Immunohistochemical Evaluation of Necrotic Malignant Melanomas

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1 Anatomic Pathology / EVALUATION OF NECROTIC MALIGNANT MELANOMAS Immunohistochemical Evaluation of Necrotic Malignant Melanomas Daisuke Nonaka, MD, Jordan Laser, MD, Rachel Tucker, HTL(ASCP), and Jonathan Melamed, MD Key Words: Malignant melanoma; Necrosis; HMB-45; Melan-A; Tyrosinase; PNL2; S-100 protein; Microphthalmia transcription factor; MITF DOI: /WKEN4ER9GXJ9GG31 Abstract We evaluated 35 cases of malignant melanomas with substantial necrosis immunostained with S-100, HMB-45, Melan-A, tyrosinase, PNL2, and microphthalmia transcription factor (MITF). Staining patterns were evaluated in viable and necrotic areas of the tumors. S-100 was the most sensitive marker (97%) in the viable tumors, but necrotic areas demonstrated nonspecific staining. Viable tumors stained variably for HMB-45 (25 [71%]), Melan-A (28 [80%]), tyrosinase (30 [86%]), and PNL2 (23 [66%]). Necrotic areas focally reacted to the same antibodies. The necrotic areas that retained immunoreactivity for these markers corresponded to areas where the outline of the tumor cells could still be recognized as ghost cells on the H&E-stained section. Areas that showed complete coagulative necrosis were negative for melanoma markers. MITF variably stained in the viable tumors but was completely negative in necrotic areas. Our study demonstrated that a combination of antibodies to HMB-45, tyrosinase, and PNL2 detected melanocytic differentiation in necrotic areas in 80% of cases. In diagnostic surgical pathology, it is not uncommon to see a tumor with extensive or even complete necrosis. Among common malignant neoplasms, malignant melanoma is notorious for its capability of metastasizing to any organs and sites and mimicking various types of benign and malignant neoplasms, and the history of cutaneous melanocytic lesions is not always evident in metastatic melanoma cases. In addition, it is not unusual for a metastatic melanoma to manifest as a completely necrotic mass. Thus, malignant melanoma is always a consideration in the differential diagnosis for tumors with extensive necrosis. A few immunohistochemical markers such as cytokeratins, particularly AE1/AE3, and thyroglobulin have been reported as useful in identifying the lineage of necrotic tumors. 1,2 The usefulness of immunohistochemical study for the diagnosis of necrotic melanoma has not been extensively investigated. 2,3 Therefore, we evaluated the diagnostic usefulness of 6 immunohistochemical markers of melanocytic differentiation by applying them to 35 melanoma specimens with extensive necrosis. Materials and Methods We retrieved 35 cases of primary (2 cases) and metastatic (33 cases) malignant melanomas with substantial necrosis, ie, the necrotic component comprising more than half of the entire tumor, from our archives. All the cases showed extensive necrosis adjacent to the viable tumor. The following 6 immunohistochemical stains were performed: S-100 protein (polyclonal, dilution 1:12,000; DAKO, Carpinteria, CA; no epitope retrieval), HMB-45 (HMB-45, Am J Clin Pathol 2007;127: DOI: /WKEN4ER9GXJ9GG31 787

2 Nonaka et al / EVALUATION OF NECROTIC MALIGNANT MELANOMAS dilution 1:50; DAKO; heat-induced epitope retrieval [HIER] in citrate buffer), Melan-A (A103, dilution 1:400; Novocastra, Newcastle upon Tyne, England; HIER in citrate buffer), tyrosinase (T311, dilution 1:200; Novocastra; HIER in EDTA buffer), PNL2 (PNL2, dilution 1:50; DAKO; HIER in DakoCytomation target retrieval solution), and microphthalmia transcription factor (MITF; D5, dilution 1:100; DAKO; HIER in citrate buffer). Ten cases of various extensively necrotic lesions were also stained with HMB-45, Melan-A, tyrosinase, and PNL2. Those lesions include 4 cases of high-grade sarcoma (leiomyosarcoma, gastrointestinal stromal tumor, and undifferentiated pleomorphic sarcoma), 2 cases of cutaneous squamous cell carcinoma, 1 case each of