Modern Pathology (2010) 23, 1151–1156 & 2010 USCAP, Inc. All rights reserved 0893-3952/10 $32.00 1151 Vascular invasion is an early event in pathogenesis of Merkel cell carcinoma Heli M Kukko1, Virve SK Koljonen1, Erkki J Tukiainen1, Caj H Haglund2 and Tom O Bo¨hling3 1Department of Plastic Surgery, Helsinki University Hospital, Helsinki, Finland; 2Department of Gastroenterological Surgery, Helsinki University Hospital, Helsinki, Finland and 3HUSLAB and Department of Pathology, Helsinki University and Helsinki University Hospital, Helsinki, Finland This study investigated vascular and especially lymphovascular invasion in primary Merkel cell carcinoma and its value as a prognostic factor. Paraffin-embedded blocks prepared from tumor samples obtained from126 patients diagnosed with Merkel cell carcinoma in 1979–2004 were immunohistochemically stained using antibodies CD31 and D2-40 to detect intravascular tumor emboli. This finding was compared with the clinical data and the disease outcome. Intravascular tumor cells were observed in 117 (93%) of the samples. The majority, 83 (66%), showed only lymphovascular invasion. Only blood vascular invasion was seen in four (3%) samples. In all, 30 (24%) samples demonstrated both lymphovascular invasion and blood vascular invasion. In only nine (7%) samples, there was no invasion within the vascular structures. The tumors lacking invasion were significantly smaller (Po0.01 and a ¼ 0.050) than those with vascular invasion, although lymphovascular invasion was observed even in the smallest tumor (0.3 cm) of this study. Already in the early stages of the disease, Merkel cell carcinoma seems to have the capacity to penetrate vessel walls. Our finding of the high frequency of lymphovascular invasion might therefore explain the extremely aggressive clinical behavior of Merkel cell carcinoma. This may support the role of sentinel node biopsy even in the case of very small primary Merkel cell carcinoma tumors. Modern Pathology (2010) 23, 1151–1156; doi:10.1038/modpathol.2010.100; published online 14 May 2010 Keywords: Merkel cell carcinoma; vascular invasion; lymphovascular; CD31; D2-40 Merkel cell carcinoma is a rare and potentially four-level staging system divides patients into ones aggressive neuroendocrine carcinoma of the skin. It having either a local disease with a primary tumor has a poorer prognosis than epidermal skin tumors, o2cm or Z2 cm (stages I and II), a lymph node entailing a high risk of local recurrences and early positive disease (stage III), or a distant metastatic regional lymph node involvement and distant disease (stage IV).16–19 Patients with tumors o2cm metastases.1–3 The etiology of Merkel cell carcinoma have better prognosis.13,20 may be multifactorial. The exposure to ultraviolet Thus far, the most consistent predictor of survival radiation,4–6 as well as immunosuppression7–10 is in Merkel cell carcinoma is the presence or absence associated with Merkel cell carcinoma. Recently, a of lymph node metastases.21 Pre-operative lymphos- novel polyomavirus, named Merkel cell polyoma- cintigraphy has been shown to reliably detect virus, was identified in Merkel cell carcinoma tumor sentinel nodes also in Merkel cell carcinoma22,23; tissue, suggesting that a viral infection might also be sentinel lymph node biopsy is thus recommended an etiological factor.11 by many authors.17,24,25 Tumor size has been shown to be a strong Vascular invasion is frequently seen in Merkel cell prognostic factor.12–16 The most commonly adopted carcinoma tumor samples. In routine hematoxylin- eosin staining, vascular invasion has been observed in 38–60% of the samples.14,15,26 The presence of Correspondence: Dr HM Kukko, MD, Department of Plastic lymphovascular invasion has been reported to Surgery, Helsinki University Hospital, To¨o¨lo¨ Hospital, P.O. Box indicate a poorer prognosis,16,27 although in these 266, Helsinki 00029 HUS, Finland. studies, the detection of lymphovascular structures E-mail: [email protected] or [email protected] 16 Received 4 March 2010; revised and accepted 19 April 2010; was based on hematoxylin-eosin staining only or published online 14 May 2010 on stainings with panvascular CD34 antibody.27 www.modernpathology.org Merkel cell carcinoma 1152 HM Kukko et al CD31, also known as platelet endothelial cell Table 1 Clinical characteristics of Merkel cell carcinoma patients adhesion molecule 1, is a type I integral membrane at diagnosis glycoprotein and a member of the immunoglobulin No. of patients (n ¼ 126) % superfamily of cell surface receptors. It is found essentially on the surface of endothelial cells. It is a Sex panvascular endothelial marker expressed in both Female 90 71 lymphatic and blood endothelial cells.28 Male 36 29 D2-40 is a monoclonal antibody directed against Age (years) human podoplanin, a transmembrane mucoprotein Mean 77.6 expressed in lymphatic endothelial cells. It is a Median 79 sensitive and specific marker of lymphatic endothe- Range 50–100 lium, and does not react with blood vessel endothe- Tumor location lium.29,30 Head