original articles 12. Grotz TE, Huebner M, Pockaj BA et al. Limitations of lymph node ratio, evidencebased benchmarks, and the importance of a thorough lymph node dissection in melanoma. Ann Surg Oncol 2013 September 18 [epub ahead of print], doi: 10.1245/s10434-013-3186-0. 13. Chan AD, Essner R, Wanek LA et al. Judging the therapeutic value of lymph node dissections for melanoma. J Am Coll Surg 2000; 191: 16–22; discussion 22–13. 14. Galliot-Repkat C, Cailliod R, Trost O et al. The prognostic impact of the extent of lymph node dissection in patients with stage III melanoma. Eur J Surg Oncol 2006; 32: 790–794. 15. Wong SL. Lymph node evaluation in colon cancer: assessing the link between quality indicators and quality. JAMA 2011; 306: 1139–1141. 16. Cascinelli N, Bombardieri E, Bufalino R et al. Sentinel and nonsentinel node status in stage IB and II melanoma patients: two-step prognostic indicators of survival. J Clin Oncol 2006; 24: 4464–4471. 17. Gervasoni JE, Jr, Sbayi S, Cady B. Role of lymphadenectomy in surgical treatment of solid tumors: an update on the clinical data. Ann Surg Oncol 2007; 14: 2443–2462.
Annals of Oncology 18. Balch CM, Gershenwald JE, Soong SJ et al. Final version of 2009 AJCC melanoma staging and classification. J Clin Oncol 2009; 27: 6199–6206. 19. Spillane AJ, Cheung BL, Stretch JR et al. Proposed quality standards for regional lymph node dissections in patients with melanoma. Ann Surg 2009; 249: 473–480. 20. Pasquali S, Spillane AJ, de Wilt JH et al. Surgeons’ opinions on lymphadenectomy in melanoma patients with positive sentinel nodes: a worldwide web-based survey. Ann Surg Oncol 2012; 258: 152–157. 21. Spillane AJ, Pasquali S, Haydu LE et al. Patterns of recurrence and survival after lymphadenectomy in melanoma patients: clarifying the effects of timing of surgery and lymph node tumor burden. Ann Surg Oncol 2013 September 20 [epub ahead of print], doi: 10.1245/s10434-013-3253-6. 22. Mocellin S, Lens MB, Pasquali S et al. Interferon alpha for the adjuvant treatment of cutaneous melanoma. Cochrane Database Syst Rev 2013; 6: CD008955. 23. Burmeister BH, Henderson MA, Ainslie J et al. Adjuvant radiotherapy versus observation alone for patients at risk of lymph-node field relapse after therapeutic lymphadenectomy for melanoma: a randomised trial. Lancet Oncol 2012; 13: 589–597.
Metastatic melanoma of unknown primary resembles the genotype of cutaneous melanomas F. Egberts1*, I. Bergner1, S. Krüger2, J. Haag2, H. M. Behrens2, A. Hauschild1 & C. Röcken2 1 Department of Dermatology, Schleswig-Holstein University Hospital, Campus Kiel, Kiel; 2Department of Pathology, Schleswig-Holstein University Hospital, Campus Kiel, Kiel, Germany
Received 7 June 2013; revised 16 August 2013; accepted 22 August 2013
Background: Although 90% of all melanomas are of cutaneous origin, some patients present with melanoma metastases of unknown origin (MUP). Commonly, in these patients an extensive search for the primary tumor is carried out. In the past, genetic analyses have shown substantial differences in pathogenetic mutations among cutaneous, acral and mucosal melanomas. The aim of this study was to assess the mutational status of MUP in order to better characterize the putative origin of the primary tumor and to evaluate potential prognostic factors. Patients and methods: The medical records of 44 patients with MUP were analyzed and a survival analysis was conducted. In total, 66 paraffin samples of 44 patients were analyzed, and in 15 patients multiple metastases were tested. Mutational analysis of the BRAF, NRAS and KIT genes was carried out. Results: Twenty-three patients (52.3%) had a mutation in the BRAF gene and 12 patients (23.8%) had a mutation in the NRAS gene. There were neither mutations in the KIT gene. In patients with multiple samples, there was 100% consistency regarding mutational status among the different metastases. The median overall survival (OS) was 86.4 months (39–134). The American Joint Committee on Cancer stage at first diagnosis of metastatic melanoma (stage III versus IV) was significantly associated with OS (P < 0.001), BRAF or NRAS mutation status had no significant prognostic impact on clinical outcomes. Conclusions: MUP resembles the genotype of cutaneous melanoma and not that of mucosal melanomas. Key words: melanoma of unknown primary, genotype, BRAF, metastatic melanoma
*Correspondence to: Dr Friederike Egberts, Department of Dermatology, University Hospital Schleswig-Holstein, Campus Kiel, Schittenhelmstr.7, 24105 Kiel, Germany. Tel: +49-431-597-1512; Fax: +49-431-597-1853; E-mail: [email protected]
© The Author 2013. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: [email protected].