adrenocortical carcinoma, ovarian serous papillary carcinoma, and pulmonary adenocarcinoma, and 1 case of abscess. For 10 selected cases of necrotic melanoma, cytokeratin AE1/AE3 (dilution 1:20; Zymed, San Francisco, CA; protease digestion), CD31 (JC70A, dilution 1:600, DAKO; HIER in citrate buffer), CD45 (2B11 + PD7/26, dilution 1:250; DAKO; HIER in citrate buffer), CD68 (KP1, dilution 1:500; DAKO; HIER in citrate buffer), chromogranin A (LK2H10, dilution 1:50; Novocastra; HIER in citrate buffer), and smooth muscle actin (SMA; 1A4, dilution 1:400; DAKO; HIER in citrate buffer) were also performed. Staining patterns were evaluated in viable and necrotic areas of the tumors. Results Immunohistochemical Results on Viable Tumor S-100 protein was the most sensitive marker (34/35 [97%]) in the viable tumors, as expected, and was positive in all cases except 1 case, with a positive ratio ranging from 5% to 100%. The S-100 protein negative case was positive for the other 5 markers in the viable areas. Viable tumors stained variably for HMB-45 (25/35 [71%]), Melan-A (28/35 [80%]), tyrosinase (30/35 [86%]), PNL2 (23/35 [66%]), and MITF (24/35 [69%]) Table 1. Two cases (6%) were completely negative for all 5 markers, whereas 17 cases (49%) were positive for all 5 markers. Three cases expressed only tyrosinase. Two cases expressed 2 markers, ie, tyrosinase and Melan-A. Six cases expressed 3 of 5 markers: tyrosinase, Melan-A, and MITF in 3 cases; tyrosinase, HMB-45, and PNL2 in 1 case; Melan-A, HMB-45, and PNL2 in 1 case; and HMB-45, PNL2, and MITF in 1 case. Five cases expressed 4 of 5 markers, with no reaction to one of the following markers: HMB-45 in 1 case, PNL2 in 2 cases, and MITF in 3 cases. The viable tumors were completely negative for AE1/AE3, CD31, CD45, CD68, chromogranin, and SMA. In addition, viable portions of all 10 cases of extensively necrotic nonmelanocytic lesions were completely negative for HMB-45, Melan-A, tyrosinase, and PNL2. Immunohistochemical Results on Necrotic Tumor Of the cases, 17 (49%) demonstrated diffuse but weak nonspecific staining for S-100 protein in the necrotic areas Image 1B. Necrotic components also stained for HMB-45 (23/35 [66%]) Image 1C, Melan-A (20/35 [57%]), tyrosinase (23/35 [66%]) Image 1D, and PNL2 (19/35 [54%]). The intensity and distribution of staining were weak and focal compared with the viable areas. MITF showed a completely negative reaction in necrotic areas Image 1E. Individual tumors expressed the same immunohistochemical markers in the viable and necrotic areas, with the exception of MITF, which was negative in the necrotic areas of all 35 specimens. Generally, the necrotic areas that reacted to the melanoma markers corresponded to areas where the outline of the tumor cells could still be recognized as ghost cells on the H&E-stained section Image 1A. Areas that showed complete coagulative necrosis were negative for the melanoma markers except for occasional nonspecific staining with S-100 protein. A combination of 3 markers, HMB-45, tyrosinase, and PNL2 or HMB-45, tyrosinase, and Melan-A, could detect melanocytic differentiation in the necrotic areas of 80% (n = 28) and 77% (n = 27) of cases, respectively. A combination of the 2 common markers, HMB-45 and Melan-A, could detect melanocytic differentiation in 74% of cases (n = 26). The necrotic areas were completely negative for AE1/AE3, CD31, chromogranin, and SMA. The CD45 reaction was predominantly present at borders between the viable and necrotic areas, and the positive cells were morphologically consistent with infiltrating lymphocytes. Of 10 Table 1 Immunohistochemical Results in 35 Malignant Melanomas * S-100 HMB-45 Melan-A Tyrosinase PNL2 MITF Viable area Necrotic area MITF, microphthalmia transcription factor. * Data are given as number of positive cases. 788 Am J Clin Pathol 2007;127: DOI: /WKEN4ER9GXJ9GG31

