Detection of Merkel cell virus and correlation with histologic presence of Merkel cell carcinoma in sentinel lymph nodes

British Journal of Cancer (2012) 106, 1314 – 1319 & 2012 Cancer Research UK All rights reserved 0007 – 0920/12 www.bjcancer.com Detection of Merkel cell virus and correlation with histologic presence of Merkel cell carcinoma in sentinel lymph nodes M Loyo*,1, J Schussel1, E Colantuoni2, J Califano1, M Brait1, S Kang3, WM Koch1, D Sidransky1, WH Westra1,4 and JM Taube3,4 1 Division of Head and Neck Cancer Research, Department of Otolaryngolgy Head and Neck Surgery, The Johns Hopkins School of Medicine, 1550 Orleans Street CRBII 5NC, Baltimore, MD 21321, USA; 2The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA; 3 Department of Dermatology, The Johns Hopkins School of Medicine, Baltimore, MD, USA; 4Department of Pathology, The Johns Hopkins School of Medicine, Baltimore, MD, USA Molecular Diagnostics BACKGROUND: Adjuvant treatment can dramatically improve the survival of patients with metastatic Merkel cell carcinoma (MCC), making early, accurate detection of nodal disease critical. The purpose of this study was to correlate Merkel cell virus (MCV) detection with histopathologic disease in sentinel lymph nodes (SLNs) of MCC. METHODS: Merkel cell carcinoma cases with SLN (n ¼ 25) were compared with negative controls (n ¼ 27). Viral load was obtained by quantitative polymerase chain reaction (PCR) for regions VP1 and LT3 of MCV. Histopathologic disease and viral load were correlated. RESULTS: Merkel cell virus was detected in 16 out of 17 (94%) of primary MCC (mean viral load (MVL) ¼ 1.44 copies per genome). Viral load in the negative controls was o0.01 copies per genome. Merkel cell carcinoma was present in 5 out of 25 (20%) SLN by histopathology, and MCV was detected in 11 out of 25 (44%) MCC SLN (MVL ¼ 1.68 copies per genome). In all, 15 out of 25 (60%) SLN showed correlation between histologic and MCV results. In all, 2 out of 25 (8%) samples were histopathologically positive and PCR negative. Of note, 8 out of 25 (32%) samples had detectable MCV without microscopic disease. CONCLUSION: Patients with positive SLN for MCV even if negative by histopathology were identified. The application of molecular techniques to detect subhistologic disease in SLN of MCC patients may identify a subset of patients who would benefit from adjuvant nodal treatment. British Journal of Cancer (2012) 106, 1314 – 1319. doi:10.1038/bjc.2012.73 www.bjcancer.com Published online 13 March 2012 & 2012 Cancer Research UK Keywords: Merkel cell carcinoma; Merkel cell polyomavirus; sentinel lymph node; tumour staging; RT – PCR Merkel cell carcinoma (MCC) is a rare, aggressive neuroendocrine carcinoma of the mechanoreceptors in the skin. It has a high propensity for early, regional lymph node metastases. Merkel cell carcinoma has been associated with a newly described polyoma virus, the Merkel cell polyomavirus (MCV or MCPyV) (Feng et al, 2008). Merkel cell virus is detected in as many as 80 – 90% of MCC studied (Feng et al, 2008; Busam et al, 2009; Shuda et al, 2011). It has also been detected in low levels in normal skin, in other inflammatory and neoplastic cutaneous diseases, and in nonlesional skin from patients with MCC (Dworkin et al, 2009; Andres et al, 2010; Foulongne et al, 2010). Despite the seemingly ubiquitous nature of the virus, a combination of findings implicates the virus in the tumourigenesis of MCC. These include the significantly higher prevalence and viral load of MCV DNA in MCC compared with other diverse benign and malignant human tissue samples (Loyo et al, 2010), the epidemiologic association with elderly and immunosuppressed patients (Penn and First, 1999; Engels et al, 2002; Rubel et al, 2002; Albores-Saavedra et al, 2010), the integration of virus before clonal expansion of tumour *Correspondence: Dr M Loyo; E-mail: Received 15 November 2011; revised 14 February 2012; accepted 17 February 2012; published online 13 March 2012 (Feng et al, 2008), the presence of signature viral mutations in tumours (Shuda et al, 2009), and the expression of viral oncoproteins such as large and small T antigen (Sastre-Garau et al, 2009; Shuda et al, 2009, 2011). Evidence suggests that sentinel lymph node biopsy (SLNB) has both prognostic and therapeutic implications for patients with MCC. Patients with lymph node metastases demonstrate a two- to three-fold higher mortality rate when compared with those without nodal involvement (Shaw and Rumball, 1991; Yiengpruksawan et al, 1991). Sentinel lymph node biopsy aids in the detection of microscopic nodal disease, identifying an additional one-third of patients with nodal involvement who would have been understaged by clinical staging or imaging alone (Gupta et al, 2006). Such detection is critical as there is a significant survival benefit for patients receiving adjuvant nodal therapy when there is histologic evidence of lymph node involvement (Gupta et al, 2006). This finding has been further substantiated in a recent study of 5823 MCC patients using data from the National Cancer Data Base, which demonstrated that node-negative status as demonstrated by pathologic evaluation was a better predictor of survival than nodenegative status by clinical nodal evaluation alone (Lemos et al, 2010), suggesting that a proportion of patients in the latter group actually had occult microscopic nodal involvement. The difference MCV correlation with MCC histology in SLNs M Loyo et al 1315 PATIENTS AND METHODS Patient specimens Study approval was obtained from the Johns Hopkins Institutional Review Board (IRB 00034420). The Johns Hopkins Hospital surgical pathology archives were searched for cases of MCC where an SLN biopsy had been performed (n ¼ 25). The SLN biopsy protocol consists of an intradermal injection of technectium-99mlabelled sulphur colloid to the primary tumour site about 2 h prior to the surgical procedure to allow for the detection of nodal drainage. Intraoperatively, a g detector is used to plan the surgical incision and locate the SLN. Isosulfan blue dye may also be used at the discretion of the operating surgeon at the time of surgery to aid in identification. Sentinel lymph node was defined as the LN that concentrated the highest radiolabel colloid (‘hottest node’). In cases with multiple lymph nodes designated as ‘sentinel’, the one labelled #1 by the surgeon was studied in an effort to include the node with the highest chance of harbouring metastatic disease and decreasing surgeon variability. Routine haematoxylin and eosin (H&E) staining was performed on the formalin-fixed, paraffin-embedded SLNs from each patient. In addition, before classifying a case as either positive or negative for MCC, at least one immunostain (AE1/AE3, Cam5.2, CK20, synaptophysin, or chromogranin) was performed to confirm the anticipated paranuclear dot-like pattern in positive cases (shown in Supplementary Figure 1) or to exclude subtle lymph node metastases in cases that were negative by H&E. Tissue blocks from the SLN and the cor (...truncated)