Figure 1 Double immunostaining CC5013 for CD8 (red) and CK22 (brown): (A) Overview ( �� 10) and (B) high power field ( �� 40) of the invasive front of colorectal cancer showing CK22 positive tumour buds surrounded by CD8+ T-lymphocytes. Evaluation of tumour buds and CD8+ T-lymphocytes Tumour budding was defined as an isolated single cancer cell or a cluster of up to five cells at the invasive front of colorectal cancer (Prall, 2007). The tumour border was scanned at a �� 100 magnification and the area of most intense budding was identified (Ueno et al, 2002). After selecting this field, the number of buds was counted using a 40 �� objective lens to focus specifically on the presence of CD8+ T-cells most highly related to the microenvironment surrounding the tumour buds.

Within this same field, all CD8+ lymphocytes were individually counted. In 10 cases, the abundance of CD8+ infiltrate led to cell counts exceeding 200 cells per field, and thus single cell counting was not feasible. These cases were assigned a CD8+ score of 200 cells. The ratio of CD8+ T-lymphocytes to the number of tumour buds (CD8+/buds index) was obtained. In 13 cases when zero buds were identified, the count of CD8+ lymphocytes was not carried out. Clinico-pathological characteristics Of these 300 cases, 279 were evaluable for CD8 and CK22 protein expression, simultaneously. Hematoxylin and eosin (H&E) stained slides were reviewed and histomorphological data included histological subtype, pT stage, pN stage and tumour grade. The tumour border configuration and the presence of conspicuous peritumoural lymphocytic infiltration were defined according to Jass et al (1986).

Clinical data were retrieved from patient records and included age at diagnosis, gender, tumour location and follow up. Clinical outcome of interest was disease-specific survival time, which was available for all 279 patients. Median follow-up time was 60 months. In all, 128 patients died of disease. Patient characteristics are listed in Table 1. Table 1 Characteristics of patients in Cohort 1 (n=279) Microsatellite instability (MSI) status, KRAS and BRAF gene status Genomic DNA was obtained from primary tumours using NucleoMag 96 Tissue Kit (Macherey Nagel, Oensingen, Switzerland) protocol and processed in the Xiril X-100 robot (Xiril, Hombrechtikon, Switzerland). Briefly, punched tissue was lysed in proteinase K.

B-beads and MB2 buffer were added to the cleared lysate and shaken for 5min at room temperature. The supernatant was removed and MB3 was added followed by shaking and supernatant Brefeldin_A removal. The genomic DNA was eluted with MB6 buffer. Genomic DNA was amplified by PCR using AmpliTaq Gold polymerase (Applied Biosystem, Foster City, CA, USA). KRAS (exon 2, codon 12 and 13) and BRAF (exon 15, codon 600) were amplified by a first and a nested PCR.