Downloaded from http://annonc.oxfordjournals.org/ at Oakland University on June 10, 2015
Annals of Oncology 25: 246–250, 2014 doi:10.1093/annonc/mdt411 Published online 24 November 2013
original articles
Annals of Oncology
mutation testing and DNA sequence analysis
Although 90% of all melanomas are of cutaneous origin, some patients present with melanoma metastases without evident primary tumor site. These melanomas of unknown primary (MUP) were first described by Das Gupta in 1963 [1]. Usually, these patients present with locoregional disease in the lymph nodes, the soft tissue or with disseminated distant metastases. Rarely, the primary melanoma can be detected at extracutaneous sites like the eyes, sinonasal, vulvovaginal or gastrointestinal areas of the body. In most of the cases, however, the primary melanoma cannot be found. In 2005, Curtin et al. described four types of melanomas at different sites of the body and with different levels of sun exposure, which could be distinguished by distinct mutation patterns [2]. Activating oncogenic mutations in the BRAF gene were reported in ∼47% of all cutaneous melanomas and somatic mutations of NRAS in 20% [3]. In mucosal melanomas, however, BRAF mutations are usually absent [4, 5], but NRAS mutations can be found in 10% of all mucosal melanomas and, notably, in up to 43% of all vaginal melanomas [6]. Moreover, mutations and/or copy number increases of KIT were found in 20%–40% of mucosal melanomas [4, 5]. Since the pathogenesis and origin of MUP are poorly understood, the genotype of these tumors is of great interest and may help to fill this gap of information. In this study, we assessed the genotype of occult metastatic melanomas with regard to the following: (i) does the genotype help to identify the putative primary tumor site of MUP, (ii) is the genotype of MUP prognostically relevant with regard to progression-free (PFS) and overall survival (OS).
Paraffin-embedded melanoma tissue from every patient was analyzed for the presence of genetic alterations. In total, 66 paraffin samples of 44 patients were analyzed, and in 15 patients, two or more samples were tested. Genomic DNA was extracted from FFPE tissue using the QIAamp DNA mini kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. To ensure a tumor cell content of >40% in the analyzed specimens, the tissue sections were manually microdissected before DNA extraction. Mutational analyses of codons 12, 13 and 61 of the NRAS gene and codon 600 of the BRAF gene were carried out by pyrosequencing as described previously [8]. Mutational analyses of the KIT gene (exons 9, 11, 13 and 17) were done by dye terminator cycle sequencing. The complete exons were amplified using a nested polymerase chain reaction approach. Primer sequences used for amplification are shown in supplementary Table S1, available at Annals of Oncology online. PCR products were purified using the Nucleospin Extract II kit (Macherey-Nagel, Düren, Germany) and both DNA strands sequenced with standard M13 forward and reverse sequencing primers and the BigDye Terminator v1.1 Cycle Sequencing kit (Life Technologies, Darmstadt, Germany). The sequencing products were purified using the NucleoSEQ kit (Macherey-Nagel) and analyzed on a 3500 Genetic Analyzer (LifeTechnologies).
patients and methods patients and data collection Forty-four patients with metastatic MUP, treated at the Department of Dermatology, University Hospital of Schleswig-Holstein, Campus Kiel, from 1998 until 2011, formed the study cohort. In all patients, no primary melanoma was found on thorough clinical examination, including total skin, ocular, otolaryngologic, gynecologic examination and colonoscopy. None of the patients had a history of a previously removed mole or birthmark. Basic clinical and tumour-specific data were collected from clinical charts. The following examinations were routinely carried out in all patients: computed tomography of the chest, abdomen and pelvis, magnetic resonance imaging of the brain. Localized nodal and/or skin disease was classified as American Joint Committee on Cancer (AJCC) stage III, and visceral or widespread metastases were classified as AJCC stage IV disease [7]. All patients provided written informed consent.
histology Histological diagnosis was based on the assessment of formalin-fixed and paraffin-embedded (FFPE) tumor samples using hematoxylin and eosinstaining. All tumor samples were reviewed before study inclusion by board certified histopathologists and the diagnosis of a malignant melanoma was confirmed in every tumor sample analyzed genetically.