or neck 73 58 This study focused on the vascular and especially Upper extremities 21 17 lymphovascular invasion in primary Merkel cell Trunk 14 11 Lower extremities 18 14 carcinoma samples. Immunohistochemistry using antibodies CD31 and D2-40 was applied to analyze Tumor size (cm) the frequency of this phenomenon and its value as a Mean 1.86 Median 1.5 prognostic factor. Range 0.3–8.5 Stage at diagnosis I Local only o2cm 75 59 Materials and methods II Local only Z2cm 36 29 III Nodala 11 9 Data on altogether 207 patients diagnosed with IV Distant metastatic 4 3 Merkel cell carcinoma during 1979–2004 were obtained from the Finnish Cancer Registry and the aNodal disease assessed by palpation or histological evaluation (when Helsinki University Hospital files. Detailed data on performed). the course of the disease were obtained from the files of the district hospitals and primary health ethanol series. Slides for CD31 staining were treated care centers. Only patients with a known primary in a microwave oven in Tris-EDTA buffer (pH 9.0) tumor, sufficient clinical data, and a tissue sample for 2 Â 7 min and 2 Â 5 min, and then cooled at room of adequate quality were included in the study. temperature for 20 min. The slides were then treated The patients’ clinical characteristics are shown in with 0.3% Dako REAL Peroxidase-Blocking Solu- Table 1. The mean follow-up time was 52.3 (range 0– tion for 5 min to block endogenous peroxidases. The 296) months. immunostaining was performed by adding mono- Paraffin-embedded blocks prepared from the clonal mouse anti-human CD31, endothelial cell, tumor samples of 126 patients were available for clone JC70A (DakoCytomation, Glostrup, Denmark) re-evaluation. The diagnoses were confirmed in a antibody (1:25 diluted in Dako REAL Antibody blinded manner by two researchers (TB and HK). Diluent, Biohit, Helsinki, Finland) for 1 h, followed The diagnoses were based on typical histological by incubating for 30 min with Dako REAL Envision/ morphology in the hematoxylin-eosin slides, and HRP detection system, and finally visualized by confirmed by immunohistochemical analysis using Dako REAL DAB þ Chromogen for 10 min. Between cytokeratin 20 and thyroid transcription factor 1 each step, the slides were washed with PBS-0.04% antibodies; the latter was negative in all samples. In Tween20. The slides were counterstained with five cases, cytokeratin 20 was negative, and the Mayer’s hematoxylin and mounted in mounting diagnosis was further confirmed with synaptophysin medium (Aquamount, BDH, Poole, UK). and chromogranin A, which gave positive results. Slides for D2-40 staining were treated in a Tumor size (the greatest surface dimension) was microwave oven in Tris-EDTA buffer (pH 9.0) for measured from the hematoxylin-eosin-stained slides. 24 min and cooled at room temperature for 20 min. Immunohistochemistry for cytokeratin 20, thyroid Immunostaining was performed adding monoclonal transcriptase factor 1, synaptophysin, and chromo- mouse anti-human D2-40 (code M3619, Dako, Glostrup, granin A were performed according to the routines Denmark) antibody diluted 1:50 (Labvision) for 30 min, of the immunohistochemical laboratory of the followed by 30 min incubation with the Envision Department of Pathology, Helsinki University Hos- (K5007, Dako) detection system, and finally visualized pital. Immunohistochemical staining for detecting by DAB Chromogen for 10 min. vascular structures was performed using antibodies CD31 and D2-40. Formalin-fixed and paraffin-embedded tumor Interpretation of Immunohistochemical Stainings samples were cut into 4 mm thick sections. Before immunohistochemistry, the sections were deparaffi- One slide of each sample was observed under a light nized in xylene and rehydrated through a graded microscope for intravascular tumor invasion. The Modern Pathology (2010) 23, 1151–1156 Merkel cell carcinoma HM Kukko et al 1153 observers were blinded regarding clinical data and death was analyzed with a log rank test and disease outcome. Vascular invasion was defined as a overall survival analyses were performed using the cluster of tumor cells within a vascular lumen, Kaplan–Meier method (NCSS 2000, Kaysville, identified by CD31 or D2-40 staining. Lymphovas- UT, USA). cular invasion was identified as a tumor embolus The study was approved by the Ethics Committee within a D2-40 positive lining (Figure 1a and b). of the Helsinki University Central Hospital. The Blood vascular invasion was identified as a tumor Ministry of Health and Social Affairs granted embolus within a CD31 positive structure that was permission to collect the patient data, and the negative in D2-40 staining (Figure 2a and b). Nikon National Authority for Medicolegal Affairs granted DS-5M-L2 camera was used for microscope image permission to collect the tissue samples for study acquisition. purposes. The correlation between tumor size and vascular invasion status was calculated using analysis of variance after logarithmic transformation, because Results the study population was not in a normal distribu- tion.