Comparison of DNA Ploidy Status and DNA Ploidy-Related Parameters in Malignant Melanoma Tissue Microarrays and Full Sections MONIKA KORABIOWSKA, MD, PHD, CARLOS CORDON-CARDO, MD, PHD, NADINE BUSCHMANN, MD, JERZY STACHURA, MD, PHD, GO ¨ STA FISCHER, MD, PHD, AND ULRICH BRINCK, MD, PHD A new high-throughput tissue-arraying technique, now frequently used in tumor pathology, requires standardization of methods of DNA analysis, previously applied in full histological sections. The main objectives of this study were to evaluate DNA ploidy status and DNA ploidy-related parameters using the CAS200 image analyzer in malignant melanoma tissue microarrays and to compare them with full histological sections. Comparison of DNA ploidy-related param- eters, including percentage of diploid cells, percentage of aneuploid cells between 2c and 4c, percentage of tetraploid cells, percentage of aneuploid cells between 4c and 8c, percentage of octaploid cells, percentage of 16-ploid cells, and 5c exceeding rate, did not reveal any significant differences between malignant melanoma tissue microar- rays and full sections. The DNA ploidy status according to Auer differed in 1 out of 59 cases investigated. Our study demonstrated that it is possible to evaluate DNA ploidy status and DNA ploidy- related parameters in tissue microarrays, which is of practical rele- vance to tumor pathology. HUM PATHOL 35:887-891. © 2004 Elsevier Inc. All rights reserved. Key words: melanoma, tissue array, ploidy status, Feulgen stain, CAS200. Image cytometry and flow cytometry are among the methods used to quantify the DNA contents in normal and malignant tissues. These 2 methods are both used in tumor pathology. Comparisons of them have shown that they are similarly effective in quantify- ing DNA for diagnostic purposes. In many studies, how- ever, priority has been given to image cytometry, which is a rapid, cost-effective, and user-friendly method for determining DNA contents. 1-5 Tissue microarrays permit high-throughput molec- ular profiling of tissue specimens using various tech- niques, including immunohistochemistry and in situ hybridization. They thus may be useful for the evalua- tion of large numbers of different molecules potentially involved in tumor development and progression. 6,7 Sev- eral studies have demonstrated a high degree of con- cordance between immunohistochemical data derived from tissue microarrays and full sections. 8,9 Similar studies to evaluate DNA contents and DNA ploidy- related parameters have not yet been performed. The principal objectives of this study were to establish whether tissue microarrays can be used to assess the DNA ploidy status and to compare the DNA ploidy status and DNA ploidy-related parameters in malignant melanoma tissue microarrays and full sections of different stages. MATERIALS AND METHODS Patients The cohort analyzed consisted of 59 patients with malig- nant melanoma with the following distribution of pT stadia: pTis, 6 patients; pT1, 5 patients; pT2, 12 patients; pT3, 14 patients; and pT4, 22 patients. All patients were treated at the University Clinic of the Jagiellonian University, Cracow, Po- land. There were 27 men and 32 women, with an average age of 52 years (range, 31 to 87). Construction of Tissue Microarrays Normal and tumor tissue were embedded in paraffin, then 5-m sections stained with hematoxylin and eosin were obtained to identify viable, morphologically representative areas of the specimen from which core biopsy specimens could be obtained. A 0.2-cm-diameter tissue core was punched from each specimen and arrayed on a recipient paraffin block (Fig 1). Then 5-m sections of these tissue microarray blocks were cut and placed on charged polylysine- coated slides. Analysis of DNA Contents After deparaffinization, 5-m-thick paraffin sections were placed in 5N hydrochloric acid for 60 minutes and stained with Feulgen stain for 1 hour (CAS DNA staining kit; Becton Dickinson, Hamburg, Germany). The slides were then rinsed in acid alcohol, cleaned in xylene, and covered with synthetic medium (Fig 2). Rat hepatocytes stained with Feul- gen were used as control cells. Slides prepared in this way were evaluated with a CAS200 image analyzer (Becton Dick- inson) and quantitative DNA analysis software. The DNA content was quantified by assigning an optical density to each pixel in the image and summing the optical density values for From the Department of Cytopathology, University of Go ¨ttingen, Go ¨ttingen, Germany; Division of Molecular Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY; Department of Pathol- ogy, Jagiellonian University, Cracow, Poland; and Department of Pathology, Reinhard Nieter Hospital, Wilhelmshaven, Academic Hos- pital of the University of Go ¨ttingen, Go ¨ttingen, Germany. Accepted for publication March 17, 2004. Address correspondence and reprint requests to Dr. Monika Korabiowska, Department of Cytopathology, University of Go ¨ttingen, Robert Koch Str. 40, 37075 Go ¨ttingen, Germany. 0046-8177/$—see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2004.03.015 887