Tumor Biol. (2014) 35:12015–12020 DOI 10.1007/s13277-014-2500-1
RESEARCH ARTICLE
α-Methylacyl-coenzyme A racemase (AMACR, p504s) is a marker to distinguish malignant melanomas from dysplastic nevi and melanocytic nevi M. Abbas & E. M. Ploch & J. Wehling & E. Schipper & S. Janciauskiene & H. H. Kreipe & D. Jonigk
Received: 10 June 2014 / Accepted: 13 August 2014 / Published online: 24 August 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Routinely processed skin biopsies are still the mainstay for the diagnosis of melanocytic skin neoplasms (MSNs) and are considered the “gold standard” for individual patient management and clinical trials. The diagnostic challenge of melanocytic lesions of the skin prompts histopathologists to consider new diagnostic tools; among these, immunohistochemistry. We aimed to find putative new immunohistochemical markers, which can supplement the histological criteria used to detect dysplasia. In this immunohistochemical study, we chose a panel of promising biomarkers which could
Mahmoud Abbas and Eva Maria Ploch have contributed equally to this work and are considered the corresponding authors. Electronic supplementary material The online version of this article (doi:10.1007/s13277-014-2500-1) contains supplementary material, which is available to authorized users. M. Abbas (*) : J. Wehling : E. Schipper : H. H. Kreipe : D. Jonigk Institute of Pathology, Hannover medical School (MHH), Carl-Neuberg-Str. 1, 30625 Hannover, Germany e-mail: [email protected] J. Wehling e-mail: [email protected] E. Schipper e-mail: [email protected] H. H. Kreipe e-mail: [email protected] D. Jonigk e-mail: [email protected] E. M. Ploch KRH Hospital Großburgwedel, Department of Anaesthesia, Intensive and Emergency Medicine, Hannover, Germany e-mail: [email protected] S. Janciauskiene Pneumology research department, Hannover medical School (MHH), Hannover, Germany e-mail: [email protected]
potentially differentiate between different MSN entities. These included α-methylacyl-coenzyme A racemase (AMACR; p504s), which is involved in the degradation of branched chained fatty acid derivates. We analysed a cohort of benign nevi and malignant melanomas. The design of the study included 78 melanocytic skin neoplasms (26 malignant melanomas and 52 benign nevi) in a tissue microarray. Immunohistochemistry of cyclin-dependent kinase inhibitor 2A (p16Ink4a), methylacyl-coenzyme A racemase (AMACR), cyclin D1, and E-cadherin was performed and assessed. We have observed that the p16Ink4a, AMACR, cyclin D1, and Ecadherin showed no exclusive staining for nevi or melanomas. However, a significant overexpression of AMACR was found in malignant melanomas compared to benign nevi. AMACR overexpression was also associated with an increased p16Ink4a staining. Our results suggest AMACR as an immunohistochemical marker for distinguishing malignant melanomas and dysplastic nevi from conventional melanocytic nevi. Keywords α-Methylacyl-CoA racemase . p16Ink4a . Benign and dysplastic nevi . Melanoma . Immunocytochemistry
Introduction The risk factors for developing malignant melanoma (MM) are both genetic and environmental. Especially, multiple melanocytic nevi (MN) are common and can be important simulants of malignant melanoma (MM). Although most MN are benign and do not progress to malignancy, some forms of nevi are risk markers and potential precursors of MM. Malignant transformation of MN is typically associated with additional risk factors [1]. Of these, increased sun exposure is considered the main risk factor for developing MM. Through the presence of such a risk factor, MN will be considered a precursor lesion of MM [2]. Hence, only those MN at highest
12016
risk for developing MM should be excised, which presents challenges for pathologists in making the diagnosis. These latter include dysplastic nevi (DN), congenital MN, and, to a lesser extent, acquired (common) MN [3]. DN still represents a controversial entity. On clinical grounds, the term “MN with atypical features” is more appropriate, while histopathologically, the term DN is more widely used. It is widely accepted that DN has diameter >5 mm, irregular margin, and varying shades in the lesion, whereas histologically, they are characterized by intradermal lentiginous hyperplasia, cytological and architectural atypia, and a fibrosing stromal response [4]. Association between DN and MM in one lesion is also common. Clinical trials and smaller investigations have been performed to differentiate between MN, DN, and MM using immunohistochemistry, fluorescence in situ hybridization (FISH), gene sequencing, etc. Nevertheless, immunohistochemistry is still the most widely used and relatively cost-effective method. The problem, however, is that histological criteria of DN are not standardized and vary (subjectively) from one center to another. Therefore, in this study, we aimed to find putative immunohistochemical markers, which could supplement the histological criteria used to detect dysplasia. Specifically, we focused on P16Ink4a, an indicator of a worse prognosis in different cancers [5], α-methylacylcoenzyme A racemase (AMACR, p504s), a mitochondrial and peroxisomal enzyme essential for the completion of the β-oxidation pathway, which is a useful marker for prostate carcinoma in clinical pathology practice [6], cyclin D1, a cell cycle regulator, which has previously been studied in nevi and malignant melanomas [7], and E-cadherin, a tumor suppressor gene, which has been previously analysed in MSN [8].