3 Anatomic Pathology / ORIGINAL ARTICLE B C D E Image 1 A, Melanoma with coagulative necrosis containing ghost cells, so-called ongoing necrosis (H&E, 20). B, S-100 protein often demonstrates diffuse, nonspecific staining in the necrotic area of the tumor ( 20). C, HMB-45 focally stains in the ongoing necrotic area of the tumor ( 20). D, Tyrosinase focally stains in the ongoing necrotic area of the tumor ( 20). E, Microphthalmia transcription factor is negative in both ongoing and complete necrotic areas of the tumor ( 20). Am J Clin Pathol 2007;127: DOI: /WKEN4ER9GXJ9GG31 A

4 Nonaka et al / EVALUATION OF NECROTIC MALIGNANT MELANOMAS cases, 5 demonstrated a weak CD45 reaction in the necrotic tumor cells immediately adjacent to the CD45+ infiltrating lymphocytes. A similar finding was also observed with CD68 staining, which highlighted the infiltrating macrophage population and, in addition, the surrounding necrotic tumor cells at a weaker intensity in 3 of 10 cases. The necrotic portions of all 10 cases of extensively necrotic nonmelanocytic lesions were completely negative for HMB-45, Melan-A, tyrosinase, and PNL2. Discussion Extensively or completely necrotic tumors can be diagnostically challenging. This situation may be encountered in a surgical resection specimen for malignant neoplasm, a lymph node biopsy for malignant lymphoma or metastatic malignant neoplasm, or a small biopsy specimen from the necrotic part of the tumor. The potential situations that could cause such necrosis are as follows: (1) adjuvant therapy before removal of the tumor 4 ; (2) a rapidly growing tumor that is susceptible to tumor apoptosis and necrosis; (3) certain tumors, particularly oncocytic neoplasms represented by Hürthle cell neoplasm of the thyroid, with a known propensity for spontaneous infarct or infarct induced by procedures such as fine-needle aspiration (FNA); and (4) FNA performed as an initial diagnostic modality; various studies have reported histomorphologic alterations including sclerosis, vascular and spindle cell proliferation, and partial to complete infarction or necrosis. 3 It is also known that melanoma cells can undergo self-destruction via programmed cell death, ie, apoptosis, regulated by multiple molecules that exert proapoptotic or antiapoptotic effects. The former include p53, Bax, tumor necrosis factor, and tumor necrosis factor related apoptosisinducing ligand, whereas the latter include bcl-2, bcl-x L, Mcl-1, and survivin. 5 A few studies have evaluated the usefulness of immunohistochemical staining in identifying the lineage of necrotic tumors. A study by Judkins et al 2 found high sensitivity and specificity in cytokeratin antibodies such as AE1, AE1/AE3, S903, and PANCK. Of 2 cases of malignant lymphoma, both retained CD45 (leukocyte common antigen), but this marker was also expressed in 3 of 14 cases of necrotic carcinoma. S- 100 protein was positive in 1 of 2 cases of malignant melanoma, but 1 of 14 cases of carcinoma was also positive for this marker. Judkins et al 1 reported the usefulness of cytokeratins such as AE1/AE3 in thyroid tumors with spontaneous or post-fna necrosis, including papillary carcinomas, Hürthle cell neoplasms, and follicular adenomas. Thyroglobulin was also reported to be expressed by papillary carcinomas and Hürthle cell neoplasms but not follicular adenomas. S-100 protein immunoreactivity was identified only in viable areas of some Hürthle cell neoplasms and papillary carcinoma but not in necrotic components. Nasuti et al 3 described 3 cases of lymphadenopathy for which FNA was performed. After diagnoses of metastatic squamous cell carcinoma for 2 cases and metastatic malignant melanoma for 1 case were made, lymph node dissection was performed in each case, which showed only coagulative necrosis with ghost cells and no viable tumor. However, immunostains for cytokeratin (AE1/AE3) and melanoma markers (HMB45, Melan-A, and S-100 protein) confirmed the diagnoses of metastatic squamous cell carcinoma and metastatic melanoma, despite extensive necrosis. Generally, expressions of HMB-45, Melan-A, and tyrosinase in