Clinical Usefulness of Immunostaining with CEA in Distinction between Malignant Melanoma and Spitz Nevus

Background: Malignant melanoma (MM) is one of the most fatal cancers in the field of dermatology. On the other hand, Spitz Nevi (SN) is a benign tumor of melanocytes. Prognosis of these neoplastic tumors is completely different, but their histopathological findings are very similar. CEA (carcinoembryonic antigen) is one of the most familiar tumor markers in colon cancer. But it has been found to be expressed in many diseases and various tissues. In human skin, CEA is expressed in sebaceous glands and sweat glands. Objectives: The aim of this study is to investigate the expression of CEA in MM and SN. Materials and Methods: Six-teen tissues of MM and Eleven tissues of SN were immunohistochemically stained with polyclonal antibody against CEA. Results: Strong positive staining was observed in MMs, on the other hand, we could not observe strong staining in SN. We suggest that immunohistochemical staining of CEA may distinguish MM from SN. *Corresponding author: Prof. Hironobu Ihn, MD, PhD, Department of Dermatology and Plastic Surgery, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan, Tel: + 81-96-373-5233/ Fax: + 81-96-373-5235; E-mail: ihn-der@kumamoto-u.ac.jp Citation: Ihn, H., et al. Clinical Usefulness of Immunostaining with CEA in Distinction between Malignant Melanoma and Spitz Nevus. (2016) Invest Dermatol Venereol Res 2(1): 14. Clinical Usefulness of Immunostaining with CEA in Distinction between Malignant Melanoma and Spitz Nevus Johno T, Egawa K, Jinnin M, Fukushima S, Masuguchi S, Inoue Y, Ihn H* Received date: November 21, 2015 Accepted date: January 25, 2016 Published date: January 29, 2016 DOI: 10.15436/2381-0858.16.013 Invest Dermatol Venereol Res | Volume 2: Issue 1 Ihn, H 2 of significant pleomorphism[7]. The defferential diagnosis of SN includes MM. These features are insufficient for discriminating SN from MM. Furthermore, SN cells are also stained positive with immunostainings which are positive for MM. Prognosis of these neoplastic tumors is completely different, but their histopathological findings are very similar. It is needed to find the way to distinguish MM from SN clearly and simply. CEA (carcinoembryonic antigen) is one of the most familiar tumor markers in colon cancer. It has been known that CEA has many subgroups, and works in connection with cell adherence, tumor immunity and so on[8]. However, anti-polyclonal-CEA antibody (poly-CEA) which reacts with almost all human CEA family as an antigen is only used in immunostaining. It has been found to be expressed in many diseases and various tissues. In human skin, CEA is expressed in sebaceous glands and sweat glands[9]. In 1993, Sanders et al. reported that MMs were positive for poly-CEA in immnostaining[10]. But, it has not used so much in clinical areas. In this report, we investigated the poly-CEA immunoreactivity for MM and SN, and clarity its usefulness to distinguish MM from SN. Materials and Methods Tissues The surgically resected specimens were used for this study. They included 16 of MM: 12 primary lesions, 2 LN metastatic lesions and 2 skin metastatic lesions, and 11 of SN. All specimens were diagnosed by clinical and histopathological findings already known. All samples were fixed in neutral buffered formalin, embedded in paraffin, and prepared for hematoxylin-eosin examination. Immunohistochemical stainings Immunohistochemical staining on paraffin-embedded section was performed using the standard streptavidin-biotin-peroxidase complex method with VECTASTAIN Elite ABC Kit. Four μm thick sections were mounted on silane-coated slide (Dako), then deparaffinized with xylene and rehydrated through a graded series of ethyl alcohol and PBS. Next, antigen-retrieval was performed using microwave for 5 minutes. After washing in PBS, non-specific binding sites were blocked with 10% normal rabbit serum at room temperature for 1 hour. Excess serum was removed from the sections. Then, the samples were incubated with the primary antibody at 4°C overnight. Following washing with PBS, they were incubated with biotinylated rabbit-anti goat IgG for 30 minutes at room temperature. The slides were rinsed and incubated with the avidin/biotin complex at room temperature for 1 hour. Visualization of the peroxidase reaction was achieved with diaminobenzidine (DAB), followed by counterstaining with Giemsa for decolorizing melanin.

2 of significant pleomorphism [7] . The defferential diagnosis of SN includes MM. These features are insufficient for discriminating SN from MM. Furthermore, SN cells are also stained positive with immunostainings which are positive for MM.
Prognosis of these neoplastic tumors is completely different, but their histopathological findings are very similar. It is needed to find the way to distinguish MM from SN clearly and simply.
CEA (carcinoembryonic antigen) is one of the most familiar tumor markers in colon cancer. It has been known that CEA has many subgroups, and works in connection with cell adherence, tumor immunity and so on [8] . However, anti-polyclonal-CEA antibody (poly-CEA) which reacts with almost all human CEA family as an antigen is only used in immunostaining. It has been found to be expressed in many diseases and various tissues. In human skin, CEA is expressed in sebaceous glands and sweat glands [9] . In 1993, Sanders et al. reported that MMs were positive for poly-CEA in immnostaining [10] . But, it has not used so much in clinical areas.
In this report, we investigated the poly-CEA immunoreactivity for MM and SN, and clarity its usefulness to distinguish MM from SN.