Tumor Biol. (2014) 35:12015–12020
Fig. 1 MM (HE ×4)
Tissue microarray construction Representative MN and MM were marked on the respective HE (hematoxylin and eosin-stained) slides (Figs. 1 and 2). TMA construction was performed essentially as previously described [9]. In total, the tissue microarray construction (TMA) included 78 MSN specimens. Immunohistochemistry of TMA Four micrometer sections of TMAs were mounted on Superfrost slides (Thermo Fisher Scientific, Pittsburgh PAUSA). Slides were deparaffinized and rehydrated conventionally. Staining was performed on a Benchmark Ultra (Ventana, Tucson, AZ, USA) automated stainer using the CC1 mild protocol for antigen retrieval. Specific monoclonal antibodies and the ultraView DAB kit (Germany) for signal detection were used (Table 1). Morphological evaluation
Material and methods
All immunostains were evaluated by at least by two pathologists. Positive immunostaining of AMACR was in some cases cytoplasmic and in others was cell membrane. Cyclin D1 was
Selected MN and MM cases Seventy-eight surgical biopsy specimens with MNs and MMs (26 MM and 52 MN) from 2005 to 2011 were retrieved from the archives of the Institute of Pathology, Hannover Medical School (MHH) and handled anonymously according to the requirements of the local ethics committee. The specimens were re-evaluated by the two experienced dermatopathologists (AM and KH). MN group included [age: mean (range)] 19 males 39 (18– 75) years and 33 females 45 (15–76) years. MM group included 19 males, 73 (48–93) years and 7 females, 72 (55–84) years. The mean age of patients with MM was significantly higher than those with MN (P
RESEARCH ARTICLE
α-Methylacyl-coenzyme A racemase (AMACR, p504s) is a marker to distinguish malignant melanomas from dysplastic nevi and melanocytic nevi M. Abbas & E. M. Ploch & J. Wehling & E. Schipper & S. Janciauskiene & H. H. Kreipe & D. Jonigk
Received: 10 June 2014 / Accepted: 13 August 2014 / Published online: 24 August 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Routinely processed skin biopsies are still the mainstay for the diagnosis of melanocytic skin neoplasms (MSNs) and are considered the “gold standard” for individual patient management and clinical trials. The diagnostic challenge of melanocytic lesions of the skin prompts histopathologists to consider new diagnostic tools; among these, immunohistochemistry. We aimed to find putative new immunohistochemical markers, which can supplement the histological criteria used to detect dysplasia. In this immunohistochemical study, we chose a panel of promising biomarkers which could
Mahmoud Abbas and Eva Maria Ploch have contributed equally to this work and are considered the corresponding authors. Electronic supplementary material The online version of this article (doi:10.1007/s13277-014-2500-1) contains supplementary material, which is available to authorized users. M. Abbas (*) : J. Wehling : E. Schipper : H. H. Kreipe : D. Jonigk Institute of Pathology, Hannover medical School (MHH), Carl-Neuberg-Str. 1, 30625 Hannover, Germany e-mail: [email protected] J. Wehling e-mail: [email protected] E. Schipper e-mail: [email protected] H. H. Kreipe e-mail: [email protected] D. Jonigk e-mail: [email protected] E. M. Ploch KRH Hospital Großburgwedel, Department of Anaesthesia, Intensive and Emergency Medicine, Hannover, Germany e-mail: [email protected] S. Janciauskiene Pneumology research department, Hannover medical School (MHH), Hannover, Germany e-mail: [email protected]
potentially differentiate between different MSN entities. These included α-methylacyl-coenzyme A racemase (AMACR; p504s), which is involved in the degradation of branched chained fatty acid derivates. We analysed a cohort of benign nevi and malignant melanomas. The design of the study included 78 melanocytic skin neoplasms (26 malignant melanomas and 52 benign nevi) in a tissue microarray. Immunohistochemistry of cyclin-dependent kinase inhibitor 2A (p16Ink4a), methylacyl-coenzyme A racemase (AMACR), cyclin D1, and E-cadherin was performed and assessed. We have observed that the p16Ink4a, AMACR, cyclin D1, and Ecadherin showed no exclusive staining for nevi or melanomas. However, a significant overexpression of AMACR was found in malignant melanomas compared to benign nevi. AMACR overexpression was also associated with an increased p16Ink4a staining. Our results suggest AMACR as an immunohistochemical marker for distinguishing malignant melanomas and dysplastic nevi from conventional melanocytic nevi. Keywords α-Methylacyl-CoA racemase . p16Ink4a . Benign and dysplastic nevi . Melanoma . Immunocytochemistry
