Detection ofMelanoma Cells DisplayingMultiple Genomic Changes in Histopathologically Negative Sentinel Lymph Nodes

Purpose: Improved detectionof early-disseminatedmelanoma cells may eventually translate into more effective patient care.Wepresent a novel strategy for detectionof melanoma cells in sentinel lymphnodes and confirm their malignant descent by genomic characterization. Experimental Design: In sentinel lymph nodes from 358 melanoma patients, we prospectively compared the rates of tumor cell detection between immunocytochemistry using HMB45 and Melan A antibodies on disaggregated lymph node samples and standard histopathology (H&E staining and immunostaining on tissue sections). Immunocytochemical melanoma cell detection was controlled by testing lymph node samples from 59 nonmelanoma patients and by isolation and comparative genomic analysis of 30 antigen-positive cells. Results: Of the 358 patients, 43 (12%) were positive by standard histopathology, whereas HMB45 immunocytochemistry detected159 of 358 (44%) positive patients. None of the control samples reacted with the HMB45 antibody. Reexamination of samples that were classified as negative by histopathology revealed that extensive serial sectioning would be necessary to achieve sensitivity similar to HMB45 immunocytochemistry. Interestingly, both the number of immunocytochemically positive samples and the number of positive cells in the sentinel node correlated with the thickness of the primary tumor (r = 0.34; P = 0.001and P < 0.0001, respectively). Twenty-four of 30 isolated immunocytochemically positive cells (80%) displayed chromosomal aberrations, some of whichwere isolated fromhistopathologically negative nodes. Conclusion: Immunocytochemical detection of melanoma cells in sentinel lymph nodes is superior to standard histopathology. It remains to be determined whether the detection and genomic characterization of isolated melanoma cells in sentinel lymphnodes will provide relevant prognostic information. Sentinel lymph node biopsy is a surgical technique for detecting micrometastatic disease in draining regional lymph nodes. It has been widely accepted as the preferred method to determine the pathologic status of the regional lymph nodes in patients with malignant melanoma and the resulting information has been incorporated into the latest version of the American Joint Committee on Cancer staging system for cutaneous melanoma (1). Once the sentinel lymph node is removed, accurate nodal staging depends on the ability to detect metastatic disease in the lymphatic tissue. To this end, mainly two approaches have been used thus far, histopathologic examination of tissue sections and reverse transcriptase-PCR detection of melanoma specific mRNA sequences. The detection rate for melanoma cells in tissue sections of sentinel lymph nodes by histopathology ranges between 9% and 34% depending on patient characteristics, the number of tissue sections examined, and whether or not immunohistochemistry is applied (2–5). Therefore, whereas histopathologic screening of 139 serial tissue sections enables the detection of f10 tumor cells in a lymph node (6, 7), it would be too laborious to be done routinely. It is evident that the drawback of histopathology limited to a few sections depends on the unpredictable spatial distribution of the tumor within the sentinel node. In theory, PCR-based methods should overcome this problem by completely homogenizing the sample. They rely on mRNA isolation from lysed lymph node tissue and on targeting of melanoma associated mRNA sequences such as tyrosinase, Melan A (Mart 1), and gp100 (HMB45). However, although reverse transcriptase-PCR methods result in higher detection rates than histopathology (up to 70%; refs. 8, 9), they are prone to false positives due to marker gene expression by melanophages, intranodal nerves, benign naevus cells, and illegitimate gene expression of marker genes by other nonmelanocytic cells. Imaging, Diagnosis, Prognosis Authors’ Affiliations: Universit@ts-Hautklinik, Eberhard-Karls-Universit@t, Tˇbingen, Germany Institut fˇr Immunologie, Ludwig-Maximilians-Universit@t, Munich, Germany Institut fˇr Pathologie, Eberhard-Karls-Universit@t, Tˇbingen, Germany and Institut fˇr Medizinische Biometrie, Eberhard-Karls-Universit@t, Ť bingen, Germany Received 9/29/04; revised 3/2/05; accepted 3/9/05. Grant support: University of Tˇbingen grants 787-0-0 and 78-0-0, theWilhelm Sander Stiftung grant 2003.005. 1, and the BioFuture grant 0311884 from the Bundesministerium fˇr Bildung und Forschung. The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Note: J. Fischer is presently at the Universit@ts-Hautklinik, Eberhard-KarlsUniversit@t, 72076 Ť bingen, Germany. Requests for reprints: Christoph Klein, Institut fˇr Immunologie, Goethestr. 31, 80336 Mˇnchen, Germany. Phone: 89-5996-696; Fax: 011-49-89-5996-696; E-mail: christopher.klein@med.uni-muenchen.de. F2005 American Association for Cancer Research. www.aacrjournals. org Clin Cancer Res 2005;11(15) August1, 2005 5425 Research. on August 4, 2017. © 2005 American Association for Cancer clincancerres.aacrjournals. org Downloaded from Here we present a novel strategy for melanoma cell detection that uses immunocytochemical, microscopic identification of melanoma cells in disaggregated sentinel lymph nodes, thus avoiding false positives resulting from illegitimate mRNA transcription and sampling errors resulting from examination of a limited number of slides. We verify the neoplastic origin by isolation of single positive cells and their genomic analysis using comparative genomic hybridization (10, 11). Thereby, we show that in a substantial number of clinically early-stage melanoma patients genomically aberrant tumor cells can be detected in sentinel lymph nodes that were classified to be free of tumor cells by routine histopathology. Patients andMethods Patients. We included 494 sentinel lymph nodes from 358 patients with clinical early-stage cutaneous malignant melanoma who underwent lymphatic mapping and sentinel lymph node biopsy at the University of Tuebingen from February 2000 until July 2003. Patient data and characteristics of the primary tumor are summarized in Table 1. Staging was done according to the American Joint Committee on Cancer guidelines from 2001 (1). The Tuebingen Ethics Committee approved all aspects of the presented study. Lymphatic mapping and sentinel lymph node biopsy. Preoperatively, a cutaneous lymphoscintigraphy was done after intracutaneous injection of 100 MBq (3 mCi) Tc-labeled nanocolloids. The technique was supplemented by injection of 1 to 2 mL isosulfan blue dye (Patent blue-V, Altana Pharma, Konstanz, Germany). Sentinel lymph nodes were detected by significant in vivo radioactivity and/or by the presence of blue dye in the afferent lymphatic vessels of the sentinel lymph node or the sentinel lymph node itself. For accurate localization of radiolabeled lymph nodes, a hand-held g probe (C-Trak, Care, Wise Medical Products, Morgan Hill, CA) was used intraoperatively. Preparation of the lymphatic tissue and histopathologic examinations. The lymph node was cut along its longitudinal axis. One half of the sentinel lymph node was fixed in 3.5% formaldehyde, paraffinembedded, and subjected to standard histopathologic work-up which included H&E staining and immunohistochemistry on three paraffin sections from one central level. For immunostaining, antibodies against HMB-45 or Melan A and S-100 (DAKO, Hamburg, Germany) were used and detected according to the avidin-biotin-peroxidase