Between 1 and 33 lymph nodes per patient (Table 1) were analysed with a Zeiss microscope (Carl Zeiss Co., Oberkochen, Germany) in their entirety
to eliminate regional variation due to the complex architecture of lymph nodes. Each field was recorded using SpotOn software (Brookvale, Australia) and CD4, CD8 and Foxp3+ cells quantified using Image J software (NIH, USA). Frequency of positively stained cells compared with total cells was acquired for each field. All samples were analysed in a double-blinded fashion. Statistical analysis Frequency counts of CD4, CD8 and Foxp3 stained cells from each field were logged to reduce data skewness, with an offset used to adjust zero counts. For each T-cell marker the R statistical software [22] was used to fit a linear mixed model to the logged count data, with a fixed effect term used to represent clinical variables, Bortezomib in vivo and random effects for patient number and lymph node. A separate model was used for each of the available clinical variables: (HDAC inhibitor disease status, differentiation, lymphatic invasion, margin, tumour site). Selleckchem Sotrastaurin In each model linear contrasts were used to assess the presence of differences in logged counts between each of the three disease status groups for each T-cell marker. An identical approach was taken in the analysis of log-ratio data for pairs of T-cell markers (CD4:Foxp3, CD8:Foxp3), with
the log-ratios of counts derived using matched fields from within each lymph node. Results Thirty three patients with stage II colon cancer were included; 13 with and 18 without recurrence after 5 years of follow up. Of the 13 patients with recurrent disease, four recurred locally and nine had systemic
http://www.selleck.co.jp/products/Vorinostat-saha.html disease (seven liver, one lung, and one lung and brain). Patient characteristics are summarised in Table 1. For each patient, between 1 and 33 lymph nodes were available for analysis (median = 10). Within each lymph node, between one and 15 sections were examined for CD4, CD8 and FoxP3 percentage (median = 10). For those nodes for which multiple sections were available, the “”within-node”" standard deviation was calculated to assess the consistency of immunological signal being obtained. Similarly, for those patients from whom multiple lymph nodes were sampled, the “”within-patient”" (i.e., “”between-node”" for the same patient) standard deviation was calculated. Finally the average immunological “”signal “” was calculated for each patient (for each of FoxP3, CD8 and CD4) and used to assess inter-patient variability by determining the “”between patient”" standard deviation. Figure 1 shows immunohistochemical staining for CD4, CD8 and Foxp3 respectively. For all three measures of immunological activity (CD4, CD8 and FoxP3), the within-node variability was around half the level of the within-patient (between-node) variability (CD4: 5.81% vs 10.