somatic activation of KIT in distinct subtypes of melanoma. J Clin Oncol. 2006, 24: 4340-6. 10.1200/JCO.2006.06.2984.View ArticlePubMedGoogle Scholar
- Helmke BM, Mollenhauer J, Herold-Mende C, Benner A, Thome M, Gassler N, Wahl W, Lyer S, Poustka A, Otto HF, Deichmann M: BRAF mutations distinguish anorectal from cutaneous melanoma at the molecular level. Gastroenterology. 2004, 127: 1815-20. 10.1053/j.gastro.2004.08.051.View ArticlePubMedGoogle Scholar
- Kimura ET, Nikiforova MN, Zhu Z, Knauf JA, Nikiforov YE, Fagin JA: 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: 1454-7.PubMedGoogle Scholar
- Xing M: BRAF mutation in papillary thyroid cancer: pathogenic role, molecular bases, and clinical implications. Endocr Rev. 2007, 28: 742-62. 10.1210/er.2007-0007.View ArticlePubMedGoogle Scholar
- Li WQ, Kawakami K, Ruszkiewicz A, Bennett G, Moore J, Iacopetta B: BRAF mutations are associated with distinctive clinical, pathological and molecular features of colorectal cancer independently of microsatellite instability status. Mol Cancer. 2006, 5: 2-10.1186/1476-4598-5-2.View ArticlePubMedGoogle Scholar
- Jass JR: Classification of colorectal cancer based on correlation of clinical, morphological and molecular features. Histopathology. 2007, 50: 113-30. 10.1111/j.1365-2559.2006.02549.x.View ArticlePubMedGoogle Scholar
- Nagasaka T, Koi M, Kloor M, Gebert J, Vilkin A, Nishida N, Shin SK, Sasamoto H, Tanaka N, Matsubara N, Boland CR, Goel A: Mutations in both KRAS and BRAF may contribute to the methylator phenotype in colon cancer. Gastroenterology. 2008, 134: 1950-60. 10.1053/j.gastro.2008.02.094.View ArticlePubMedPubMed CentralGoogle Scholar
- Calhoun ES, Jones JB, Ashfaq R, Adsay V, Baker SJ, Valentine V, Hempen PM, Hilgers W, Yeo CJ, Hruban RH, Kern SE: BRAF and FBXW7 (CDC4, FBW7, AGO, SEL10) mutations in distinct subsets of pancreatic cancer: potential therapeutic targets. Am J Pathol. 2003, 163: 1255-60. 10.1016/S0002-9440(10)63485-2.View ArticlePubMedPubMed CentralGoogle Scholar
- Schönleben F, Qiu W, Bruckman KC, Ciau NT, Li X, Lauerman MH, Frucht H, Chabot JA, Allendorf JD, Remotti HE, Su GH: BRAF and KRAS gene mutations in intraductal papillary mucinous neoplasm/carcinoma (IPMN/IPMC) of the pancreas. Cancer Lett. 2007, 249: 242-8. 10.1016/j.canlet.2006.09.007.View ArticlePubMedGoogle Scholar
- Singer G, Oldt R, Cohen Y, Wang BG, Sidransky D, Kurman RJ, Shih IeM: Mutations in BRAF and KRAS characterize the development of low-grade ovarian serous carcinoma. J Natl Cancer Inst. 2003, 95: 484-6. 10.1093/jnci/95.6.484.View ArticlePubMedGoogle Scholar
- Mayr D, Hirschmann A, Löhrs U, Diebold J: KRAS and BRAF mutations in ovarian tumours: a comprehensive study of invasive carcinomas, borderline tumours and extraovarian implants. Gynecol Oncol. 2006, 103: 883-7. 10.1016/j.ygyno.2006.05.029.View ArticlePubMedGoogle Scholar
- Rees JL: The genetics of sun sensitivity in humans. Am J Hum Genet. 2004, 75: 739-51. 10.1086/425285.View ArticlePubMedPubMed CentralGoogle Scholar
- Aust DE, Haase M, Dobryden L, Markwarth A, Löhrs U, Wittekind C, Baretton GB, Tannapfel A: Mutations of the BRAF gene in ulcerative colitis-related colorectal carcinoma. Int J Cancer. 2005, 115: 673-7. 10.1002/ijc.20925.View ArticlePubMedGoogle Scholar
- Olsha O, Mintz A, Gimon Z, Gold Deutch R, Rabin I, Halevy A, Reissman P: Anal melanoma in the era of sentinel lymph node mapping: a diagnostic and therapeutic challenge. Tech Coloproctol. 2005, 9: 60-2. 10.1007/s10151-005-0196-3.View ArticlePubMedGoogle Scholar
- Yap LB, Neary P: A comparison of wide local excision with abdominoperineal resection in anorectal melanoma. Melanoma Res. 2004, 14: 147-50. 10.1097/00008390-200404000-00012.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/343/prepub
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