Introduction The risk factors for developing malignant melanoma (MM) are both genetic and environmental. Especially, multiple melanocytic nevi (MN) are common and can be important simulants of malignant melanoma (MM). Although most MN are benign and do not progress to malignancy, some forms of nevi are risk markers and potential precursors of MM. Malignant transformation of MN is typically associated with additional risk factors [1]. Of these, increased sun exposure is considered the main risk factor for developing MM. Through the presence of such a risk factor, MN will be considered a precursor lesion of MM [2]. Hence, only those MN at highest
12016
risk for developing MM should be excised, which presents challenges for pathologists in making the diagnosis. These latter include dysplastic nevi (DN), congenital MN, and, to a lesser extent, acquired (common) MN [3]. DN still represents a controversial entity. On clinical grounds, the term “MN with atypical features” is more appropriate, while histopathologically, the term DN is more widely used. It is widely accepted that DN has diameter >5 mm, irregular margin, and varying shades in the lesion, whereas histologically, they are characterized by intradermal lentiginous hyperplasia, cytological and architectural atypia, and a fibrosing stromal response [4]. Association between DN and MM in one lesion is also common. Clinical trials and smaller investigations have been performed to differentiate between MN, DN, and MM using immunohistochemistry, fluorescence in situ hybridization (FISH), gene sequencing, etc. Nevertheless, immunohistochemistry is still the most widely used and relatively cost-effective method. The problem, however, is that histological criteria of DN are not standardized and vary (subjectively) from one center to another. Therefore, in this study, we aimed to find putative immunohistochemical markers, which could supplement the histological criteria used to detect dysplasia. Specifically, we focused on P16Ink4a, an indicator of a worse prognosis in different cancers [5], α-methylacylcoenzyme A racemase (AMACR, p504s), a mitochondrial and peroxisomal enzyme essential for the completion of the β-oxidation pathway, which is a useful marker for prostate carcinoma in clinical pathology practice [6], cyclin D1, a cell cycle regulator, which has previously been studied in nevi and malignant melanomas [7], and E-cadherin, a tumor suppressor gene, which has been previously analysed in MSN [8].
Tumor Biol. (2014) 35:12015–12020
Fig. 1 MM (HE ×4)
Tissue microarray construction Representative MN and MM were marked on the respective HE (hematoxylin and eosin-stained) slides (Figs. 1 and 2). TMA construction was performed essentially as previously described [9]. In total, the tissue microarray construction (TMA) included 78 MSN specimens. Immunohistochemistry of TMA Four micrometer sections of TMAs were mounted on Superfrost slides (Thermo Fisher Scientific, Pittsburgh PAUSA). Slides were deparaffinized and rehydrated conventionally. Staining was performed on a Benchmark Ultra (Ventana, Tucson, AZ, USA) automated stainer using the CC1 mild protocol for antigen retrieval. Specific monoclonal antibodies and the ultraView DAB kit (Germany) for signal detection were used (Table 1). Morphological evaluation
Material and methods
All immunostains were evaluated by at least by two pathologists. Positive immunostaining of AMACR was in some cases cytoplasmic and in others was cell membrane. Cyclin D1 was
Selected MN and MM cases Seventy-eight surgical biopsy specimens with MNs and MMs (26 MM and 52 MN) from 2005 to 2011 were retrieved from the archives of the Institute of Pathology, Hannover Medical School (MHH) and handled anonymously according to the requirements of the local ethics committee. The specimens were re-evaluated by the two experienced dermatopathologists (AM and KH). MN group included [age: mean (range)] 19 males 39 (18– 75) years and 33 females 45 (15–76) years. MM group included 19 males, 73 (48–93) years and 7 females, 72 (55–84) years. The mean age of patients with MM was significantly higher than those with MN (P
