ORIGINAL ARTICLE Interval sentinel lymph nodes in melanoma: a digital pathology analysis of Ki67 expression and microvascular density Christian Marinaccio 1 • Giuseppe Giudice 2 • Eleonora Nacchiero 2 • Fabio Robusto 3 • Giuseppina Opinto 4 • Gaetano Lastilla 4 • Eugenio Maiorano 4 • Domenico Ribatti 1,5 Received: 17 June 2015 / Accepted: 22 July 2015 Ó Springer-Verlag Italia 2015 Abstract The presence of interval sentinel lymph nodes in melanoma is documented in several studies, but con- troversies still exist about the management of these lymph nodes. In this study, an immunohistochemical evaluation of tumor cell proliferation and neo-angiogenesis has been performed with the aim of establishing a correlation between these two parameters between positive and negative interval sentinel lymph nodes. This retrospective study reviewed data of 23 patients diagnosed with mela- noma. Bioptic specimens of interval sentinel lymph node were retrieved, and immunohistochemical reactions on tissue sections were performed using Ki67 as a marker of proliferation and CD31 as a blood vessel marker for the study of angiogenesis. The entire stained tissue sections for each case were digitized using Aperio Scanscope Cs whole-slide scanning platform and stored as high-resolu- tion images. Image analysis was carried out on three selected fields of equal area using IHC Nuclear and Microvessel analysis algorithms to determine positive Ki67 nuclei and vessel number. Patients were divided into positive and negative interval sentinel lymph node groups, and the positive interval sentinel lymph node group was further divided into interval positive with micrometastasis and interval positive with macrometastasis subgroups. The analysis revealed a significant difference between positive and negative interval sentinel lymph nodes in the percentage of Ki67-positive nuclei and mean vessel number suggestive of an increased cellular proliferation and angiogenesis in positive interval sentinel lymph nodes. Further analysis in the interval positive lymph node group showed a significant difference between micro- and macrometastasis subgroups in the percentage of Ki67-positive nuclei and mean vessel number. Per- centage of Ki67-positive nuclei was increased in the macrometastasis subgroup, while mean vessel number was increased in the micrometastasis subgroup. The results of this study suggest that the correlation between tumor cell proliferation and neo-angiogenesis in interval sentinel lymph nodes in melanoma could be used as a good predictive marker to distinguish interval positive sentinel lymph nodes with micrometastasis from interval positive lymph nodes with macrometastasis subgroups. Keywords Angiogenesis Á Interval lymph node Á Melanoma Á Metastasis Á Tumor progression Introduction The sentinel lymph node (SLN) is the first lymph node receiving direct lymphatic drainage from a primary cancer site [1]. In melanoma, lymphoscintigraphy is used to identify the diffusion of the sequential lymph node basins & Domenico Ribatti ribatti@anatomia.uniba.it; domenico.ribatti@uniba.it 1 Department of Basic Medical Sciences, Neurosciences and Sensory Organs, University of Bari Medical School, Policlinico - Piazza G. Cesare, 11, 70124 Bari, Italy 2 Department of Plastic and Reconstructive Surgery, University of Bari Medical School, Bari, Italy 3 Mario Negri Sud Foundation, Santa Maria Imbaro, Italy 4 Department of Emergency and Organ Transplantation, University of Bari Medical School, Bari, Italy 5 National Cancer Institute ‘‘Giovanni Paolo II’’, Bari, Italy 123 Clin Exp Med DOI 10.1007/s10238-015-0379-9