ICAM-2 Limits Metastasis Figure 6. Immunohistochemical analyses ICAM-2 expression with a tissue microarray containing primary tumor specimens from 90 individual patients showed that a relatively high level of ICAM-2 expression was associated with primary tumor cells recognized to have limited metastatic potential. ICAM-2 antibody binding was detected using a peroxidase-tagged secondary antibody and DAB substrate (brown), by standard methods. Specimens were counterstained with hematoxylin/eosin, to visualize morphology of tumor and stromal cells. Staining was scored as: percent of stained cells6staining intensity (0, 1+, 2+, or 3+). The scores for ICAM-2 expression of the Stage 4, Stage 2, and Stage 4S tumor specimens pictured were 0, 270, and 300, respectively. Normal lymphocytes (a positive control) received a score of 100 (100% of lymphocytes6a staining intensity of 1+). Each specimen is described in more detail in the text. doi:10.1371/journal.pone.0003629.g006 neuroblastoma arising in a child younger than 18 months with limited tumor spread to the skin and/or liver that is amenable to surgical resection. Despite its favorable prognosis, stage 4S neuroblastoma is comprised of immature neuroblastoma cells that are microscopically indistinguishable from immature cells seen in high-risk neuroblastoma tumors. The immature neuroblasts (100%) of this stage 4S tumor exhibit strong (3+) ICAM-2 expression (Expression Score = 100%63+ = 300), again reflecting a correlation between relatively high level of expression of ICAM-2 and low potential for progressive metastatic disease. Notably, the contrast between results for ICAM-2 expression of immature neuroblasts in the stage 4 tumor compared to the 4S tumor indicates that ICAM-2 is not simply a differentiation marker. Correlation between ICAM-2 expression and favorable clinicopathologic features While long-term survival data are not yet available for the specific patients whose tumors are included in the COG microarray, the outcome for neuroblastoma patients can be reasonably predicted on the basis of well-characterized criteria that include histology, PLoS ONE | www.plosone.org 8 November 2008 | Volume 3 | Issue 11 | e3629