A hematoxylinnuclear counterstain was also applied.This was followed by the monoclonal antibody KP utilizing the standardalkaline phosphataseantialkalinephosphatasemethod and new fuchsin substrate as the chromogen.This produced single sections with blood vessels stained brown and macrophages stained red, and a hematoxylin nuclear counterstain was also applied.The EGFR level was determined using a ligand binding method described previously. Tumors with an EGFR level of greater than fmolmg of protein were considered positive.Microvessel density was assessed in the three most vascular areas or hotspots following a brief scan of the entire section at low power.The vascular and macrophage hot spots are chosen rather than random fields, because these areas are thought to be the most biologically important regions.The doublestained sections were assessed for vascular index and M Betamethasone simultaneously by first scanning the section at low power to find the three most highly vascularized areas.The section was then scanned again to determine the three densest areas of macrophage infiltration; each area was then assessed for M and then assessed immediately for vascularity.Data from the series of breast tumors examined was divided by tertiles into three catego ries corresponding to low, medium, and high vascular grade.We have reported previously that the upper third group by vascular grade has a worsened prognosis when compared to the middle and lower groups. The three most dense areas of macrophage content were enumerated simultaneously for macrophage and vascular density; this was followed by the three most dense areas of vascular activity being evaluated in the same manner.That is, the highly angiogenic areas were poorly populated with macro phages when compared to the more macrophagedense fields, which were similarly poorly vascularized.Macrophage density in lowan giogenesis areas was higher than in high angiogenesis areas. This inverse relationship was maintained when highly vascularized and poorly vascularized tumors were examined separately.In this series of cases, a total of patients were evaluable for survival.When examining the effect of M on Silver sulfadiazine overall survival, a similar effect was observed with higher death rates at above the median and in the upper third M class. Examining nodenegative tumors separately, using the median cut point for M, it was found that M,I was a significant predictor of poorer outcome for relapsefree survival in this group. A similar finding was observed when examining overall survival, with M again splitting the lowvascular grade group. However, in the highvascular grade group, M was unable to split the series into two more prognostically significant subgroups in either relapsefree. Dividing the series by high and low MI, it was found that vascular grade was able to further stratify the lowMI group into two prog nostically different subgroups for both relapsefree, with the high vascular grade category having the worse prognosis.This was not observed when vascular grade was used to stratify the highMI group of cases. M was compared to other patient and tumor variables, including age, tumor size, nodal status, grade, ER, and EGFR.Split by median, using v tests at cut point for M, no significant associations were evident.This obser vation could be due either to the presence of more vessels, allowing the infiltration of more macrophages, or the proangiogemc activity of macrophages inducing the proliferation, migration, and differentiation of endothelial cells.