metastatic melanomas are weaker in intensity and patchier in distribution than those in primary melanoma, as our study showed. It has been reported that metastatic melanomas subsequent to multiple primary melanomas or multiple recurrent melanomas significantly lose expression of the melanoma common tumor antigens such as Melan-A. 6 It has been postulated that tumor antigens can be recognized by cell-mediated immunity during development of the primary cancer by the formation of antigen-specific cytotoxic lymphocytes and that such recognition leads to the destruction of the tumor cells expressing the antigen (tumor regression); tumor cells with antigen loss then have a selective survival advantage accounting for the subsequent predominance of neoplastic cells with antigen loss. 6 The areas of necrotic tumor that reacted to the melanoma markers were located between the viable tumors and areas of the complete coagulative necrosis, and the necrotic tumor cells could still be recognized as ghost cells. These ghost cells were characterized by cytoplasmic eosinophilia, variable preservation of nuclear chromatin, and obscured cytoplasmic borders. These cells have been described as necrobiosis or ongoing necrosis. 4 The study on these ghost cells in hepatocellular carcinomas treated by radiofrequency ablation demonstrated that antibody to detect single-strand DNA labeled the nuclei in the ghost cells but not in the viable or completely necrotic cells, whereas mitochondrial antibody labeled the cytoplasm of viable cells but not cells of ongoing or complete necrosis. Necrotic areas of melanomas were negative for nonmelanocytic markers such as AE1/AE3, CD31, CD45, CD68, chromogranin A, and SMA, whereas extensively necrotic nonmelanocytic lesions were negative for melanocytic markers such as HMB-45, Melan-A, tyrosinase, and PNL2. Both results support the sensitivity and specificity of HMB-45, Melan-A, tyrosinase, and PNL2 in the diagnosis of necrotic melanoma. Given that many metastatic melanomas immunohistochemically demonstrate variable immunoreactivity to melanoma 790 Am J Clin Pathol 2007;127: DOI: /WKEN4ER9GXJ9GG31

5 Anatomic Pathology / ORIGINAL ARTICLE markers, a panel of markers is recommended for the diagnosis of metastatic melanoma with extensive necrosis. Our study demonstrated that a combination of 3 markers, HMB-45, tyrosinase, and PNL2 or HMB-45, tyrosinase, and Melan-A, could detect melanocytic differentiation in the necrotic areas of 80% and 77% of cases, respectively. A combination of the 2 common markers, HMB-45 and Melan-A, could detect melanocytic differentiation in 74% of cases. S-100 protein and MITF are not reliable markers in this context; the former has a tendency for nonspecific staining, and the latter lacks sensitivity in necrotic areas. From the Department of Pathology, New York University Medical Center, New York, NY. Address correspondence to Dr Nonaka: Dept of Pathology, New York University Medical Center, TCH-461, 560 First Ave, New York, NY References 1. Judkins AR, Roberts SA, LiVolsi VA. Utility of immunohistochemistry in the evaluation of necrotic thyroid tumors. Hum Pathol. 1999;30: Judkins AR, Montone KT, LiVolsi VA, et al. Sensitivity and specificity of antibodies on necrotic tumor tissue. Am J Clin Pathol. 1998;110: Nasuti JF, Gupta PK, Baloch ZW. Clinical implications and value of immunohistochemical staining in the evaluation of lymph node infarction after fine-needle aspiration. Diagn Cytopathol. 2001;25: Itoh T, Orba Y, Takei H, et al. Immunohistochemical detection of hepatocellular carcinoma in the setting of ongoing necrosis after radiofrequency ablation. Mod Pathol. 2002;15: Hussein MR, Haemel AK, Wood GS. Apoptosis and melanoma: molecular mechanisms. J Pathol. 2003;199: Saleh FH, Crotty KA, Hersey P, et al. Autonomous histopathological regression of primary tumours associated with specific immune responses to cancer antigens. J Pathol. 2003;200: Am J Clin Pathol 2007;127: DOI: /WKEN4ER9GXJ9GG31 791