Volume 25 | No. 1 | January 2014
external quality assurance The BRAF mutational assay was certified successfully by the quality assurance program of the German Society of Pathology and the Bundesverband Deutscher Pathologen e.V.
statistical methods The data were analyzed using SPSS for Windows (Version 20.0, IBM, New York). Baseline descriptive statistics included proportions and mean or median values, as appropriate by data distribution. Fisher’s exact test was used to test for correlations. In the case of ordinal variables, Kendall’s tau test was used. Estimated survival curves were constructed by the Kaplan–Meier method. Differences between the curves were evaluated using the log-rank test. Probabilities of
Annals of Oncology 18. Balch CM, Gershenwald JE, Soong SJ et al. Final version of 2009 AJCC melanoma staging and classification. J Clin Oncol 2009; 27: 6199–6206. 19. Spillane AJ, Cheung BL, Stretch JR et al. Proposed quality standards for regional lymph node dissections in patients with melanoma. Ann Surg 2009; 249: 473–480. 20. Pasquali S, Spillane AJ, de Wilt JH et al. Surgeons’ opinions on lymphadenectomy in melanoma patients with positive sentinel nodes: a worldwide web-based survey. Ann Surg Oncol 2012; 258: 152–157. 21. Spillane AJ, Pasquali S, Haydu LE et al. Patterns of recurrence and survival after lymphadenectomy in melanoma patients: clarifying the effects of timing of surgery and lymph node tumor burden. Ann Surg Oncol 2013 September 20 [epub ahead of print], doi: 10.1245/s10434-013-3253-6. 22. Mocellin S, Lens MB, Pasquali S et al. Interferon alpha for the adjuvant treatment of cutaneous melanoma. Cochrane Database Syst Rev 2013; 6: CD008955. 23. Burmeister BH, Henderson MA, Ainslie J et al. Adjuvant radiotherapy versus observation alone for patients at risk of lymph-node field relapse after therapeutic lymphadenectomy for melanoma: a randomised trial. Lancet Oncol 2012; 13: 589–597.
Metastatic melanoma of unknown primary resembles the genotype of cutaneous melanomas F. Egberts1*, I. Bergner1, S. Krüger2, J. Haag2, H. M. Behrens2, A. Hauschild1 & C. Röcken2 1 Department of Dermatology, Schleswig-Holstein University Hospital, Campus Kiel, Kiel; 2Department of Pathology, Schleswig-Holstein University Hospital, Campus Kiel, Kiel, Germany
Received 7 June 2013; revised 16 August 2013; accepted 22 August 2013
Background: Although 90% of all melanomas are of cutaneous origin, some patients present with melanoma metastases of unknown origin (MUP). Commonly, in these patients an extensive search for the primary tumor is carried out. In the past, genetic analyses have shown substantial differences in pathogenetic mutations among cutaneous, acral and mucosal melanomas. The aim of this study was to assess the mutational status of MUP in order to better characterize the putative origin of the primary tumor and to evaluate potential prognostic factors. Patients and methods: The medical records of 44 patients with MUP were analyzed and a survival analysis was conducted. In total, 66 paraffin samples of 44 patients were analyzed, and in 15 patients multiple metastases were tested. Mutational analysis of the BRAF, NRAS and KIT genes was carried out. Results: Twenty-three patients (52.3%) had a mutation in the BRAF gene and 12 patients (23.8%) had a mutation in the NRAS gene. There were neither mutations in the KIT gene. In patients with multiple samples, there was 100% consistency regarding mutational status among the different metastases. The median overall survival (OS) was 86.4 months (39–134). The American Joint Committee on Cancer stage at first diagnosis of metastatic melanoma (stage III versus IV) was significantly associated with OS (P < 0.001), BRAF or NRAS mutation status had no significant prognostic impact on clinical outcomes. Conclusions: MUP resembles the genotype of cutaneous melanoma and not that of mucosal melanomas. Key words: melanoma of unknown primary, genotype, BRAF, metastatic melanoma
*Correspondence to: Dr Friederike Egberts, Department of Dermatology, University Hospital Schleswig-Holstein, Campus Kiel, Schittenhelmstr.7, 24105 Kiel, Germany. Tel: +49-431-597-1512; Fax: +49-431-597-1853; E-mail: [email protected]
© The Author 2013. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: [email protected].