Tissues
The surgically resected specimens were used for this study. They included 16 of MM: 12 primary lesions, 2 LN metastatic lesions and 2 skin metastatic lesions, and 11 of SN. All specimens were diagnosed by clinical and histopathological findings already known. All samples were fixed in neutral buffered formalin, embedded in paraffin, and prepared for hematoxylin-eosin examination.

Immunohistochemical stainings
Immunohistochemical staining on paraffin-embedded section was performed using the standard streptavidin-biotin-peroxidase complex method with VECTASTAIN Elite ABC Kit. Four μm thick sections were mounted on silane-coated slide (Dako), then deparaffinized with xylene and rehydrated through a graded series of ethyl alcohol and PBS. Next, antigen-retrieval was performed using microwave for 5 minutes. After washing in PBS, non-specific binding sites were blocked with 10% normal rabbit serum at room temperature for 1 hour. Excess serum was removed from the sections. Then, the samples were incubated with the primary antibody at 4°C overnight. Following washing with PBS, they were incubated with biotinylated rabbit-anti goat IgG for 30 minutes at room temperature. The slides were rinsed and incubated with the avidin/biotin complex at room temperature for 1 hour. Visualization of the peroxidase reaction was achieved with diaminobenzidine (DAB), followed by counterstaining with Giemsa for decolorizing melanin.

Results
At first, we reconfirmed the result of general staining for melanocytic lesions, HMB-45, S-100 protein and MART-1. Positive staining for HMB-45, S-100 protein and melan A was observed in all MMs and SNs (Table1).  Next, we found strong positive staining for poly-CEA in all MMs included metastatic lesions. In 12 primary lesions, 5 cases were ++, 7 cases were + . In 4 metastatic lesions, we could see all positive poly-CEA staining much stronger ( Figure  1 -3). At least we would suggest that specimens positive for poly-CEA have the possibility of MM. Previous reports indicated that strong positive staining for poly-CEA was observed in MM specimens, similar to our result.   On the other hand, we could observe little poly-CEA staining in SN. One specimen of SN showed a little positive staining for poly-CEA, but the others were almost negative (4 cases -, 6 cases ±) ( Figure 4, Table 2). In any cases, the density of staining was all intentionally weaker compared with MM.   Gambichler et al. suggested that expression of CEA-CAM1 in MM is an independent factor for metastasis risk [11] . We found that the frequency of SN positive for poly-CEA was much less than that of MMs. This suggests that MM have capability of metastasis, but SN do not.

Discussion
MM is a serious form of skin cancer, and the prevalence of MM annually is increasing faster than that of any other cancers. In Caucasians, exposure to the sunlight may be an important causal factor of MM. On the other hand, in more heavily pigmented races, many of MM occurs on the skin of the unexposed soles. MM may be divided into four clinical types: lentigo maligna melanoma, superficial spreading melanoma, nodular melanoma, and acral lentiginous melanoma. In Eastern Asia including Japan, acral lentiginous type occurr more frequently than any other types [11,12] .
The 10-years survival rate in the early stage of MM is over 80%. But, the patients with MM in stage III B and stage IV have an unfavorable outcome, with 10-year survivals of less than 50% and less than 10%, respectively [12] . Therefore, we have to find and treat MM as early as possible.
Although the 'gold standard' for melanoma diagnosis remains histopathological analysis the dermoscopists play a significant role in the diagnostic process at present. But the judgement is sometimes difficult. Therefore, many researchers have investigated the diagnostic methods of MM.
The recent studies have shown that melanomas differ from nevi by the presence of frequent genes or losses of particular chromosomal regions, for example 6p25-RREB1, 6q23-Myb, 11q13-CCND1 and so on [13][14][15][16] . We will be able to detect melanomas using a multi-colored fluorescence in situ hybridization (FISH) which detects copy number changes of chromosomal regions commonly found to be aberrant in melanoma. Takata et al. showed clearly that MM essentially differs from the SN in respect of variation or deficiencies, such as RAS, BRAF and CDKN2A genes [17] . Kozubek et al. reported that the miRNA profiling could distinguish MM from nevi [18] .

Conclusion
It is thought that the judgment of MM and SN would be possible by these techniques. But these new methods are not common and not available in general clinical laboratory.
On the other hand, poly-CEA is one of the most available antibody. Moreover, this antigen-retrieval method and staining method are also simple. Limitations of our study include the absence of functional studies. But, our data may give supports to distinguish MM from SN clearly and simply.
A small number of the patients with MM and those with SN were included in these studies. Further studies are required to clarify the clinical usefulness of immunohistochemical staining of CEA that may distinguish MM from SN.