Downloaded from http://annonc.oxfordjournals.org/ at Oakland University on June 10, 2015
Annals of Oncology 25: 246–250, 2014 doi:10.1093/annonc/mdt411 Published online 24 November 2013
original articles
Annals of Oncology
mutation testing and DNA sequence analysis
Although 90% of all melanomas are of cutaneous origin, some patients present with melanoma metastases without evident primary tumor site. These melanomas of unknown primary (MUP) were first described by Das Gupta in 1963 [1]. Usually, these patients present with locoregional disease in the lymph nodes, the soft tissue or with disseminated distant metastases. Rarely, the primary melanoma can be detected at extracutaneous sites like the eyes, sinonasal, vulvovaginal or gastrointestinal areas of the body. In most of the cases, however, the primary melanoma cannot be found. In 2005, Curtin et al. described four types of melanomas at different sites of the body and with different levels of sun exposure, which could be distinguished by distinct mutation patterns [2]. Activating oncogenic mutations in the BRAF gene were reported in ∼47% of all cutaneous melanomas and somatic mutations of NRAS in 20% [3]. In mucosal melanomas, however, BRAF mutations are usually absent [4, 5], but NRAS mutations can be found in 10% of all mucosal melanomas and, notably, in up to 43% of all vaginal melanomas [6]. Moreover, mutations and/or copy number increases of KIT were found in 20%–40% of mucosal melanomas [4, 5]. Since the pathogenesis and origin of MUP are poorly understood, the genotype of these tumors is of great interest and may help to fill this gap of information. In this study, we assessed the genotype of occult metastatic melanomas with regard to the following: (i) does the genotype help to identify the putative primary tumor site of MUP, (ii) is the genotype of MUP prognostically relevant with regard to progression-free (PFS) and overall survival (OS).
Paraffin-embedded melanoma tissue from every patient was analyzed for the presence of genetic alterations. In total, 66 paraffin samples of 44 patients were analyzed, and in 15 patients, two or more samples were tested. Genomic DNA was extracted from FFPE tissue using the QIAamp DNA mini kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. To ensure a tumor cell content of >40% in the analyzed specimens, the tissue sections were manually microdissected before DNA extraction. Mutational analyses of codons 12, 13 and 61 of the NRAS gene and codon 600 of the BRAF gene were carried out by pyrosequencing as described previously [8]. Mutational analyses of the KIT gene (exons 9, 11, 13 and 17) were done by dye terminator cycle sequencing. The complete exons were amplified using a nested polymerase chain reaction approach. Primer sequences used for amplification are shown in supplementary Table S1, available at Annals of Oncology online. PCR products were purified using the Nucleospin Extract II kit (Macherey-Nagel, Düren, Germany) and both DNA strands sequenced with standard M13 forward and reverse sequencing primers and the BigDye Terminator v1.1 Cycle Sequencing kit (Life Technologies, Darmstadt, Germany). The sequencing products were purified using the NucleoSEQ kit (Macherey-Nagel) and analyzed on a 3500 Genetic Analyzer (LifeTechnologies).
patients and methods patients and data collection Forty-four patients with metastatic MUP, treated at the Department of Dermatology, University Hospital of Schleswig-Holstein, Campus Kiel, from 1998 until 2011, formed the study cohort. In all patients, no primary melanoma was found on thorough clinical examination, including total skin, ocular, otolaryngologic, gynecologic examination and colonoscopy. None of the patients had a history of a previously removed mole or birthmark. Basic clinical and tumour-specific data were collected from clinical charts. The following examinations were routinely carried out in all patients: computed tomography of the chest, abdomen and pelvis, magnetic resonance imaging of the brain. Localized nodal and/or skin disease was classified as American Joint Committee on Cancer (AJCC) stage III, and visceral or widespread metastases were classified as AJCC stage IV disease [7]. All patients provided written informed consent.
histology Histological diagnosis was based on the assessment of formalin-fixed and paraffin-embedded (FFPE) tumor samples using hematoxylin and eosinstaining. All tumor samples were reviewed before study inclusion by board certified histopathologists and the diagnosis of a malignant melanoma was confirmed in every tumor sample analyzed genetically.
Volume 25 | No. 1 | January 2014
external quality assurance The BRAF mutational assay was certified successfully by the quality assurance program of the German Society of Pathology and the Bundesverband Deutscher Pathologen e.V.
statistical methods The data were analyzed using SPSS for Windows (Version 20.0, IBM, New York). Baseline descriptive statistics included proportions and mean or median values, as appropriate by data distribution. Fisher’s exact test was used to test for correlations. In the case of ordinal variables, Kendall’s tau test was used. Estimated survival curves were constructed by the Kaplan–Meier method. Differences between the curves were evaluated using the log-rank test. Probabilities of
